Cancer Associated Viruses, (2012) (UnitedVRG)

Current Cancer Research
Series Editor
Wafik El-Deiry
UnitedVRG
Medical Release Group
For further volumes:

http://www.springer.com/series/7892
UnitedVRG
Erle S. Robertson
Editor
Cancer Associated Viruses
UnitedVRG
Editor
Erle S. Robertson
Professor of Microbiology
Director of the Tumor Virology Training Program
Perelman School of Medicine
University of Pennsylvania
Philadelphia, PA, USA
erle@upenn.edu
UnitedVRG
ISBN 978-1-4419-9999-3
e-ISBN 978-1-4614-0016-5
DOI 10.1007/978-1-4614-0016-5
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011940811
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In Memoriam
Baruch S. Blumberg, M.D., D.Phil.
Baruch Samuel (Barry) Blumberg died suddenly on April 5, 2011 shortly after giving
a presentation on citizen science at the NASA Lunar Science Institute in Ames,
California. Barrys talk concerned making spacecraft data available to the public so
that ordinary people could contribute to its interpretation. That final talk reflected
his deep belief that anyone who was willing to invest time and thought could have
ideas that would lead to new understandings of research information.
This volume is about viruses and cancer, but Barry was neither a virologist nor an
oncologist. However, he contributed fundamentally to both. Barry began his research
career as a medical student at Columbia University when he took an elective in
v
vi
In Memoriam
Tropical Medicine in Suriname. There, he observed that filariasis was rampant, but
that only some of the many infected people showed signs of disease. Suriname had
many different ethnic groups and some of the diversity in responses was associated
with ethnicity. That led him to wonder for the rest of his life why humans living in
the same environment responded so differently to infectious agents.
After an internship and assistant residency in medicine at Bellevue Hospital in
New York City and a fellowship in rheumatology, Barry went to Oxford University
to study biochemistry with Alexander Ogston. He was in the lab at the same time as
Oliver Smithies who also went on to win a Nobel Prize. In England, he was exposed
to the history of scientific discovery and began formulating his scientific ideas. It
was there that he found his scientific inspiration in the lives and work of the nineteenth-century naturalists Charles Darwin and Alfred Russell Wallace. He began to
apply the principles of evolution to his research.
After receiving a doctorate, Barry went to an obscure unit called Geographic
Medicine and Genetics of the National Institute of Arthritis and Metabolic Disease
(NIAMD) in Bethesda, Maryland. He chose this hidden corner because he thought
it would allow him to pursue his own ideas and travel wherever he pleased. He
began a lifelong pattern of collecting blood samples everywhere he went, making
observations about the people from whom they were drawn and often collecting
samples of vegetation from their environments. It was at the NIH that Barry honed
his interest in genetic polymorphisms in human blood, inherited variants of proteins
or blood groups, which he believed were likely to be associated with human diseases. As a believer in the central importance of natural selection, he thought all
such variants had to be important. Otherwise, they would not have persisted in
human populations.
In 1964, the Director of the Institute for Cancer Research (ICR, precursor to the
Fox Chase Cancer Center) recruited Barry to become the head of a new Division of
Clinical Research. The lure was that Barry was promised he could do whatever he
wanted as long as his research ultimately had consequences for disease in humans.
Barry was intrigued and immediately began assembling a small group of physicians
to staff this new enterprise. He had complete faith that his approach: identifying
variants in human blood and then finding out what they meant, would be much more
informative than starting with a disease and trying to identify its causes. It was this
approach that first resulted in identifying an antigen on a lipoprotein in serum, and
subsequently to a different antigen, the Australia antigen.
Barry focused the efforts of his new division on understanding the biological
significance of Australia antigen. In a series of studies of diseases associated with
the antigen, the group found that Australia antigen was closely associated with one
form of viral hepatitis, later called hepatitis B. At the same time, they observed that
Australia antigen was a particle similar in appearance to a virus. That was enough
information for Barry to begin to develop a unique vaccine, one that was prepared
from antigenic particles in human blood. The patent for the vaccine was submitted
in 1969 and granted in 1971. By 1975, before the vaccine had even been tried in
humans, Barry predicted in print that the vaccine would not only prevent infection
In Memoriam
vii
with the hepatitis B virus but that it would also prevent liver cancer. Therefore, it
would be the first cancer vaccine.
In 1976, Barry was awarded the Nobel Prize in Physiology or Medicine for discoveries concerning new mechanisms for the origin and dissemination of infectious
diseases. Very few people are privileged to win a Nobel Prize. Barry made his success a celebration for everyone in his group. He took as many of his colleagues and
staff in the division to the ceremony as he was permitted, and least 15 and their
spouses.
In 1989, he returned to Oxford to become the Master of Balliol College serving
until 1994. Balliol was founded in 1263 and Barry was its first American Master.
From 1999 to 2002, he was director of the NASA Astrobiology Institute. He continued his affiliation with NASA and in 2008 became a Senior Scientist at the NASA
Lunar Science Institute. In 2005, he became the President of the American
Philosophical Society, founded by Benjamin Franklin, and the oldest learned society in the Americas.
Barry was always a happy person. He celebrated his own life by living it to the
fullest. He cycled, hiked, ran, rock-climbed, canoed, and kayaked until the end of
his life. Barry had a long and happy marriage. He was always proud of the accomplishments of his wife, Jean, his four children and nine grandchildren. He left a
legacy of accomplishments that saved an enormous number of lives and prevented
hundreds of millions of people from becoming ill with the hepatitis B virus. On
every continent, his many friends and colleagues mourn his loss.
Fox Chase Cancer Center
W. Thomas London
Preface
For almost 30 years, there has been no comprehensive text covering the many viral
agents and their contributions to cancers or cell proliferation. The goal was to provide a relatively up-to-date tome, which would be a wonderful resource for the
many investigators in the field of viral oncology. The chapters are meant to be a
thorough review of the literature, which covers specific viruses as well as provide
some synthesis of what we now know about viruses, cell proliferation and the genes
that target specific cellular pathways. The previous works are outdated, and as this
book is put to press, we would still have additional works to be published that will
certainly be missed. We have tried to be as comprehensive as possible within the
guidelines of the text without sacrificing the science and we have allowed authors
much flexibility that would only be fitting if one takes on a job to complete a chapter
that is as comprehensive and current as we have attempted in this book.
The primary goal here was to provide the most comprehensive version of chapters covering the majority of viruses and cancers, which will be a major resource
for all trainees in the field of viral oncology from undergraduates and graduate
students to post-doctoral fellows in basic science and translational or clinical studies, as well as investigators related to viral oncology. This approach was certainly
limited as we will, without a doubt, be missing some of the detail and intricate
nuances of each viral system. That said, we certainly tried to encourage a general
theme throughout and so the experienced readers in the field may find some aspects
of it less inviting. Nevertheless, I think that overall, we have attained a level of
scientific sophistication for each of the chapters that it would be worth the time of
experts in the field to read and it would be a solid contribution enjoyed by all
including novices, as well as the experts. Thus, I take full responsibility for any
omission that may have occurred, unintentionally. I also want to say that each contributor has done a fantastic job in completing his or her chapter, and that one
would have to give them all a tremendous thank-you for their efforts in making this
project happen even with the burdensome task of meeting deadlines and responding
to my many emails in nudging them along.
The book begins with a chapter that introduces the father of viral oncology
Professor Peyton Rous with some of his many interesting findings and his training,
ix
Preface
as well as his international interactions with many scientists across the world.
This year would be the 100th anniversary of the initial discovery of the Rous
Sarcoma Virus. This is followed by a chapter contributed by Baruch Bloomberg,
one of the many Nobel laureates whose contributions to the field has made a huge
impact in saving lives throughout the world. He was passionate about pushing for
the development of the hepatitis B vaccine and in doing so, led to the vaccination of
millions throughout the world who would have been infected and would have a
higher probability of developing hepatocellular carcinoma. He presents a historical
perspective on viruses and cancer. The chapters by Drs. Jae Jung, Blossom Damania
and Robin Weiss present a broad outline of how viruses can contribute or drive the
oncogenic process and the potential for cancer transmission. Dr. Alwine wrote the
introductory chapter for the DNA tumor viruses and suggests that while some large
DNA viruses may not be able to directly transform a cell, they can certainly alter
signaling and metabolism in ways that can certainly drive the transformation and
possible immortalization of the infected cells. The chapter by Dr. Bala Chandran
brings together the many contributions by the large DNA herpesviruses and their
ability to induce the oncogenic process. Further on this theme, we also cover specific chapters on viruses in herpesviridae with my group looking at the
Lymphocryptoviruses; Dr. Schultz on the Rhadinoviruses; Dr. Rose on the contribution of the retroperitoneal fibromatosis herpesvirus to retroperitoneal fibromatosis,
a Kaposis sarcoma-like disease in macaques with simian AIDS; Dr. Wong exploring the viruses in nonhuman primates; Dr. Speck presenting the murine herpesvirus
model of tumorigenesis; and Drs. Parcells and Morgan who describe the Mareks
disease virus and its contribution to T-cell lymphomas in chickens. These chapters
provide an in-depth analysis of these viral agents and their similarities and differences in driving the oncogenic process.
We have had a great deal of success in bringing in a number of talented investigators looking at the small DNA tumor viruses, in particular the Polyoma and Papilloma
viruses as well as the Adenoviruses. Dr. Gjoerup did a fantastic job in describing the
many facets of the Polyomas and their contribution to cancer and this was followed
by chapters from Drs. Butel, Hirsch, Khalili and Becker who provided a thorough
review of the SV40 as a model system, the BK virus, the JC virus and the new
Merkel cell Polyoma virus, respectively. I should also mention at this stage, that
more recently in July 2010, there was a report of another virus, which belongs to the
Polyoma virus family now called Trichodysplasia Spinulosa-associated Polyoma
virus (TSV), which was identified in a rare skin disease called Trichodysplasia
Spinulosa, exclusively seen in immunocompromised patients. It is yet to be seen if
this is a ubiquitous virus in the population, which becomes opportunistic in these
group of patients. A review of the Papilloma viruses covering the HPV and BPV
systems was completed by Drs Jianxin You and Suzannne Wells. Adenoviruses,
another group of oncogenic DNA viruses, were also included, although to date there
has been no direct association with Adenoviruses and human cancers. However,
there has been a wealth of information over more than 30 years showing that
Adenoviruses are fully capable of meeting the major criteria for driving the oncogenic process using in vitro studies and also inducing tumors in an animal model.
Preface
xi
The Hepadnaviruses have also been addressed in the compendium, where we

take a closer look at the contributions of the hepatitis viruses B and C. Professor
Tim Block has done a marvelous job of reviewing the many general attributes of the
hepatitis viruses and the cancers they are associated with from a virological to a
more molecular perspective. This is followed by chapters on hepatitis B virus by
Dr. Mason from the Fox Chase Cancer Center, where Barry Bloomberg spent a
great many of his years as a scientist working on the hepatitis virus. Dr. Mason did
a fantastic job in getting us up to date on the causes of chronic liver disease, cirrhosis and many years later HBV-induced hepatocellular carcinoma, which takes at
times as long as 40 years. Dr. Bret Lindenbach explores the contributions of hepatitis C virus to the development of hepatocellular carcinoma and summarizes the
clinical and molecular virology links between the HCV virus and HCC. Dr. Kathleen
Boris-Lawrie finds an interesting angle to explore further the role of HIV-1 as a risk
factor for the development of malignancies in AIDS patients by describing why
Kaposis sarcoma, non-Hodgkins lymphoma and cervical carcinomas can function
as prognostic indicators of AIDS and begins to suggest the relationship of coinfection and how this may contribute to the oncogenic process. We also cover the
HTLV-1 and HTLV-2 where their contributions to cell proliferation were well
described. Dr. Chou-Zen Giam focuses on the role of HTLV-1 in causing adult T-cell
leukemia paying attention to the role of two viral proteins Tax and HBZ in viral
replication and leukemogenesis. Dr. Patrick Green focuses on the biology and
pathogenesis of HTLV-2 and further dissects the various cellular processes utilized
by the virus in contributing to cell proliferation. The chapter on avian and murine
retroviruses was skillfully put together by Drs. Karen Beemon and Naomi Rosenberg.
This chapter provides information on viral oncogenes and the cooperation between
these viral oncogenes as a major step in the development of cancer. They also
describe the potential role of these viruses as vector systems. Dr. Leslie Parent goes
into further detail in contributing the chapter on the Rous Sarcoma Virus which
takes the reader from the provirus concept and how the integrated provirus, which
certainly has transforming activities, led to the identification of the a cellular gene
highly homologous to the viral transforming gene. Dr. Susan Ross thoroughly presents the mouse mammary tumor viruses (MMTV), which can cause breast cancer in
mice by causing insertional activation of mutation of cellular oncogenes and provides an extremely useful model for understanding human breast cancer. Another
very interesting virus is the Jaagsiekte Sheep retrovirus, which is associated with
lung cancer. This virus causes ovine pulmonary adenocarcinoma which is derived
from the secretory lung epithelial cells. Dr. Hung Fan describes here how the pathology of the virus is directly linked to the envelope protein (Env) and that JSRV is
mostly pathogenic in the lung as the viral LTR is transcriptionally active only in
differentiated airway epithelial cells. Drs. Renne and Swaminathan now contributed
a chapter on the small RNAs and their role in viral-mediated cancers. The final
chapter was a wonderful contribution by Dr. Charles Wood, who explores the role
of immunodeficiency and opportunistic infections and their cooperation in driving
the oncogenic process.
xii
Preface
Finally, I wanted to personally thank all the authors for their patience and their
hard work in getting their contributions to me as timely as can be expected. Even the
folks who were somewhat tardy in their delivery have made it worthwhile, and I can
say overall that I am personally happy with the final product. I hope this project
provides a renewed vigor to our community of scientists to explore the contributions
of viruses to cancer. In my many discussions with investigators in the cancer field it
is still amazing and a bit puzzling to me that a great many are still hesitant to
acknowledge that viruses or infectious agents on a whole have much to do with
cancer, even though it is well known that about 20% of all known cancers are associated with infectious agents. I hope this renewed thrust will minimize these concerns and provide new support for the many investigators who have spent their
entire lives working towards understanding how viruses contribute to the oncogenic
process. Happy reading.
Cheers
Erle S. Robertson
Contents
Peyton Rous: A Centennial Tribute to the Founding

Father of Cancer Virology......................................................................
Volker Wunderlich and Peter Kunze
Viruses and Cancer: A Historical Perspective HBV

and Prevention of a Cancer ....................................................................
Baruch S. Blumberg
25
Virus-Mediated Cell Proliferation .........................................................

Sun-Hwa Lee, Stacy Lee, and Jae Ung Jung
45
Viral-Encoded Genes and Cancer .........................................................

Blossom Damania
81
Oncogenic Viruses and Cancer Transmission ...................................... 101

Robin A. Weiss
DNA Viruses and Cancer: Taking a Broader Look ............................. 119

James C. Alwine
Herpesviruses and Cancer ..................................................................... 133

David Everly, Neelam Sharma-Walia, Sathish Sadagopan,
and Bala Chandran
Lymphocryptoviruses: EBV and Its Role in Human Cancer ............. 169

Santosh Kumar Upadhyay, Hem Chandra Jha, Abhik Saha,
and Erle S. Robertson
Nonhuman Primate Gamma-herpesviruses and Their Role

in Cancer .................................................................................................. 201
Ryan D. Estep and Scott W. Wong
10
Rhadinoviruses: KSHV and Associated Malignancies ........................ 215

Susann Santag and Thomas F. Schulz
xiii
xiv
Contents
11
Retroperitoneal Fibromatosis Herpesvirus and Kaposis

Sarcoma-Like Tumors in Macaques ...................................................... 251
Laura K. DeMaster and Timothy M. Rose
12
Murine Gammaherpesvirus-Associated Tumorigenesis...................... 267

Kathleen S. Gray and Samuel H. Speck
13
Mareks Disease Virus-Induced T-Cell Lymphomas ........................... 307

Mark S. Parcells, Joan Burnside, and Robin W. Morgan
14
Polyomaviruses and Cancer ................................................................... 337

Ole Gjoerup
15
Polyomavirus SV40: Model Infectious Agent of Cancer ..................... 377

Janet S. Butel
16
BK Polyomavirus and Transformation ................................................. 419

Tina Dalianis and Hans H. Hirsch
17
Polyomavirus JC and Human Cancer: Possible Role

of Stem Cells in Pathogenesis ................................................................. 433
Kamel Khalili, Martyn K. White, Jennifer Gordon,
and Barbara Krynska
18
Merkel Cell Polyomavirus ...................................................................... 449

David Schrama and Jrgen C. Becker
19
Human Papillomaviruses and Cancer................................................... 463

Jianxin You and Susanne Wells
20
Tumorigenesis by Adenovirus Type 12 E1A ......................................... 489

Hancheng Guan and Robert P. Ricciardi
21
Overview of Hepatitis Viruses and Cancer ........................................... 509

Timothy M. Block, Jinhong Chang, and Ju-Tao Guo
22
Hepadnaviruses and Hepatocellular Carcinoma ................................. 531

William S. Mason
23
Hepatitis C Virus and Hepatocellular Carcinoma ............................... 571

Brett Lindenbach
24
Human and Animal Retroviruses: HIV-1 Infection

Is a Risk Factor for Malignancy ............................................................ 585
Amy M. Hayes and Kathleen Boris-Lawrie
25
HTLV-1 and Oncogenesis ....................................................................... 613

Chou-Zen Giam
26
Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology

and Pathogenesis ..................................................................................... 647
Rami Doueiri and Patrick L. Green
Contents
xv
27
Mechanisms of Oncogenesis by Avian and Murine

Retroviruses ............................................................................................. 677
Karen Beemon and Naomi Rosenberg
28
Rous Sarcoma Virus: Contributions of a Chicken Virus

to Tumor Biology, Human Cancer Therapeutics,
and Retrovirology.................................................................................... 705
Leslie J. Parent
29
Mouse Mammary Tumor Virus and Cancer ........................................ 739

Susan R. Ross
30
Jaagsiekte Sheep Retrovirus and Lung Cancer ................................... 755

Chassidy Johnson and Hung Fan
31
Small RNAs and Their Role in Herpesvirus-Mediated Cancers ........ 793

Sankar Swaminathan and Rolf Renne
32
Viral Malignancies in HIV-Associated Immune Deficiency ................ 819

Pankaj Kumar, Veenu Minhas, and Charles Wood
Index ................................................................................................................. 853
Contributors
James C. Alwine Department of Cancer Biology,

Abramson Family Cancer Research Institute, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA
alwine@mail.med.upenn.edu
Jrgen C. Becker Director, Division of General Dermatology,
Department of Dermatology, Medical University of Graz,
Auenbruggerplatz 8, A-8036, Graz, Austria
juergen.becker@medunigraz.at
Karen Beemon Department of Biology, Johns Hopkins University,
Baltimore, MD 21218, USA
KLB@JHU.edu
Timothy M. Block Department of Microbiology and Immunology,
Drexel University College of Medicine, Pennsylvania Biotechnology Center,
Doylestown, PA, USA
Hepatitis B Foundation, Pennsylvania Biotechnology Center,
Doylestown, PA, USA
timothy.block@drexelmed.edu
Baruch S. Blumberg Fox Chase Cancer Center, Philadelphia, PA 19111, USA
baruch.blumberg@fccc.edu
Kathleen Boris-Lawrie Department of Veterinary Biosciences,
Center for Retrovirus Research, Center for RNA Biology, Comprehensive
Cancer Center, Ohio State University, Columbus, OH 43210, USA
boris-lawrie.1@osu.edu
Joan Burnside Department of Animal and Food Sciences,
University of Delaware, Newark, DE 19716, USA
xvii
xviii
Contributors
Janet S. Butel Department of Molecular Virology and Microbiology,

Dan L. Duncan Cancer Center, Baylor College of Medicine,
Houston, TX 77030, USA
jbutel@bcm.edu
Bala Chandran H.M. Bligh Cancer Research Laboratories, Department of
Microbiology and Immunology, Chicago Medical School, Rosalind Franklin
University of Medicine and Science, North Chicago, IL 60064, USA
bala.chandran@rosalindfranklin.edu
Jinhong Chang Department of Microbiology and Immunology,
Doylestown, PA, USA
Jinhong.Chang@drexelmed.edu
Tina Dalianis Department of Oncology-Pathology, Karolinska Institutet,
Cancer Center Karolinska R8:01, Karolinska University Hospital,
171 76, Stockholm, Sweden
tina.dalianis@ki.se
Blossom Damania Department of Microbiology and Immunology
and Lineberger Comprehensive Cancer Center, University of North Carolina
at Chapel Hill, Chapel Hill, NC 27599, USA
blossom_damania@med.unc.edu
Laura K. DeMaster Department of Global Health, University of Washington,
Seattle Childrens Research Institute, Seattle, WA, USA
Rami Doueiri Department of Veterinary Biosciences, The Ohio State University,
Columbus, OH 43210, USA
Ryan D. Estep Vaccine and Gene Therapy Institute, Oregon Health
and Science University, Beaverton, OR, USA
David Everly H.M. Bligh Cancer Research Laboratories, Department of
Microbiology and Immunology, Chicago Medical School,
Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
Hung Fan Department of Molecular Biology and Biochemistry,
Cancer Research Institute, University of California, Irvine, CA 9269, USA
hyfan@uci.edu
Chou-Zen Giam Department of Microbiology and Immunology, Uniformed
Services University of the Health Sciences, Bethesda, MD 20814, USA
cgiam@usuhs.mil
Ole Gjoerup Cancer Virology Program, University of Pittsburgh Cancer Institute,
Research Pavilion Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA
ovg27@pitt.edu
Contributors
xix
Jennifer Gordon Department of Neuroscience and Center for Neurovirology,

Temple University School of Medicine, 3500 N. Broad Street, Philadelphia,
PA 19140, USA
Kathleen S. Gray Department of Microbiology & Immunology,
Emory Vaccine Center, Emory University School of Medicine, Atlanta,
GA 30322, USA
Patrick L. Green Center for Retrovirus Research, The Ohio State University,
Department of Veterinary Biosciences, The Ohio State University, Columbus,
OH 43210, USA
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio
State University, Columbus, OH 43210, USA
Comprehensive Cancer Center and Solove Research Institute, The Ohio State
University, Columbus, OH 43210, USA
green.466@osu.edu
Hancheng Guan Department of Microbiology, School of Dental Medicine,
University of Pennsylvania, Philadelphia, PA 19104, USA
Ju-Tao Guo Department of Microbiology and Immunology,
Doylestown, PA, USA
Amy M. Hayes Department of Veterinary Biosciences, Center for Retrovirus
Research, Center for RNA Biology, Comprehensive Cancer Center,
Ohio State University, Columbus, OH 43210, USA
Hans H. Hirsch Department of Biomedicine, Clinical and Transplantation
Virology, Institute for Medical Microbiology, University of Basel,
Basel, Switzerland
Infectious Disease and Hospital Epidemiology, University Hospital Basel,
Basel, Switzerland
hans.hirsch@unibas.ch
Hem Chandra Jha Department of Microbiology, University of Pennsylvania
Abramson Comprehensive Cancer Center, University of Pennsylvania
Chassidy Johnson Department of Molecular Biology and Biochemistry,
Cancer Research Institute, University of California, Irvine, CA 9269, USA
Jae Ung Jung Department of Molecular Microbiology & Immunology,
University of Southern California, School of Medicine, 2011 Zonal Avenue,
HMR401, Los Angeles, CA 90033, USA
jaeujung@usc.edu
xx
Contributors
Kamel Khalili Department of Neuroscience and Center for Neurovirology,

Temple University School of Medicine, 3500 N. Broad Street, Philadelphia,
PA 19140, USA
kamel.khalili@temple.edu
Barbara Krynska Center of Neural Repair and Rehabilitation and Shriners
Hospitals Pediatric Research Center, Temple University School of Medicine,
3500 N. Broad Street, Philadelphia, PA 19140, USA
Department of Neurology, Temple University School of Medicine,
3500 N. Broad Street, Philadelphia, PA 19140, USA
Pankaj Kumar Nebraska Center for Virology and the School
of Biological Sciences, Morrison Center, University of Nebraska-Lincoln,
Lincoln, NE 68583, USA
Peter Kunze Institute of Pathology Georg Schmorl, 01067, Dresden, Germany
peterku34@web.de
Sun-Hwa Lee Department of Molecular Microbiology & Immunology,
sunhlee@usc.edu
Stacy Lee Department of Molecular Microbiology & Immunology,
Brett Lindenbach Section of Microbial Pathogenesis, Yale University School
of Medicine, 354C Boyer Center for Molecular Medicine, New Haven, CT
06536-0812, USA
brett.lindenbach@yale.edu
William S. Mason Fox Chase Cancer Center, Philadelphia,
PA 19111, USA
ws_mason@fccc.edu
Veenu Minhas Nebraska Center for Virology and the School
Robin W. Morgan Department of Animal and Food Sciences,
Mark S. Parcells Department of Animal and Food Sciences,
Parcells@UDel.edu
Contributors
xxi
Leslie J. Parent Department of Medicine, Penn State College of Medicine,

Hershey, PA 17033, USA
Department of Microbiology and Immunology, Penn State College of Medicine,
Hershey, PA 17033, USA
lparent@psu.edu
Rolf Renne Department of Molecular Genetics and Microbiology,
UF Shands Cancer Center, University of Florida, Gainesville,
FL 32610-3633, USA
rrenne@ufl.edu
Robert P. Ricciardi Department of Microbiology, School of Dental Medicine,
University of Pennsylvania, Philadelphia, PA 19104, USA
Abramson Cancer Center, University of Pennsylvania School of Medicine,
Philadelphia, PA 19104, USA
ricciard@upenn.edu
Erle S. Robertson Department of Microbiology, University of Pennsylvania
erle@upenn.edu
Timothy M. Rose Department of Pediatrics, University of Washington,
Seattle Childrens Research Institute, Seattle, WA, USA
trose@u.washington.edu
Naomi Rosenberg Sackler School of Graduate Biomedical Sciences,
Tufts University School of Medicine, Boston, MA 02111, USA
naomi.rosenberg@tufts.edu
Susan R. Ross Department of Microbiology, University of Pennsylvania
rosss@mail.med.upenn.edu
Sathish Sadagopan H.M. Bligh Cancer Research Laboratories, Department
of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin
University of Medicine and Science, North Chicago,
IL, USA
Abhik Saha Department of Microbiology, University of Pennsylvania
xxii
Contributors
Susann Santag Institute of Virology, Hannover Medical School,

Hannover, Germany
David Schrama Director Division of General Dermatology,
Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8,
A-8036, Graz, Austria
Thomas F. Schulz Institute of Virology, Hannover Medical School,
Hannover, Germany
Schulz.Thomas@mh-hannover.de
Neelam Sharma-Walia H.M. Bligh Cancer Research Laboratories, Department
of Microbiology and Immunology Facilities, Chicago Medical School, Rosalind
Franklin University of Medicine and Science, North Chicago, IL, USA
Samuel H. Speck Department of Microbiology & Immunology,
Emory Vaccine Center, Emory University School of Medicine, Atlanta,
GA 30322, USA
sspeck@emory.edu
Sankar Swaminathan Division of Infectious Diseases, Department of Medicine,
University of Utah School of Medicine, Salt Lake City, UT 84132, USA
sankar.swaminathan@hsc.utah.edu
Santosh Kumar Upadhyay Department of Microbiology,
University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
Robin A. Weiss Division of Infection & Immunity, University College London,
London, UK
r.weiss@ucl.ac.uk
Susanne Wells Department of Microbiology, University of Pennsylvania
Division of Hematology/Oncology, Cincinnati Childrens Hospital,
Cincinnati, OH, USA
Susanne.Wells@cchmc.org
Martyn K. White Department of Neuroscience and Center for Neurovirology,
Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA
19140, USA
Contributors
xxiii
Scott W. Wong Vaccine and Gene Therapy Institute, Oregon Health & Science
University, 505 NW 185th AvenueBeaverton, OR 97006, USA
Division of Pathobiology and Immunology, Oregon National Primate
Research Center, Beaverton, OR, USA
Department of Molecular Microbiology and Immunology,
Oregon Health & Science University, Portland, OR, USA
wongs@ohsu.edu
Charles Wood Nebraska Center for Virology and the School
cwood1@unl.edu
Volker Wunderlich Max Delbrck Center for Molecular Medicine, 13125,
Berlin, Germany
vwunder@mdc-berlin.de
Jianxin You Department of Microbiology, University of Pennsylvania
jianyou@mail.med.upenn.edu
Chapter 1
Peyton Rous: A Centennial Tribute

to the Founding Father of Cancer Virology
Volker Wunderlich and Peter Kunze
Introduction
On December 10, 1966, the American pathologist and cancer researcher Francis
Peyton Rous (18791970) (Fig. 1.1), professor emeritus at the Rockefeller Institute
for Medical Research, New York, was awarded the Nobel Prize for Physiology or
Medicine for his discovery of tumor-inducing viruses. He received the award in
Stockholm from the hands of the Swedish King Gustav VI Adolf (18821973).
Fifty-five years after his discovery (Rous 1911a, b) and forty years after his first
nomination by Karl Landsteiner (18681943) (Nomination Database 19011951),
one of the great scientists of the twentieth century was awarded this long-deserved
honor. His work launched a new era of medicine (Vogt 1996). Amazingly, however,
up to now, science historians have not written a biography of Rous.
Just a few months before the Nobel ceremony, Rous had received the prestigious
Paul Ehrlich and Ludwig Darmstaedter Prize (Germanys supreme medical accolade)
in St. Pauls Church in Frankfurt am Main on March 14, 1966. There, he began his
award acceptance speech with the words:
The joy that moves me on this festive occasion is particularly great because it brings to
mind some personal memories. When I studied at The Johns Hopkins School of Medicine
at the beginning of this century, all young physicians looked to Germany as a model. The
dean of the school, Professor William Welch, an eminent pathologist, had many years earlier
worked with Paul Ehrlich in Breslau (today Wroclaw) [both under the supervision of the
pathologist Julius Cohnheim (18391884)], and upon his return to the United States had
informed the American medical community of the major progress made in Germany during
this time. (Rous 1966: 20) [German in original]
V. Wunderlich (*)
Max Delbrck Center for Molecular Medicine, 13125 Berlin, Germany
e-mail: vwunder@mdc-berlin.de
P. Kunze
Institute of Pathology Georg Schmorl, 01067 Dresden, Germany
E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research,
DOI 10.1007/978-1-4614-0016-5_1, Springer Science+Business Media, LLC 2012
UnitedVRG - Medical Release Group
V. Wunderlich and P. Kunze
Fig. 1.1 Photograph

of Peyton Rous, 1959
in Israel, courtesy of
Dr. Inge Graffi, Berlin.
Photo credit: Weizmann
Institute of Science,
Rehovot, Israel
In fact, during those years, German science (and in a broader sense European
science) was a world leader in many fields within and outside of medicine, not only
in the inner circle of Paul Ehrlich. As a result, it was extremely attractive for young
scientists from other countries to come to Germany to work here temporarily. For a
future career in the USA, proof of European experience could be quite helpful, not
unlike as it is today with many appointments of professors in Germany, where a
previous work stay in the USA is regarded a conditio sine qua non.
If the young Rous, while still a student, dreamed of a sojourn in Germany, this
dream was soon to become reality. In the official Nobel biography, which is based
on Rouss own autobiographical notes, an additional personal recollection of the
laureate is recorded:
[At The Johns Hopkins Medical School] he graduated in 1905 [Doctor of Medicine] and
became an intern in its hospital. Then, finding himself unfit to be a real doctor, he turned
to medical research instead, and for this purpose became an instructor in pathology at the
University of Michigan on a beggarly salary. His work in the laboratory turned out to be
mainly that of a technician because the University had small funds only, but with noble
generosity Professor Alfred [sic] Warthin, head of the Department, came to his rescue, actually
offering to teach summer school in his stead, and give Peyton the sum thus earned, if he
would study German hard and use the money to go for the summer to a certain hospital in
Dresden where morbid anatomy was taught. Dresden in 1907! Exquisite city in an exquisite
land, with no hint of war in the air! (Anonymous 1966)
Rous, who in 1966 was well along in years, could still remember Dresden,
although he seemed to have forgotten the names of the host institution (the specific
hospital) and his Dresden mentor. Thus, these details remained largely unclear and
Peyton Rous
were mentioned only very briefly in the Biographical Memoirs (Andrewes 1971;
Dulbecco 1976) dedicated to Rous. However, his sojourn in Dresden was the only
one abroad before Rous began his exceptionally successful career as independent
research scientist at the Rockefeller Institute, and apparently even in retrospect the
time spent in Dresden was very important to him. In this paper, we report on new
research inquiries concerning Warthin, Schmorl, and Rous. But first, we shall briefly
present some of the pathologists who influenced Rous before his stay in Dresden.
In subsequent sections, we shall briefly present several aspects of Rous work in the
years immediately following his Dresden stay that were crucial for tumor virology.
A Fresh Age of Medical Endeavor in America:

William Henry Welch
Welch and Warthin influenced the career of the young Rous in different ways. He
studied at the School of Medicine at The Johns Hopkins University, Americas first
research university, which opened in 1892. Apparently, William Henry Welch
(18501934) (Fig. 1.2), the founding dean and professor of pathology at the medical
school and at that time academic teacher, was able to spark Rous interest in experimental medicine in general and in pathology in particular. In Baltimore, Welch
established the first pathology teaching laboratory in the USA. In general, Welch
dedicated himself to an extraordinary degree to the modernization of American
medicine in teaching and research [a fresh age of medical endeavor came in an
Fig. 1.2 Photograph

of William H. Welch. Photo
credit: Courtesy of the
Rockefeller Archive Center
age in which experiment largely took over from observation, Rous wrote, in retrospect
(Rous 1948: 611)], whereby the experience gained during his research stays in
Europe helped him (18751878, 1884) (MacCallum 1936; Flexner and Flexner
1941; Flexner 1943; Brieger 1970). As first president of the Board of Scientific
Directors at the Rockefeller Institute for Medical Research in New York (19011933),
Welch took a keen interest in Rous subsequent rise to become a distinguished
scientist. Just a few years after his death, Rous had the honor of presenting a William
Henry Welch Lecture (Rous 1941). On another occasion, Rous noted: It is not too
much to say that modern scientific medicine reached America through William
Henry Welch (Rous 1949: 411). The Journal of Experimental Medicine was
launched in 1896 by Welch as founding editor of a new type of medical publication,
which was then developed into a highly prestigious journal by Rous during his
extremely long tenure (19221970, until 1945 together with Simon Flexner).
Aldred Scott Warthin: A Consummate Pathologist

As a young, freshly graduated medical doctor, Rous came to Aldred Scott Warthin
(18661931) (Fig. 1.3) at the University of Michigan with the aim of doing experimental work and obtaining training in pathology. He could not have made a better
choice.
Warthin was initially trained as church musician before turning to the study of
medicine. He received his MD in 1891 and his PhD in 1893. He worked temporarily
in the Department of Internal Medicine at the University of Michigan before he
Fig. 1.3 Photograph

of Aldred S. Warthin. Photo
credit: A.S. Warthin Papers,
Bentley Historical Library,
University of Michigan
Peyton Rous
started his academic career there as pathologist. Following various positions from
1896 on (among them as instructor, a position Rous later held under him), he was
appointed professor and director of the Pathological Laboratories at the University
of Michigan in Ann Arbor in 1903. Between 1893 and 1900, Warthin regularly traveled
to Europe during the summer months in order to work at the institutes of pathology
in Vienna, Freiburg, and Dresden and at the same time to pursue his multifaceted
interests (music, art history, collection of old books, particularly of medical incunabula).
While doing so, he in no way neglected his extensive pathological research. A number
of eponyms are today associated with the name Warthin: Warthins sign (exaggeration
of pulmonary sounds in acute pericarditis), Warthins tumor (benign salivary gland
tumor with lymphoid tissue covered by epithelium), and WarthinFinkeldey giant
cells (multinucleated giant cells seen in the lymphoid tissues of patients with measles).
Moreover, he translated Ernst Zieglers (18491905) Lehrbuch der allgemeinen und
speziellen pathologischen Anatomie und Pathogenese [Text-Book of Pathological
Anatomy and Pathogenesis] (two volumes; Jena, 18811882) from German into
English. Warthin was, as one would say today, very well-networked and held important
posts in numerous medical societies.
In person and manner, Warthin was a model of virile fastidiousness. [] Warthins approach
to pathology was based upon a familiarity with and keen interest in internal medicine.
He had a full appreciation of the biological significance of pathology, but to him, study in
this field represented a particular opportunity for advancement of medicine as a science.
(Anonymous 1932: 13435) [And Rous later noted]: Warthin was bright-eyed and fresh-colored, quick and strong. He was drastic yet kind, earnest yet cheerful, and most sensitive to
beauty. He loved music, gardens, books, and friends. (Rous 1936: 494)
Warthins research on the familial incidence of cancer had a particularly lasting

impact. In 1895, he initiated one of the most thoroughly documented and longest
family histories ever recorded (Warthin 1913, 1925). Recently, a new update of this
family, originally referred to as Warthins family G and subsequently described as
Lynch syndrome family, has been published (Douglas et al. 2005). He [Warthin]
can properly be called the father of cancer genetics, Henry T. Lynch affirmed,
90 years after the beginning of this study (Lynch 1985).
In his later years, Rous drew attention to some forgotten, yet at their time very
far-sighted, works of his teacher.
In 1904 and again in 1906 Professor Aldred Warthin of the University of Michigan, a
consummate pathologist whose abilities I came to know through serving under him as
instructor, reported facts making plain that human leukemia is a neoplastic disease; and in
1907 he published a study showing that this held true of a leukemia he came upon in a
chicken [Warthin 1907. At the end of this paper he stated: The problem of leukemia, then,
becomes identical with that of malignant neoplasms in general. Not bad for 1907]. No
causative agent was then perceptible within the neoplastic tissue, but in 1908 two Dutch
[sic, correct would be Danish] workers, Ellermann and Bang, reported on a virus as causing
a chicken leukemia [Ellermann and Bang 1908]. Soon after, they procured another agent
from a leukemia of a differing sort, and by means of these agents they transferred the two
diseases in fowl after fowl. Their findings were wholly convincing yet were written off
because leukemia was not generally realized then to be a neoplastic disease. Indeed, this did
not come about until the 1930s. Warthins papers had been completely overlooked. He and
the [Danish] workers were more than 20 years ahead of their time. (Rous 1967a: 844)
In the summer of 1898, Warthin was a guest of Schmorl at Dresden Friedrichstadt

Municipal Hospital. During these months, he performed a number of dissections,
the protocols of which are still preserved today in the journals of the institute
(Fig. 1.4). Warthin must have enjoyed his stay so much that he several years later
recommended his protg Rous to go to Dresden, too, and work with Schmorl.
From his own experience, he knew that for this purpose a sufficient command of the
German language was required. As mentioned before, he proposed to Rous to
finance his trip and stay in Dresden. Rous gladly accepted this suggestion and
remained grateful to his mentor throughout his life.
One can assume that Warthin also saw the famous Dresden Dance of Death while
he was in Dresden, an over 12-m long sandstone relief with 27 figures dating from
the year 1534 (Dresdner Totentanz). He must have pointed this out to Rous too. In any
case, Rous titled his later lecture in Oxford, named after the British humanist and
physician Thomas Linacre (14601524), The Modern Dance of Death with a picture
of The Physician from Holbeins Dance of Death from 1523 to 1526 on the
cover of the printed version (Rous 1929). Warthin had been engaged with this subject
for many years and published an in-depth and famous study in 1931 which traces the
dance-of-death motif through six centuries (Warthin 1931).
Georg Schmorl: A Scientist with Most Infectious Enthusiasm

The PathologicalAnatomical Institute of the Dresden Friedrichstadt Municipal
Hospital had existed since 1849 and since 1894 was situated in a spacious new
building (the present Institute of Pathology Georg Schmorl) (Kunze 1999: 2225,
7079) (Fig. 1.5). It was the domain of well-known pathologists, among them were
Albert von Zenker (18251898), Felix Victor Birch-Hirschfeld (18421899), and
Adolf Neelsen (18541894). Since 1894, Christian Georg Schmorl (18611932)
(Fig. 1.6) had been head of the institute, which was already then very attractive, and
helped give the place its special aura. Again and again, physicians from many countries
came to Dresden to work with Schmorl. Their number reached hundreds, which is
why it was not possible to mention the names of later prominent guest researchers
in Schmorls obituaries.
For instance, the British pathologist Hubert Maitland Turnbull (18751955) was
a guest researcher at the institute from 1905 to 1906, and after him a long series of
British scientists. Under the influence of Schmorl, Turnbull later performed a great
service to British pathology (Russell 2004).
Work in morbid anatomy at Dresden under the inspiring guidance of Professor Georg Schmorl
revolutionized Turnbulls ideas about his future. Schmorl was noted for his work on bone
pathology, and Turnbull had gone to Dresden primarily to become versed in this as a prelude
to entering orthopedic surgery. In the early years of this century morbid anatomy was regarded,
at least in this country, as a subject which had nothing more to offer: it had been sucked dry
by Virchow and his school, and bacteriology was in the ascendant. But at Dresden the lively
teaching and work of Schmorl gave the lie to all this; so much so that Turnbull resolved to give
his future to morbid anatomy and to redeem its status in England. (Russell 1956)
Peyton Rous
Fig. 1.4 Excerpts from autopsy reports written by Warthin 1898 in Dresden. Reproduction with
the permission of the Archive of the Institute of Pathology Georg Schmorl, Dresden Friedrichstadt
Municipal Hospital, Germany
Fig. 1.5 View of the PathologicAnatomical Institute, Dresden Friedrichstadt Municipal Hospital,
in the year 1907, seen from Friedrichstrasse. The institutes auditorium is located on the left side
under the cupola. On the right, St. Matthews Church can be seen, which was constructed according
to the plans of Daniel Pppelmann in 1732. Contemporary postcard. Reproduction with the
permission of the archive of the Institute of Pathology Georg Schmorl, Dresden Friedrichstadt
Fig. 1.6 Portrait of Christian

Georg Schmorl. Oil painting
(118 85 cm) by Robert Sterl
(18671932), an important
representative of German
Impressionism, 1921.
Reproduction with the
permission of the Dresden
Friedrichstadt Municipal
Hospital, photo:
Martin Wrker
Peyton Rous
The high standard of British pathology that is based on the schools of Schmorl
and Turnbull (Storey 2008) certainly was a prerequisite for the remarkable performance
that was achieved in this country in the field of chemical carcinogenesis since the
1920s (Lawley 1994).
Like the famous German poet Gotthold Ephraim Lessing (17291781), Schmorl
attended the renowned Frstenschule (the Princes school) of St. Afra in Meien.
Later, he always emphasized the enduring influence of his schooling. After graduating,
he studied mathematics and natural sciences for several semesters, before deciding
to study medicine. After earning his doctorate in medicine in 1887, he became the
assistant of Birch-Hirschfeld in Leipzig. There, he qualified to become lecturer in
pathology in 1892. After Neelsens early death, he took on the position as prosector
and director of the Pathological Institute of the Dresden Friedrichstadt Municipal
Hospital in 1894, a position he held until 1932 (from 1903 on, as professor) (Turnbull
1932; Geipel 1934; Junghanns 1983; Scholz 2007). Schmorl was particularly interested
in microscope technology. He was the author of the standard textbook Pathological
Histological Examination Methods (1897), which within 37 years had seen 16
editions and throughout Germany became an indispensable reference as Der kleine
Schmorl (the little Schmorl) for generations of young pathologists. Moreover, he
had an affinity for photography, microphotography, and X-ray photography which
he had developed for use in pathology and in which he achieved true mastery; preferably, he wanted to perform all the necessary work with his own hands. Noteworthy
are his pioneering works on bone pathology, especially the work in which he for the
first time describes Nodulus intraspongiosus Schmorl. Through his unique collection
of pathological bone specimens (the Georg Schmorl Pathology Collection), he
gained an international reputation. Schmorl was possessed of tireless energy, a
most infectious enthusiasm, great diligence, an astonishing memory and a lively
imagination. To search with knife or microscope was obviously a joy to him
(Turnbull 1932: 982).
Peyton Rous in Dresden

Whether Rous only stayed a summer (Anonymous 1966), a few months (Huggins
1970), or a whole year (Andrewes 1971; Dulbecco 1976) in Dresden cannot be
concluded from the preserved documents. What can be verified is that he performed
23 dissections (Fig. 1.7) between the beginning of July until the end of August 1907
(Wunderlich and Kunze 2008). As the protocols show, he was proficient in German.
Furthermore, a photograph is preserved showing Rous and other physicians together
with Schmorl (Fig. 1.8) further evidence for Rous stay in Dresden. The picture
deserves a place in a future biography of Rous to be written by historians of science.
Schmorl, the passionate photographer, was probably responsible for the carefully
staged arrangement of people and objects in the photograph.
10
Fig. 1.7 Excerpts from autopsy reports written by Rous 1907 in Dresden. Reproduction with the
permission of the Archive of the Institute of Pathology Georg Schmorl, Dresden Friedrichstadt
Peyton Rous
11
Fig. 1.8 Christian Georg Schmorl (fourth from left, seated) surrounded by visiting researchers
(among them, the almost 28-year-old Peyton Rous, third from left) and some assistants. Pathological
Anatomical Institute of Dresden Friedrichstadt Municipal Hospital, summer 1907. Gustav Molineus
(18801954), later professor of medicine in Dsseldorf, is standing to the left of Rous. Second from
the right is Curt Oehme (18831963), later professor of internal medicine in Heidelberg. Details
regarding the other people depicted in the photograph are not preserved. The photograph is clearly
posed, as can be seen in the symmetrical arrangement of the people, among other things. The pathologists most important equipment of that time (microscope, hand microtome, staining solutions, and
solvents) round off the picture. Reproduction with the permission of the Archive of the Institute of
Pathology Georg Schmorl, Dresden Friedrichstadt Municipal Hospital, Germany
In view of the severe destruction through the devastating air raids in Dresden in
February 1945 and the worst flood of the century in August 2002, which did not
exclude Dresden Friedrichstadt Municipal Hospital, it is a stroke of good fortune
that these documents could survive the past 100 years. No hint of war in the air, is
how Rous in 1966 melancholically remembered the undamaged Dresden of 1907
(Anonymous 1966).
On June 15, 1907, an article by Rous was published in the Journal of Infectious
Diseases in which he suggested an improvement of Schmorls celloidin-plate
method (Rous 1907). This enabled the simultaneous staining of many paraffin
sections with additional dyes, making the overall staining procedure more efficient.
12
This work, which apparently already originated in Ann Arbor, must have pleased
Schmorl, the methodologist in quest of perfection. In a way, it was the admission
ticket for Rous.
From reports of other Schmorl students (Turnbull 1932; Geipel 1934; Junghanns
1983), we know a great deal about the work flow in the Schmorl Institute. Every
coworker had to prepare his microscopic specimens himself, cut and stain them as
well as obtain the necessary tools (staining solutions, microscope slides, and glass
covers) himself. Although very many autopsies had to be performed, Schmorl found
the time to discuss the results in the autopsy room every day and to give important
information to the other autopsists. On Saturday mornings, the always well-attended
demonstrations took place in the auditorium, which physicians from other institutions
were allowed to attend as well. Schmorls teaching was of the nature of personal
coaching, and his pupils, who were all graduates, had the inestimable advantage of
learning his method of working. [] He gave without stint from his store of knowledge, whether published or not, to any who wanted to learn (Turnbull 1932: 984).
Supposedly, Rous learned quite a lot about how to present results as well. Many
of his later papers were illustrated with excellent photographs and microphotographs
[]. Descriptions were detailed, for Rous liked to have his observations fully documented, and the accounts were often full of vivid imagery (Andrewes 1971: 648).
Like Schmorl, he photographed with meticulous attention to detail (ibid: 652).
Besides pathology, bacteriology was another key area in the Schmorl institute.
A bacteriological research center was opened in 1897 in the Dresden Municipal
Hospital, subordinate to the Pathological Institute and thus to Schmorl. By 1907, the
number of examinations had increased to almost 7,000 per year. Among them were
cases of important infectious diseases, such as diphtheria, typhoid fever, cholera,
infection of wounds, anthrax, tetanus, influenza, pneumonia, tuberculosis, syphilis,
and rabies. Therefore, Rous must have learned many new things about infectious
diseases. This may have been unexpected for him, since for a long time his teacher
Welch had not considered the etiological role of bacteria as having any particular
significance (Temkin 1950) and Warthin had concentrated solely on research on syphilis
and tuberculosis (Anonymous 1932; Rous 1936). It was also not clear at that time if
the still young field of bacteriology was to be considered part of pathology or rather
of hygiene. In any case, the broadening of Rous horizon very soon had consequences
for his own research. Even in 1923, Rous commented on his own inborn lack of
aptitude for bacteriology (Rous to Gye, quoted from Becsei-Kilborn 2010: 141).
As Warthin had intended, the stay in Dresden superbly rounded off Rous training.
Here, he experienced methodological perfection using state-of-the-art technology.
After Welch and Warthin, Schmorl was another research personality who had a
profound impact on his further career.
Following the extraordinary achievements of bacteriology at the end of the nineteenth century and the associated perception that many diseases had a monocausal
etiology, around 1900, many hopes were also placed on finding a rapid solution to
the problem of cancer. In the following years, it was up to Rous to work out the first
evidence of a multicausal explanation of carcinogenesis in order to later come to the
confident realization that under natural conditions malignant tumors gradually
develop in a multifactorial process (Rous 1967b).
Peyton Rous
13
Simon Flexner and Medical Discovery

During his studies, Rous was infected with tuberculosis bacteria while performing
an autopsy, developed lymph-node TB, and was forced to interrupt his studies for a
year. After his stay in Dresden, he was diagnosed with pulmonary tuberculosis,
which forced him to pause again, but fortunately he recovered quickly.
Afterward, it was Warthin, once again, who gave him a crucial bit of information:
Dr. Warthin told Peyton Rous that the Rockefeller Institute for Medical Research [in New
York] was casting a wide net of grants for beginners, and he asked him if Peyton would like
him to apply for one that would free Peyton for experimental work. That grant enabled Rous
to find out enough about lymphocytes to be deemed worth publishing in the Journal of
Experimental Medicine [Rous published in 1908 three papers on lymphocytes in this journal],
edited by Simon Flexner, who was also the director of the Institute; and after another few
months Flexner asked Rous to take over the laboratory for cancer research [at the Department
of Bacteriology and Pathology] which Flexner was quitting to learn more about poliomyelitis,
then crippling many American children. (Anonymous 1966)
The Rockefeller Institute for Medical Research (from 1964 on, Rockefeller
University) was founded in 1901 as a private institute by John D. Rockefeller
(18391937) and opened in 1904. It was the first American institute that exclusively
engaged in biomedical research. At first, research on infectious diseases and bacteriology was the focus of interest; however, the problem definition was very broad and
comprised many branches of basic biological research (Corner 1964; Hollingsworth
2004). The founding director was the pathologist Simon Flexner (18631946)
(Fig. 1.9), who held this position from 1901 to 1935 with great success. In his view,
Fig. 1.9 Photograph

of Simon Flexner. Courtesy
of the Rockefeller Archive
Center
14
the institute was an attempt to add knowledge by discovery and to apply that
knowledge to the prevention and alleviation of disease (Rous 1949: 416).
Flexner had studied pharmacy and medicine at the University of Louisville, and
earned his PhD in 1889. As 27-year-old, he came to William Welch at The Johns
Hopkins University in Baltimore and there experienced the stimulating founding
phase of the School of Medicine. Welch became his teacher, promoter, and later his
fatherly friend; Flexner soon became Welchs closest colleague. The focus of
Flexners research was on experimental pathology, bacteriology, and immunology.
Because of his expertise in these areas, he was repeatedly appointed head of
commissions to combat epidemics at home and abroad. In the years 1893 and 1909,
he spent some time in Europe in order to work with Friedrich Daniel von
Recklinghausen (18331910) in Strasbourg and later with Emil Fischer (18521919)
and Ernst Leopold Salkowski (18441923) in Berlin, among others. Prior to
becoming director of the Rockefeller Institute, he had been professor for Pathological
Anatomy at The Johns Hopkins University since 1899 and since 1900 Head of the
Department of Pathology at the University of Pennsylvania (Corner 1972).
As researcher, Flexner became known especially for his work on epidemic cerebrospinal meningitis. At the Rockefeller Institute, he developed a serum treatment
(Flexners serum), which proved successful first with monkeys and later, during an
epidemic, with humans. A treatment with serum from immunized horses was introduced simultaneously and independently of Flexner by the German physician Georg
Jochmann (18741915).
Flexners research on poliomyelitis was of particular importance for Rous and the
work that he had already begun at the Rockefeller Institute. At that time, outbreaks
of polio were a major problem, not only in America. From 1908 to 1909, the team
led by Flexner identified a filterable virus (!) as responsible agent of the disease [the
RNA virus which was discovered independently in Vienna by Karl Landsteiner
(18681943) and Erwin Popper (18791955) is today assigned to the Picornaviridae
family]. Flexner and colleagues also identified the transmission path of the virus.
They showed that it enters the body through the nose, attacking the olfactory nerve.
They could also experimentally infect monkeys with poliomyelitis by administering
the virus in the nasopharynx (Flexner and Lewis 1909). For his work on serum treatment of epidemic cerebrospinal meningitis and on transmission of poliomyelitis to
monkeys, Flexner was nominated ten times for a Nobel Prize in Physiology or
Medicine (Nomination Database 19011951), but did not get the Prize.
However, contemporaries and historians regard Flexners greatest achievement
to lie in the organization of medical research and especially in the unprecedented
success of the Rockefeller Institute (Corner 1964; Hollingsworth 2004). Perhaps
no man save Welch has done so much for American medicine (Rous [commenting
on Flexner] 1948: 613). Although many other high-ranking researchers had worked
at the Rockefeller Institute for a long time and could have been considered to pay
tribute to Flexner in obituaries, this task fell on Peyton Rous (Rous 1948, 1949).
He did it in a loving way. He [Flexner] had proved that the experimental method
can meet human needs if it be given its head, wide and free; and he had shown that
discoverers can be discovered (Rous 1948: 613).
Peyton Rous
15
At the beginning, Flexner had also been head of a laboratory for cancer research
at the Rockefeller Institute. Together with James W. Jobling (18761961), he
discovered a transplantable tumor, a rat adenocarcinoma most likely of prostatic
origin (FlexnerJobling carcinoma). This particular tumor has served for many
decades as a unique test material in cancer research (Triolo 1964: 12; Corner 1964:
59), for instance in the famous experiments of Otto Warburg on the energy metabolism
of cancer cells. Because of urgent work in other areas, particularly in the research of
poliomyelitis, Flexner gave up his position as head of the laboratory, after he had
found an appropriate successor in the then 30-year-old Rous. However, a number of
obstacles had to be overcome before Rous could take over. Rous later described that
at this time youngsters were warned off from the Institute with the slurring phrase
Rockefeller money (Rous 1949: 418). Since cancer research was considered a
futile field among scientists, even Welch had implored him: Whatever you do,
dont commit yourself to the cancer problem (Andrewes 1971: 644). Luckily, Rous
did not follow this advice. Presumably, it was again Warthin who, besides Flexner,
encouraged Rous despite all the prophecies of doom to turn toward cancer research.
Toward the end of his life, Rous was able to acknowledge that [Cancer is] the most
intellectually worthwhile of all diseases (Rous to Heagensen 1957, quoted in
Becsei-Kilborn 2010: 117).
A Discovery Greeted with Skepticism

At the Rockefeller Institute, Rous soon had the luck of the diligent: still in 1909, a
poultry farmer consulted him who had noticed a big tumor in the chest of one of his
chickens (a Plymouth Rock breed). Worrying that this could be a threat for other
chickens of his flock, the breeder consulted different pathologists without success.
Only Rous realized that this was a spindle cell sarcoma. This malignant tumor
served Rous in the following years as a model for basic research studies; it was,
therefore, later termed the Rous sarcoma (classic chicken sarcoma). To begin with,
Rous succeeded for the first time in the transmission of a chicken tumor by means
of injection into healthy animals however, only when the recipients were genetically compatible animals (Rous 1910). If a homogenate of such tumors was passed
through a filter that was not permeable to cells or bacteria, this filtrate in turn generated spindle cell sarcomas in healthy chicks after inoculation, which Rous as an
experienced pathologist could clearly identify (Rous 1911a, b). Although he initially
did not use the term virus to explain his results (he spoke cautiously only of a
filterable agent), the chosen experimental setup did not allow for any other interpretation: for the first time, it was shown that a real tumor was caused by an
infectious agent, probably a virus. Leukemias which showed similar results, however less convincing, were reported by Vilhelm Ellermann (18711924) and Oluf
Bang (18811937) (Ellermann and Bang 1908). However, at that time, leukemias
were not considered to belong to the neoplastic diseases. In the 1890s, viruses as
novel biological entities were recognized for their characteristic of passing through
16
bacteria-proof filters while preserving their biological activity. In 1911, Rous

published his findings. He soon succeeded in developing a detection method for the
agent on the chorioallantoic membrane of the chicks. Shortly thereafter, he proved
a virus etiology for two further distinct chicken sarcomas. Today, the term Rous
sarcoma virus (RSV) not only includes Rous original isolates, but numerous other
chicken viruses that have been isolated independently from each other and that
induce sarcomas through a genetic mechanism similar to the original isolate. RSV
was the first known tumor virus and the first representative of the retroviruses.
The initial euphoria about Rous discovery was soon followed by disillusionment.
Apparently, viruses were not generally responsible for the induction of tumors.
Also, the virus theory could hardly be brought into conformity with contemporary
beliefs about the causal genesis of cancer. Furthermore, with the outbreak of World
War I, Rous had to devote himself to other subjects (among them, with great success,
the conservation of blood).
Recent historical research substantiates that Rous discovery triggered a longlasting scientific discussion (van Helvoort 1999, 2004; Becsei-Kilborn 2010).
However, a detailed description of this discussion would go beyond the framework
of this article. Rous found himself facing considerable skepticism, even resistance.
Even James B. Murphy (18841950), temporarily his close colleague when carrying
out these experiments and who later gained increasing influence in the US cancer
research community, did not believe in the involvement of viruses, but interpreted
the agent as a transmissible mutagen. Later, he thought to have proved it to be a
ferment. Rous sharpest critic was James Ewing (18661943), who was very
conscious of his power as pathologist and director of research at the Memorial
Hospital for Cancer and Allied Diseases in New York City. Ewing largely rejected
experimental pathology for the research of cancer etiology and believed the origin
of cancer to be within the cell itself. Frequent points of criticism by other scientists
about Rous experiments were: (1) when preparing the filtrate, a few tumor cells
could have passed through the filter; (2) the effect might have been caused not by an
infectious agent, but by products synthesized by tumor cells; (3) it was doubted that
the induced sarcomas were real tumors, and it was suggested that they should rather
be seen as granulomas; (4) since the agent could not infect most cells with the
exception of certain chicken cells the high specificity of its effect remained enigmatic; and (5) whether tumor development could be caused by external factors at all
or if tumors develop in an endogenous way was not decided at that time. However,
the belief favoring purely endogenous reasons prevailed at that time. Although Rous
could dispel some objections, he did not succeed in convincing his critics. All in all,
the results were for a long time considered an oddity of chickens that were of no
relevance to the situation in humans.
But there were also advocates for Rous, in America most notably Flexner and the
highly respected Leo Loeb (18691959). In Japan, Akira Fujinami (18701934)
found a very similar agent in chicken sarcomas that is known today as Fujinami
virus (Fujinami and Inamoto 1914). From the mid 1920s on, much-discussed studies
by the British scientists Willam E. Gye (18841952) and Christopher H. Andrewes
(18961988) kept Rous experiments from being forgotten. Rous himself did not
take up work in this area until 1933, after Richard E. Shope (19011966) had
Peyton Rous
17
succeeded with a cell-free transmission of papillomas of cottontail rabbits (Shope

1933). Shope left the alleged virus tumor to his colleague Rous for further experiments. The model gave Rous the opportunity to study many characteristics of the
natural development of tumors. Under special conditions, real carcinoma developed
from the papilloma. Rous found that tumors develop gradually. A phase of tumor
initiation is followed by phases of promotion and progression up to the fully developed
metastasizing tumor (Rous and Kidd 1941). This may cause a synergistic effect of
viruses and chemical carcinogens. The terms latent or dormant tumor cells and
cancer as a multifactorial disease were also introduced by Rous on the basis of
these experiments (see Rous 1967b).
The real breakthrough of the virus theory of cancer came in the 1950s. In 1951
and 1954, respectively, Ludwik Gross (19041999) in New York and Arnold Graffi
(19102006) in Berlin were able to prove that viruses caused lymphatic (Gross 1951)
and myeloid leukemia (Graffi et al. 1954) in mice. Numerous other isolations of
DNA- or RNA-containing oncogenic viruses were made in a variety of animal species.
Many of these became outstanding models of the emerging molecular biology.
The golden age of tumor virology had begun.
Rous had always believed in the viral nature of his agent. In a letter to his British
colleague Stephen L. Baker (18881978), he confessed in 1930: My own belief
has always been that the agents causing these tumors are viruses. But at that time,
he had to continue carefully [] though the statement is confidential to you
(quoted from Becsei-Kilborn 2010: 132).
The Great Good Fortune of Rous

I will always consider it good fortune that just when I finished my apprenticeship in physics,
chemistry and medicine, the Kaiser Wilhelm Institute for Biology was founded in Berlin,
and that upon its founding Emil Fischer, then Vice President of the Kaiser Wilhelm Society,
forthwith appointed me as scientific member [upon the recommendation of Theodor Boveri
(18621915)]. [] I have no doubt that my scientific achievements can mainly be attributed
to the unusual degree of freedom and independence that I [] have received. [German in
original]
This is how Otto Warburg (18831970) commented in retrospect on his career,

according to his student and biographer Sir Hans Krebs (19001981) (Krebs 1978:
351; Krebs 1981). Warburg and Rous, who were almost the same age, were probably
the most important cancer researchers of the early twentieth century (Fujimura
1996a, b). Like Warburg, Rous had completed a long period of apprenticeship
served with Welch, Warthin, and Schmorl when he joined the new Rockefeller
Institute, in which he then (from 1920, as member) was to experience an impressive
career. Rous, too, always regarded these circumstances as good fortune: Environment
is everything for a scientific man, he wrote (Rous 1949: 412).
Both scientists benefited from developments that took place in these years. On the
one hand, the character of cancer research changed: Cancer research became an
experimental science at the turn of the century, along with most of the biological
18
sciences (Fujimura 1996b: 247). On the other hand, novel institutions were founded
for nonuniversity research: at first, the Rockefeller Institute for Medical Research in
New York which later served as a model for the foundation of the German Kaiser
Wilhelm Society with its various institutes. Therewith, a culture of excellence was
institutionalized (Hollingsworth 2004) that was to lead to great achievements in the
cases of Rous and Warburg: Discovery [is] the most satisfying of human experiences
(Rous 1949: 423).
However, the priorities which Flexner set from the beginning for the Rockefeller
Institute were also a stroke of good fortune for Rous. Until the end of Flexners term
of office, the role of pathology was sacrosanct: Pathology is far more important for
us than physiology and pharmacology, and the background of medicine than general
science. Our pathologists are all moving on; pathology is the fundamental branch of
medicine (Flexner 1993, quoted from Corner 1964: 187). This was in the spirit of
Rous, who told Jacob Furth (18961979) in 1942: Experimental pathology has
always been to me one of the most exciting of human activities (quoted from
Becsei-Kilborn 2010: 116).
As mentioned before, Flexner was also a major proponent of virus research. At
precisely the time when Rous began his research on chicken sarcoma, the studies of
Flexners team reached a milestone with the evidence of a virus etiology of poliomyelitis. Rous decisive proposition a cell-free transmission of chicken sarcoma
probably arose out of discussions within the Rockefeller Institute.
Fortunately, Rous could count on Flexners continuing support during the yearlong controversies on the significance of his discovery. Rous himself was very
confident early on. The identification of the chicken sarcoma had already been a
considerable achievement in 1909 (very little research had been done on poultry
tumors at that time), and he was now able to fend off his critics because of his versatility and expertise as a pathologist. He was aware that the nature of the tumors
induced by cell-free transmission was a decisive argument. Evidence of their malignancy was in every respect unambiguous: cell-free filtrates of the same tumor induced
reproducible, histologically identical neoplasms, each independently obtained agent
induced tumors of various types, and all induced tumors had the ability for invasive
growth and metastasizing, i.e., they were genuine malignant neoplasms.
At the latest during these experiments, it became clear that Peyton Rous had been
very fortunate in the choice of his teachers. He kept them in honorable memory
throughout his life. In his obituaries for Simon Flexner, he also remembered William
H. Welch, who had imparted a vision of an experimental pathology to the young
Rous and who let him take part in the vibrant ambiance of scientific discovery (Rous
1948, 1949). Rous was indebted to Aldred S. Warthin for a thorough education as
pathologist and for key professional guidance, such as providing the contact to
Schmorl and Flexner, as well as encouraging the shift to cancer research. He commemorated him in a beautifully written obituary (Rous 1936) and as late as 1967
alluded to long-forgotten work of his teacher (Rous 1967a). In Dresden, he encountered European science and culture and was full of praise even 60 years later
(Anonymous 1966). He studied the advances in bacteriology in Germany under Georg
Schmorl. In the working method of Schmorl, he saw methodological perfection
Peyton Rous
19
combined with the use of modern technical (especially photographic) equipment.

Thus, he was well-prepared to seize the opportunities that were offered to him at the
Rockefeller Institute. Among his teachers and promoters, it was Simon Flexner,
who discovered the discoverer in Rous, whom he had known the longest and probably revered the most. He commemorated him in obituaries that are well worth
reading even today (Rous 1948, 1949). Flexner was the guarantor of freedom of
research at the Rockefeller Institute, and this proved to be of crucial importance for
the seminal discovery of tumor-inducing viruses.
Epilogue
Although Peyton Rous is first and foremost known as the father of the tumor virus
(van Epps 2005; Moore and Chang 2010), his name is also associated with other
important achievements. At the Rockefeller Institute, his well-rounded education
bore rich fruit. During World War I, the preservation of living blood cells was an
urgent medical need. Together with Joseph Richard Turner, Jr. (1889?), Rous
developed a method which enabled long-term storage of blood without clotting. In
a mixture of 3 parts of human blood [], 2 parts of isotonic citrate solution [] and
5 parts of isotonic dextrose solution [], the cells remain intact for about 4 weeks
(Rous and Turner 1916). This method enabled Oswald H. Robertson (18861966),
the pioneer of transfusion medicine, to set up the worlds first blood bank behind the
front line in Belgium in 1917. Rous cooperated closely with Robertson, as a number
of joint publications demonstrate. The RousTurner solution is still in use for
human blood transfusions, and in the 90 years since its first use it has saved countless
lives. Rous was particularly proud of this achievement and rightly so.
A cell and tissue culture method which has been a laboratory standard up to the
present day can also be traced back to Rous research. To obtain a suspension of
individual living cells from the fixed tissue, he performed a digestion with trypsin
(Rous and Jones 1916). The optimal trypsin concentration had to be determined
separately for each kind of tissue. Once again, with this method, Rous proved his
talent for finding solutions that could be carried out easily. In conjunction with his
work on blood conservation, Rous also focused on the functions of the liver, gall
bladder, and kidney as well as on the permeability of small vessels. The Rous test
for hemosiderin in urine to detect hemosiderosis (Rous 1918) was one of the results
of his research at that time.
Any appraisal of Rous would be incomplete if it neglected to draw attention to
Rous great merits as long-time editor (from 1922 to 1970) of the renowned Journal
of Experimental Medicine. He invested a lot of time and energy in this task.
According to contemporary witnesses, he continued working well into old age and
was equally meticulous in revising manuscripts as he was in conducting experiments in the laboratory. Both activities along with his modesty contributed to
Rous extraordinary reputation. As a young man, Cornelius Rhoads (18981959)
experienced Rous as he revised his manuscript, and Rhoads described this later
20
quite humorously: Dr. Rous, gravely and patiently, reviewed my efforts with me,
demolished my conclusions, refuted my claims and made clear the proper use of my
native tongue. He then rebuilt on the ruins such a clear picture of the problem, and
the procedure required to solve it, that my conceit was converted almost imperceptibly to inspiration, my enthusiasm to resolution. As I left the generous, patient, and
kindly man, I was no longer the same individual (Rhoads 1959).
Time and again during the twentieth century, the RSV as a model for fundamental
studies proved to be a serendipitous choice, and its scope extended far beyond
oncology. After Rous, several scientists received the Nobel Prize in Medicine for
studies in which this virus played a decisive role. In 1974, one of the prize winners
was Albert Claude (18991983), who pioneered the fractionation of cells by
differential centrifugation. This discovery was the prerequisite for making images
of the structure of the RSV, among others, using an electron microscope the first
time for a tumor virus (Claude et al. 1947). Howard M. Temin (19341994) and
David Baltimore (born 1938) discovered reverse transcriptase and were honored for
their work in 1975. Their research was conducted with Rous sarcoma and Rauscher
murine leukemia viruses (Temin and Mizutani 1970; Baltimore 1970). J. Michael
Bishop (born 1936) and Harold E. Varmus (born 1939) received the Nobel Prize in
1989 for the discovery of cellular oncogenes. They were able to identify the cellular
origin of retroviral oncogenes, among others, first for the src gene, the viral homolog
of which is responsible for the transforming activity of the RSV (Stehelin et al. 1976).
For their discovery of human immunodeficiency virus (Barr-Sinoussi et al. 1983),
today besides the RSV the most well-known retrovirus, Franoise Barr-Sinoussi
(born 1947) and Luc Montagnier (born 1932) were awarded the Nobel Prize in 2008
along with Harald zur Hausen. Other equally important discoveries in the field can
also be mentioned, further substantiating George Kleins appraisal: Few fields in
modern biology and certainly no other field in cancer research can be traced back to
the work of one man in the same way that the foundation in the field of viral oncology can be traced back to the work of Peyton Rous in 1911 (Klein 1980).
Acknowledgments We thank Carol Oberschmidt (Berlin) for translation and Professor Manfred
F. Rajewsky (Essen) for critical reading of the manuscript.
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Chapter 2
Viruses and Cancer: A Historical

Perspective HBV and Prevention of a Cancer
Baruch S. Blumberg
Introduction
Viruses are thought to be the causative agent of about 1520% of cancers, including
some of the worlds most common cancers. As is the case for most diseases, cancers
have multiple causes or factors that contribute to etiology, pathogenesis, prognosis,
and response to treatment. In addition to the cancers with identified viral causes, there
are probably additional cancer disease entities in which viruses contribute to pathogenesis and in which prevention of infection may significantly alter the outcome.
In recent years, there have been reports, primarily in the general press, on the
disaffection with the progress of cancer control and treatment. These often refer to
the so-called War on Cancer (although that term was not the official designation)
initiated on December 23, 1971 and, as perceived by the public, its failure to have a
dramatic effect on control and treatment. There have been improvements in treatments for many cancers; for several childhood and usually relatively rare adult cancers,
therapies have been impressive. But for many cancers, treatment has resulted in only
limited increased survival and the treatments themselves may diminish the quality
of life. The search for better treatments is a major research program.
That War on Cancer campaign started with many goal-directed research programs.
Viral-caused cancer was one of the goal-directed projects based on the models from
virus/cancer relations in experimental, domestic, and wild animals. However, human
cancers did not appear to closely follow the animal models, and by the mid-1970s
support for virus-caused cancers in humans had diminished. But, as in many research
areas, interest appears to wax and wane over the years. As evidenced by this book
and others on the same topic, the increase in the number of research groups studying
cancer and infectious agents, interest is growing once again.
B.S. Blumberg (*)

Fox Chase Cancer Center, Philadelphia, PA 19111, USA
e-mail: baruch.blumberg@fccc.edu
25
26
B.S. Blumberg
Prevention has had a major effect on decreasing the load of cancer in human
populations. Primary prevention, for example smoking cessation programs, has
resulted in major decreases in the incidence of cancer of the lung and other cancer
as well as noncancer diseases. Dietary measures have probably decreased cancer of
the colon and other organs. Cancer of the stomach has decreased dramatically,
presumably as a consequence of changes in the environment, in diet, or some other
factors that are not clearly identified. Secondary prevention, i.e., early detection and
treatment, appears to have decreased the cancer load for cancers of the breast, colon,
and others. The research on cancer-causing viruses (and other infectious agents)
promises to facilitate even greater advances in prevention and treatment.
The hepatitis B vaccine was invented in 1969 two years after the discovery and
identification of hepatitis B virus (HBV). Product development began in the mid-1970s
and it was approved for use in the early 1980s. It is now one of the most commonly
used vaccines worldwide. The hepatitis B vaccination campaigns and other control
measures have dramatically reduced the incidence of infections, the HBV carrier
state, and acute and chronic liver disease. HBV, along with hepatitis C virus (HCV),
is an etiological agent for over 80% of all primary cancers of the liver (hepatocellular carcinoma, HCC) and it is expected that in time there will be dramatic drops in
the incidence of the cancer. Several studies have already shown significant decreases
in the HCC incidence in countries with early and effective vaccination programs
and a high incidence of the cancer. The CDC in its Hepatits B Vaccine Fact Sheet
stated (Hepatitis B Vaccine: Fact Sheet First Anti-cancer Vaccine (2006) http://
www.cdc.gov/hepatitis, May 17, 2006):
Hepatitis B vaccine prevents hepatitis B disease and its serious consequences like hepatocellular carcinoma (liver cancer). Therefore, this is the first anti-cancer vaccine.
The WHO has noted that, second to smoking cessation, HBV vaccination is the
major cancer interventional prevention program.
In 2007, about 25 years after the approval of HBV vaccine, a second cancer prevention
vaccine was accepted by the FDA. Vaccines for several strains of papilloma
virus have been shown to effectively prevent cancer of the cervix, other cancers, and
common diseases of the reproductive system. Its use is now spreading widely as the
appropriate populations for vaccination are being identified.
As a consequence of these practical advances and a growing understanding of the
viral-caused cancer process at the molecular level, we are now in a period of
increased interest in cancer virology, as witnessed by this book and others on the
subject, and the organization of new research groups. In this paper, I briefly review
some of the established cancer virus relations and then discuss the research, public
health, and clinical processes that led to the HBV vaccine and its consequences.
The HBV story can serve as an example that can help to advance similar mass
prevention programs.
Most cancer therapies are dependent on either removing cancer by surgery or by
the destruction of existing cancer cell with radiation, chemotherapy, or by altering
the host immune system to reject cancer cells. For viral-induced cancers, these
harsh methods may not be necessary. The cancer could be averted or delayed by the
27
antiviral treatment of those already infected before symptoms appear and, possibly,
also after clinical disease develops. This should be less toxic than many current
therapies. The effectiveness of antiviral therapy would also be another direct demonstration of the etiological role of a virus.
This is an exciting time for virus/cancer studies as the effectiveness of vaccines
for at least two common cancers inspires a search for additional viral connections
and applications.
Viral Origins of Human Cancers

There are several excellent reviews of this subject, with an emphasis on molecular
virology, for example Boccardo and Villa (2007) and DeVita et al. (2008). As Boccardo
and Villa have noted, the strongest direct evidence for a meaningful etiologic
relation is the prevention of the cancer by vaccination. This has been shown for
HBV and HCC in large national studies (see, for example, Liang et al. 2009), and
for papilloma virus [human papilloma virus (HPV)] and cancer of the cervix in field
trials. As noted above, another direct demonstration of etiologic relation would be
the prevention and/or treatment of a cancer with an antiviral.
There are many other accepted virus/cancer etiologic relations. In many cases,
they are believed to be indirect which appears to mean that they do not conform to
molecular biological models of carcinogenesis. From a practical point of view, the
question of a direct or secondary cause is not as important as an understanding of
process that allows practical prevention or treatment.
An important feature of cancer prevention by vaccination is the question of instituting universal vaccination to protect the relatively small portion of the population
who develop cancer. However, vaccination is further justified if the program also
protects against more common diseases. This is true for HBV vaccine, where protection is also offered against acute and chronic hepatitis, cirrhosis, and liver failure
and probably some forms of kidney diseases, polyarteritis nodosa, and others. The
same could be said for HCV if a vaccine is found for it. The HPV vaccine in addition to protecting against cancer of the cervix and other cancers protects against
several common sexually transmitted and other diseases, including condyloma
acuminate, warts, and recurrent respiratory papillomatosis.
Vaccines are not currently widely used for other cancer-related viruses, but there
is hope that they will be; other preventive methods against infectious agents can
currently be used. The EpsteinBarr virus (EBV) is etiologically related to several
cancers, some of them common in particular locations. They include nasopharyngeal
carcinoma, Burkitts Lymphoma one of the first virus cancer relations suspected
on epidemiologic grounds Hodgkins Disease, and several others. Intensive research
on inventing a vaccine and determining its optimal use is in progress.
Human T-cell leukemia virus (HTLV-1) is etiologically associated with adult
T-cell leukemia as well as other noncancer diseases. These include HTLV-1-associated
myelopathy/tropical spastic paraparesis (HAM/TSP), uveitis, and infective dermatitis.
28
B.S. Blumberg
There are probably other causes of death associated with chronic HTLV-1 infection.
In a historic prospective study in Zaire (now Democratic Republic of the Congo)
where HTLV-1 is common (Lechat et al. 1997), after about 20 years, individuals
who were HTLV-1 carriers in 1969 had a significantly lower survival compared
to noncarriers. These studies confirmed a similar report from Japan. If further
supported, this could justify the widespread use of an HTLV-1 vaccine when and if
one is produced, in that vaccination could prevent many diseases in addition to the
relatively rare leukemia and other identified associated diseases.
HTLV-II has been isolated from a patient with hairy cell leukemia and there may
be an etiologic relation of the virus with this disease.
The EBV is associated with several cancers that are common in particular
geographic regions. Denis Burkitt, a physician practicing and doing epidemiologic
studies in equatorial Africa, described an unusual lymphoma, soon named after
him. He showed that its distribution followed that of malaria and hypothesized
that it had an infectious origin associated with mosquito transmission. Epstein and
Barr, then, isolated a virus from the cells of a Burkitts lymphoma case and the virus
was found to be extremely common in African cases. This was not true of sporadic
cases found away from the tropics, suggesting multiple etiologies operating in
different environment. EBV is also associated with nasopharyngeal carcinoma that
is extremely common in China and elsewhere, and Hodgkins lymphoma.
Human immunodeficiency virus types I and II (HIV-I, HIV-II) are associated
with several cancers. These include other virus-related cancers: Kaposis sarcoma
[associated with human herpes virus 8 (HHV-8) or Kaposis sarcoma herpes virus
(KSHV)], Hodgkins lymphoma (associated with EBV), and cervical carcinoma
(associated with HPV). The immune deficiency that characterizes AIDS may
increase the susceptibility to these cancers, but there also appears to be an interaction
of the genome of HIV in the cancer pathogenic process. These findings illustrate
that the pathogenesis of viral-caused cancers may involve multiple viruses or other
factors in pathogenesis. Humans have many more bacterial cells in their body than
their own cells and even more viruses and one or more of these may interact
with the virus associated with the specific cancer. This may appear to make the problem
incomprehensively complex, but the history of medicine has shown that effective
measure can be found even though the entire process is not fully understood.
Discovery of the Hepatitis B Virus

The discovery of the HBV did not start as a directed search for the virus but as a
project in basic clinical research. It was a circuitous and convoluted process whose
outcome would have been difficult to predict at its onset. (This and following sections
are adapted in part from Blumberg 2002, 2010.)
A striking feature of medicine is the great variation in host response to diseasecausing agents. Starting in 1957, we set out to find inherited polymorphic and
acquired variation in the blood proteins that could be related to disease susceptibility
29
(Blumberg 1961). This is analogous, at the phenotype (proteome) level, to the

contemporary search for the relation between genomic polymorphic variation and
disease using SNIPS and other databases. It was an interesting process as it required
worldwide collections in different disease environments to compare the distribution
of the polymorphic alleles and try to understand their relation to disease. Protein
variation was determined using the recently introduced method of electrophoresis in
gel. In 1961, another technique was deployed. Many serum proteins are polymorphic;
hence, patients who had multiple transfusions would likely to be exposed to proteins
they had not inherited. If some of the polymorphic proteins were antigenic, the patients
might develop antibodies against them and their blood could be used as a reagent
to discover and study antigenic protein polymorphisms. Using double diffusion in agar
gel, we identified a complex, inherited antigenic system of the serum low-density
lipoproteins (the Ag System, Allison and Blumberg 1961). Using these and other
anti-lipoprotein antibodies identified by us (and to a greater measure by others),
associations were found with cardiovascular disease, Alzheimers, and diabetes.
We continued to test the sera of transfused patients against a panel of sera with
the expectation that we would find additional antigen-antibody systems of interest.
In 1963, a precipitin band was detected between the sera of a transfused hemophilia
patient from New York and, among others, the sera of Australians (Blumberg et al.
1965). Much of the subsequent research was done on these sera; the reactant
protein was called Australia antigen (Au). The next problem was to learn what it
was. Au was rare in normal Americans but common in leukemia patients; this
generated the hypothesis that people at high risk of leukemia might also have
high frequencies of Au. Since patients with Downs syndrome (DS, chromosome 21
trisomy associated with mental retardation) are at high risk for an unusual form of
leukemia, we predicted that they would have a high frequency of Au; this was confirmed in a series of studies in large institutions for the mentally retarded (Blumberg
et al. 1967) (Fig. 2.1).
In the meanwhile, there were a series of observations that raised the possibility
that Au was associated with hepatitis. It was found in transfused people, occasional
patients with hepatitis, and in institutionalized patients (i.e., leprosy, mental retardation), where infectious spread would be expected. But, the most convincing observation was in a DS patient. He did not have Au when first seen but did so on
subsequent testing; the appearance of Au coincided with the onset of hepatitis.
We, then, formally tested the hypothesis that Au was associated with hepatitis by
comparing the prevalence of Au in patients with and without clinical hepatitis; Au
was much more common in the patients. The next series of tests were designed to
determine if Au was a hepatitis virus or part of it. This was confirmed by clinical,
electron microscope, transmission and other studies. The initial observations were
soon confirmed by Okochi and Murakami (1968), Vierucci et al. (1968) and Prince
(1968). Prince associated Au with the HBV that had been postulated by Krugman
and other pioneers in the hepatitis field before our discovery of HBV. The identification
of one virus facilitated the identification of others (HAV, HDV, HCV, HEV, etc.) in
several other laboratories that greatly increased the probabilities of control and
treatment of viral hepatitis (Fig. 2.2).
Fig. 2.1 Scan. ppt 112K.

The first published image of
the precipitin reaction in agar
gel between Australia
antigen (the surface antigen
of HBV) in the top well, and
the antibody against it in the
bottom well. The top well
contains serum form a patient
with leukemia who is a
carrier of HBV. The bottom
well contains serum from a
patent with hemophilia who
has received many blood
transfusions, some from
blood donors who were HBV
carriers, and developed
antibodies against the surface
antigen (Australia antigen)
B.S. Blumberg
Fig. 2.2 Blumberg Surinam. ppt (299K). Surinam, South America, September 1950. The photograph
was taken in a Djuka community near the mining town of Moengo on the Cottica River (Kotikaliba) during the course of an infectious disease survey and clinical care project. It was during these
surveys that striking differences in response to infection were noted and reported
31
There were immediate applications of the discovery of HBV. The Au test, as it

was called, became widely used for the diagnosis of acute and chronic hepatitis, a
further aid to control and treatment. It was a major step forward in viral diagnosis;
a virus could be diagnosed within a few hours by direct detection. Previously, viral
diagnosis often depended on comparing the titers of specific antibodies early in
infection to titers during convalescence (Merigan 1997, personal communication).
In 1969, we suggested testing blood donors to detect asymptomatic carriers of
HBV (Blumberg et al. 1969). It was soon adapted at Philadelphia General Hospital
(Senior et al. 1974), and later, after some controversy, at many other places. Soon,
posttransfusion hepatitis due to HBV appeared to be under control. Subsequently,
the discovery of HCV further reduced the frequency of posttransfusion hepatitis to
the extent that it is no longer a major medical and surgical problem.
Invention of the Vaccine

One of the major problems in vaccine invention is the identification of the specific
antibody or cellular immune reaction that provides protection. The failure to do so
has slowed the development of vaccines against HIV, HCV, tuberculosis, malaria,
and other pathogens. The identification of a protective antibody for HBV was
possible soon after the research began. By 1968, we recognized that antibodies
(anti-HBs) against the surface antigen (HBsAg) of HBV were probably protective.
We had rarely seen individuals who had both HBsAg (indicating infection) and
anti-HBs in their blood; this is consistent with protection. Further, Okochi in
Tokyo (Okochi et al. 1970) had shown that patients, who had anti-HBs before they
were transfused with donor blood containing HBV, were much less likely to develop
hepatitis than those who did not have anti-HBs. Later, in a multiyear study in a renal
dialysis unit where HBV infection was endemic, Lustbader demonstrated a striking
level of anti-HBs protection (Lustbader et al. 1976).
A peculiar feature of HBV recognized after EM visualization of particles of the
virus (Bayer et al. 1968; Dane et al. 1970) was that, in addition to the whole virus
particles, there were very large numbers of spherical and rod-shaped particles in the
peripheral blood of carriers and other infected individuals that contained only
HBsAg. In some carriers, this amounted to more than 1% of their total serum
protein. The vaccine was prepared from these particles.
In 1969, the Institute for Cancer Research filed a patent in the USA and foreign
countries for the process for extracting surface antigen particles free from whole
virus as the source of the vaccine and for the product (Blumberg and Millman 1972;
Blumberg 1972). To quote from the summary of the patent application: a vaccine
against viral hepatitis is derived from blood containing Australia antigen, having
particles resembling viruses which are substantially free of nucleic acid, of a size
range of 180210 A, substantially free of infectious particles. The vaccine where
required is attenuated or altered. The preferred procedure of removing impurities
including infectious components involves centrifugation, enzyme digestion, column
32
B.S. Blumberg
gel filtration, differential density centrifugation in a solution of sucrose, dialysis,

differential centrifugation in a solution of cesium chloride and dialysis. At the time,
this was a unique method for producing a vaccine. None had previously been
produced from the blood of viral carriers and none has since then.
Studies by Krugman and his colleagues increased the interest in the Blumberg/
Millman vaccine (Krugman et al. 1971). They inoculated children with a preparation
of HBV-positive serum which had been boiled for 1 min. Subsequently, they evaluated
whether these children had been protected against hepatitis by injecting them with
untreated serum containing the virus. The heated serum provided substantial (but
not complete) protection against HBV infection. This experiment had the effect of
impressing potential manufacturers that the vaccine was feasible.
In 1971, we received an assurance of interest from Merck Pharmaceuticals (Hilleman
1971, personal communication). By 1975, a sufficient amount of work had been done
in laboratories in the USA and elsewhere to encourage us to recommend production
and field testing of the vaccine; a licensing agreement was concluded with Merck. The
noted vaccine expert Maurice Hilleman was given responsibility for the project and an
experimental vaccine was produced and tested in animals (Buynak et al. 1976).
Vaccine Field Trials

By the early 1980s, a series of vaccine field trials were completed, primarily by
Szmuness and colleagues (Szmuness et al. 1980, 1981). The Szmuness trial has
been described as one of the best organized and executed trials of any human vaccine
and a milestone in preventive medicine (London and Blumberg 1985). It was primarily on the basis of this trial that the vaccine was approved by the FDA; it is described
in some detail (summarized from London and Blumberg 1985).
The first problem was to choose a population with a sufficiently high risk of infection to make a vaccine trial feasible. Szmuness believed that the trial should be carried
out in a population which stood to benefit from an effective vaccine (Szmuness et al.
1980). Among the populations at high risk considered for the trial were residents of
institutions for the mentally retarded (in whom we had earlier reported a high HBsAg
frequency), patients undergoing maintenance hemodialysis, members of the medical
staff of hemodialysis centers, American Indians, and homosexual men. By the late
1970s, very few new residents were being admitted to state institutions for the retarded,
and the rate of new infections in long-term residents was quite low. Quarantine procedures had been instituted in Philadelphia (Snydman et al. 1976) and subsequently
elsewhere; they had greatly reduced the incidence of hepatitis B infections.
By 1975, Szmuness ascertained that the risk of infection among homosexual
men in New York City was high and that they were cooperative, intelligent, and
well-educated (Szmuness et al. 1975). The prevalence of hepatitis B markers was
68% among more than 10,000 men surveyed, and the annual incidence of infection
was projected to be 19.2% (Szmuness et al. 1978), later estimated at 30% (Szmuness
33
1980, personal communication). The study included 1,083 male subjects admitted
between November 1978 and October 1979, of whom 549 received vaccine and 534
a placebo. The first two inoculations were given 1 month apart, and the third was
given 6 months after the first. Follow-up was carried out for at least 6 months after
the last dose of vaccine but for 12 months for most participants. Ninety-three percent
of the subjects received all three inoculations, an outstanding compliance rate.
The results were convincing. First, there was no difference in the frequency of
severity of side effects between the vaccine and placebo groups. Second, the antibody
response in the vaccinated groups was excellent; 77% of the vaccines had significant levels of anti-HBs within 2 months of the first inoculation, and 96% had
antibody after the third dose, while only 25% of placebo recipients without
evidence of active HBV infection developed anti-HBs. Third, there was a clear
difference in the number of HBV infectious events between the vaccine and
placebo recipients. Of 122 such events, 93 (76.2%) occurred in the placebo recipients
and 29 (23.8%) in the vaccines (p < 0.0001). Fifty-two of the subjects had an event
classified as hepatitis B (alanine aminotransferase levels 90 IU plus the appearance of HBsAg in their serum). Only seven of these most serious events occurred in
vaccinated men, and all but one of these occurred prior to the third dose of vaccine.
There were 73 HBV events in the placebo group and 14 in the vaccines (p < 0.0001),
and only 4 of the 14 events occurred after the third dose of vaccine. The efficacy
ratio (incidence in placebo recipients over those in vaccines) reached 14.0 for the
period from 5 to 18 months after vaccination. HBV infections which occurred in
vaccine recipients of the full immunization schedule only happened in those
who had not produced anti-HBs antibody. Finally, an unforeseen but clinically and
biologically important result was that those vaccinated subjects who did not produce
anti-HBs were not more likely to develop persistent infections than placebo recipients
who became infected.
Thus, Szmuness and his colleagues (Szmuness 1980, personal communication)
were able to conclude that this placebo-controlled, randomized, double-blind clinical
trial, conducted in 1,083 subjects who had an unusually high risk of hepatitis B
virus infection, proves the efficacy of the vaccine . Subsequent trials supported
this conclusion (Maupas et al. 1981; Francis et al. 1982; Chan et al. 2004; Desmyter
et al. 1983; Benahamon et al. 1984). The Szmuness trial was not only effective, but
also efficient. Just over a 1,000 people were involved. Compare this to the more
than one million children involved in the testing of polio vaccine and the thousands
that have been involved in the so-far unsuccessful trials of an HIV vaccine.
Millions of doses of the plasma-derived vaccine have been used. Reports of side
effects have on occasion led to suspension of the vaccine programs, but they were
subsequently reinstated (Marshall 1998). The effectiveness of HBsAg derived from
plasma as a protection-inducing antibody validated its manufacture by recombinant
methods, and recombinant vaccine is now the major source of the vaccine (McAleer
et al. 1984). It was the first vaccine produced by the recombinant method and for
many years the only one; it has helped to make the vaccine available worldwide as
the cost of manufacture and distribution decreases.
34
B.S. Blumberg
Global Vaccination Programs

National vaccination programs began soon after the vaccine was available (see, for
example, Gatcheva et al. 1995; Bonnanni 1995; Ginsberg and Shouval 1992; De la
Torre and Esteban 1995). In February 1990, we convened The International
Conference on Prospects for Eradication of Hepatitis B Virus that that included
reports on the extent of the HBV endemic, the national resources available for
vaccination programs, and the possibilities for control and, possibly, eradication
(Blumberg 1990). In 1992, the WHO placed HBV vaccine on the Extended Program
on Vaccination setting a target of 1997 for integration into national programs. By
April of 2005, 158 of the 192 members of the World Health Organization had
national vaccination programs (Kim et al. 2007). HBV is one of the most widely
used vaccines worldwide.
The results are impressive. Newborn and childhood vaccination was started in
Taiwan in 1984 with excellent national participation and long-term reporting of the
results (Chan et al. 2004). In 1999, 15 years later, the carrier prevalence had dropped
from 9.8 to 0.7% (p < 0.001). The prevalence had also dropped significantly (but
not as much) in children in a similar cohort who had not been vaccinated. There
have been similar findings elsewhere (see, for example, Da Villa et al. 1992, 1995).
This implies a type of herd immunity that could hasten the overall effect of the
program and accelerate control and, possibly, eradication.
There was also a striking drop in deadly fulminant hepatitis in young children
(Kao et al. 2001). From 1975 to 1984, the average mortality from fulminant hepatitis
was 5.36/100,000 infants; from 1985 to 1998 after the vaccination program had
started it was 1.71/100,000.
There have been reports from elsewhere of striking drops in the prevalence of
HBV carriers and decrease in the incidence of clinical hepatitis. Several HBV
carrier surveys before and after vaccination programs have been summarized
(Blumberg 2004). In a regional study in the Peoples Republic of China, the prevalence of carriers decreased from 16.0 to 1.4%. The before and after percentages in
other countries are similar: The Gambia 10.0 and 0.6%; Japan 2.7 and 0.9%; Saudi
Arabia, 6.7 and 0.3%; Catalonia (Spain), 9.3 and 0.9%. In the USA, the rate of new
HBV infections has declined significantly since 1991. It dropped from a peak of
more than 70,000 cases in 1984 to less than 20,000 in 2006. The decline has been
greatest among children born since 1991, when routine vaccination of children was
recommended by the CDC (CDC Web site 2006). In Alaska, following an intensive
vaccination campaign among Native Americans, the incidence dropped from 215
cases/100,000 before the vaccination programs to 714 cases/100,000 in 1993 after
the program was in place. In 1995, no cases were reported (McMahon et al. 1996).
A national hepatitis serological survey was conducted in China in 1992 (Liang
et al. 2009). The authors estimated that there were 120 million HBV carriers, that
20 million patients suffered from chronic effects of HBV including chronic liver
disease (cirrhosis, liver failure, etc.) and HCC, and that about 300,000 die annually
from the consequences of late-stage HBV infection. Liver cancer and cirrhosis are
35
both on the list of the ten most common causes of fatal diseases and HBV is the
major cause of both of these. About one-third of all the HBV carriers in the world
are in Peoples Republic of China.
In 2009, a report was published on the effects of the vaccine program based on a
national epidemiological study involving 160 counties, 369 township and village
clusters, and 81,775 persons (Liang et al. 2009). The authors concluded that China
had achieved the goal of reducing HBsAg prevalence to less than 1% in the vaccinated population (mostly children under 5 years) and 1620 million HBV carriers
had been prevented as a result of the infant vaccination program. Furthermore, in
the vaccine-impacted population, i.e., young children, 2.83.5 million future
deaths have been prevented. There are about 375400 million carriers worldwide.
A tentative projection to the whole world population can be attempted using the
Chinese data. In many places worldwide, the vaccination programs began earlier
and the compliance rates were somewhat higher. The Chinese estimates were based
on the results in the childhood vaccinated population. In several studies, it has been
shown that in countries with successful childhood vaccination program there is
also a significant drop in HBV carrier incidence in the unvaccinated population,
presumably as a consequence of herd immunity (see above). Taken these factors
into account, about 1525 million future deaths have been prevented worldwide.
Plans for the continuation of the successful campaign in China are being
discussed, including the possibility of eradication (So 2006).
HBV: The First Cancer Vaccine

Perhaps, the most conceptually important outcome of the vaccination program is
the decrease of HCC, primary cancer of the liver. HCC is one of the most deadly
and common cancers worldwide. It is the third most common cause of death from
cancer in males and the seventh most common in females. Most HCCs are caused
by infection with HBV or HCV; HBV is said to account for 6575% of the cases
worldwide. In Taiwan, the yearly incidence of HCC in the vaccine-impacted
population (age 614 years) declined from 0.7/100,000 (19811986) before
vaccination programs were fully implemented to 0.36/100,000 between 1990 and
1994 after the vaccination program was in place (Huang and Lin 2000; Chan et al.
2004). A follow-up report found that the prevention of HCC has continued from
childhood to early adulthood (Chang et al. 2009). The major cause of continued
disease was inadequate vaccination and highly infective mothers. This is another
indication for the treatment of HBV carrier mothers, particularly those with titers of
HBV DNA.
HBV vaccine is the first preventive cancer vaccine with a demonstrated impact
on the worlds cancer load. In 2007, about 25 years after the introduction of the
first, the second cancer prevention vaccine was launched. It provided effective
protection against strains of papilloma virus preventing cancer of the cervix and
other cancers (Chen and Berek 2007). There are also continuing studies on the
36
B.S. Blumberg
possibility of vaccine protection against EB virus and the prevention of nasopharyngeal and other cancers (Hepeng 2008).
Secondary Antiviral Prevention: Prevention by Delay

National vaccination programs are the most effective means of controlling HCC.
But what of those already infected? There are about 400 million carriers of HBV
and some 300 million carriers of HCV at risk for chronic liver disease and HCC.
Antiviral treatment may delay or abort the risk of these diseases. We termed this
prevention by delay (Blumberg and London 1981) as the antiviral treatment can
prolong the symptom-free period until the carrier dies of other causes. Treatment of
HBV carriers with antivirals (see, for example, Liaw et al. 2004) or by other means
can greatly decrease the risk of HCC; this is discussed elsewhere in this book.
A further advantage of treatment is that decreasing titers of virus decreases the
infectivity of carriers and patients and, therefore, the risk of transmission to those
who have not been vaccinated or were inadequately vaccinated. In particular, it
could reduce the transmission of HBV from mothers to their children at the time of
birth and soon afterward. This could hasten the control of HBV and increase the
possibility of eradication.
Fortunately, prevention of HCC, and probably of other viral-caused cancers, has
the double arm of primary prevention and secondary prevention to aid in control.
The Genetics of Hepatitis B Virus

The initial research on hepatitis B began as a study in the inheritance of susceptibility
to HBV chronic infection (Blumberg et al. 1966), and more recent population and
genomic studies have added rich detail (for review, see, Blumberg 2006a, b). There
are multiple loci at which one allele increases susceptibility to chronic infection
and an alternate allele increases the probability of developing protective antibody.
An added interesting aspect of these observations is that these same alleles may
affect susceptibility to other infectious agents. For example; the DRB1*1302 allele
at the MHC Class II locus (chromosome 6) is related to susceptibility to HBV
chronic infection, response to falciparum malaria, and response to papilloma virus.
The VDR locus (chromosome 12) is related to responses to HBV, Mycobacterium
tuberculosis, and Mycobacterium leprae; there are many other examples. We have
categorized the organisms with affinities to the same genetic locus as Microorganism
Gene Affinity Clusters (MIGAC). In some cases, an allele that confers an advantage
to the host for one member of the cluster may be disadvantageous for another. These
allelic variations constitute genetic polymorphisms and as such both advantages and
disadvantages may be expected [for example, carriers of HBV bind larger quantities
of iron than uninfected people. This may be an advantage in regions with low dietary
37
iron intake (Sutnick et al. 1974; Felton et al. 1979)]. It is interesting to conjecture
how members of the cluster are affected if one of the infectious agents in the cluster
is greatly decreased as a result of a successful vaccination program.
Conjectures on the Future of Research on Viruses and Cancer

1. The Editor has asked me to comment on the possible directions for future
research, including my preferences, in respect to cancer and viruses or, more
broadly, infectious agents. Preventive measures have made an important contribution to the decrease of the load of human cancer and, obviously, research
should be encouraged and increased. The apparent success of the HBV vaccine
program in decreasing the incidence of one of the most common and deadly
cancers was an encouragement to seek others. As already noted, it required about
25 years for the introduction of the second cancer prevention vaccine, against
the papilloma virus, an indication of the lack of funding for this theater of
research in the past.
2. There are multiple factors in the pathogenesis of cancers. Independent of what
is considered the real cause, often expected to be some gene selection, introduction, or alteration, the most important cause may be distant from genetic
effects but amenable to interventional change. For example, HBV and HCV may
not be directly involved in genetic change consistent with theoretical expectations, but prevention or treatment of infection can considerably decrease
risk. Control of other contributors to advancing pathogenesis, for example the
removal of aflatoxins and/or other environmental enhancers of cancer,
decreasing iron and iron storage levels (Weinberg 1984; Hann et al. 1989) controlling excessive alcohol intake, and probably many others, can contribute significantly to decreasing the risk of HCC and other untoward consequences of HBV
infection (Blumberg 2002).
3. Viruses that are designated as the main cause of a cancer interact with other
infectious agents in the host. As noted above, there are interactions of the genome
of HIV with other cancer-associated virus. Humans are infected normally with
vast numbers of bacteria and virus. It is unlikely that they would not interact with
cancer-related viruses. Also, hosts are often coinfected if viruses have similar
transmission routes. HBV infection is associated with infection with HCV, HIV,
HTLV-1, and probably other blood-borne infectious agents. The host can respond
differently to each of the following: acute disease, carrier state, development of
chronic infection, production of protective and other antibodies, integration into
the host genome, or genomes of the other organisms. The study of the interactions
of viruses with each other and with the genome of the host can provide insight
into the process of carcinogenesis, therapy, and prevention.
4. In their review, Boccardo and Villa (2007) cite many examples in which the same
or similar viruses may cause several cancers (i.e., EBV and Burkitts Lymphoma,
nasopharyngeal carcinoma, Hodgkins disease, etc.; HPV and genital carcinoma,
38
B.S. Blumberg
carcinoma of the oropharynx, etc.). It would be fruitful to continue this search.

There are several studies showing an association of HBV with cancer of the
pancreas (see, for example, Iloeje et al. 2010). Duck HBV, a hepadnavirus
(HBV-related viruses), localizes in the pancreas and HBV, although primarily
localized to the liver, and is also found in the pancreas in humans (Coyne et al.
1970). These findings suggest that HBV has a role in cancer of the pancreas or
that another virus that is cross antigenic with HBV may be involved. It is informative to determine if the HBV vaccination campaign has had an effect of the incidence
of cancer of the pancreas. The first disease associations reported for HBV were
with leukemias, but there has been little follow-up on these leads (Blumberg
et al. 1967). HBV has recently been associated with lymphoma (Engels et al.
2010), and further studies on this connection are warranted.
5. Most cancer viruses are associated with more noncancer disease; in many cases,
they are more common than the cancer. The association (noted above) of chronic
HTLV-1 infection with decreased survival is an example of the potential public
health importance of chronic infections with viral-caused cancers. The recent
studies of zur Hausen and his colleagues (zur Hausen 2010) on the Torque Teno
Virus (TTV) that is related to cancers, MS, and other disease characterized by
inflammation is an important example of how this research is proceeding.
6. Prevention against and treatment of infectious agents has been one of the
most successful accomplishments of scientific medicine and this can be
extended to cancers caused or influenced by infectious agents. A future direction for cancer centers and research institutes, then, could or should be to
seek viruses in patients with various cancers and, perhaps more importantly,
those who are at the risk of cancer. A strategic approach would be to start
with the null hypothesis that all cancers include one or more microorganisms
(including viruses) in their etiological roster, and then use available methods
(and methods to be developed) for detecting past or present infection. This
could include infection in prior generations of the host that remain in the
hosts genome.
7. Another fruitful area for research would be to continue the search for polymorphic
genetic variation that increases the risk for and/or alters the pathogenesis and
treatment of cancer. There have been important advances in this field since the
early days of searching for polymorphic variants in serum proteins and elsewhere
that influence susceptibility to disease and affect response to drugs (see, for
example, Blumberg 1961). The use of whole genome sequences has facilitated
the identification of many sites, but it has also made it more complicated and
difficult to apply. As data accumulates, it is likely that general laws will emerge
and the use of these variations to protect the most susceptible will increase.
Identifying the genetic components of susceptibility makes it easier to find environmental agents in that a subpopulation can be identified in which the environmental
influences are strong. Further investigation of Magac clusters (see above) can
provide leads on disease connections and operations of many genes on the
same outcome.
39
8. The discovery and development of new and the improvement of current antivirals
effective against specific or many viruses should be a major research program.
The intensive research on treatments for HIV and AIDS has given strong support
to pharmaceutical companies seeking useful and profitable products, and this can
be extended to HBV and HCV. (There are worldwide about 700 million carriers
of HBV and HCV.) It was recognized early in the HBV research programs that
complete removal of the virus from its host may be difficult or impossible as the
virus is often integrated into the host genome. However, decreased titers can
considerably reduce the risk for the development of chronic liver disease and
HCC. There are now several antivirals that are effective in doing this and others
are in the process of development. In many cases, carriers of cancer-related
viruses require long-term treatment using relatively low doses as reduction of
titer rather than total elimination is the goal. This could also result in decreased
side effects. Long-term treatments are attractive to pharmaceutical companies
and could encourage research and development of the antivirals. The availability
of effective and safe antivirals encourages the detection of carriers, of whom currently
only a small percentage has been identified.
Conclusions
Viruses cause many of the cancers that afflict humans and these same viruses often
cause other noncancer diseases that may be more common than the cancers.
This provides an additional justification for national and universal vaccination
programs. Known cancer viruses are often implicated in the etiology or pathogenesis
of other cancers and it is important to find these connections. If this is common, then
the existing cancer vaccine programs and those added in the future may lead to
decreases in the incidence of other cancers not now recognized as virally caused.
There are now two vaccination programs HBV and HPV that are in place and
appear to be successful; it is expected that others will be added in time as vaccines
or other preventive measures are found for the existing virus-related cancers. It is
likely that viruses have a role in the pathogenesis of many other cancers than those
already identified and an important direction for cancer research would be to find
these and develop countermeasures, including vaccination. There is also the possibility of developing new and better antvirals to treat those chronically infected
with known cancer viruses. As experience with secondary prevention of cancer with
antivirals increases, treatment may be a useful method to determine if a cancer is
caused by a virus.
The HBV vaccine cancer prevention program has been in place for nearly
30 years. It has served as a model for future programs and has been described in
some detail in this chapter. HBV was discovered in a nongoal-directed project, as is
often the case in the solution of medical and biological problems. The original
vaccine was derived from a portion of the virus the surface antigen particles that
40
B.S. Blumberg
exist in large quantities in the blood of carriers of HBV; this was a unique method
of making a vaccine that did not require cell culture. HBV vaccine was subsequently
made by the recombinant method, the first vaccine so produced. It is now one of
the most commonly used vaccines in the world. (However, there are still regions
that are inadequately served.) The reduction in the prevalence of HBV carriers and
the incidence of acute and chronic liver disease and HCC has been profound.
Rigorous control and even eradication may be possible.
We are in a period of increased awareness of the viruscancer connection and
the major effect it has and can have on the control of several and perhaps many
cancers. Strong support for continued research and application moves these programs
forward.
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Chapter 3
Virus-Mediated Cell Proliferation

Sun-Hwa Lee, Stacy Lee, and Jae Ung Jung
Introduction
It is estimated that 1520% of all human cancers are linked to human tumor viruses,
including hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus
(HPV), human T-cell lymphotropic virus (HTLV), EpsteinBarr virus (EBV), and
Kaposis sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8).
HTLV is an RNA tumor virus associated with adult T-cell leukemia, whereas
HBV, HPV, EBV, and KSHV are DNA tumor viruses associated with liver cancer
(HBV), cervical and other anogenital cancers (HPV), Burkitts lymphoma and
nasopharyngeal carcinoma (EBV), and Kaposis sarcoma (KS) (KSHV) (Dayaram
and Marriott 2008).
Simply defined, cell proliferation is the increase in cell numbers as a result of cell
growth and division. For normal cells, entry into an active proliferative state from a
quiescent state (G0) depends on the presence of exogenous, mitogenic, growth stimulatory signals, such as diffusible/soluble growth factors, components of the extracellular matrix (ECM), and cell-to-cell adhesion/interaction molecules, since the
binding of these stimulatory signals to their receptors induces a variety of intracellular signaling transduction pathways involved in cellular proliferation (Hanahan
and Weinberg 2000). Tumor cells, however, depend less on exogenous, growth
stimulatory signals in the initiation of proliferation. Thus, the ability to proliferate
in the absence of external growth factors is suggested to be one of the hallmarks of
tumor cells and is generally achieved by either overexpression of growth receptors
and/or ligands, mutations in receptors, or downstream signaling molecules whose
activities are independent of ligand binding or defects in specific components of the
cell cycle machinery (Hanahan and Weinberg 2000).
S.-H. Lee (*) S. Lee J.U. Jung

Department of Molecular Microbiology and Immunology, University of Southern California,
School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA
e-mail: sunhlee@usc.edu
45
46
S.-H. Lee et al.
Likewise, tumor viruses appear to have evolved numerous strategies that promote
the proliferation of infected host cells in the absence of external growth stimulatory
signals for their replication and survival, thereby ultimately contributing to the transformation of infected host cells. To this end, several viral proteins encoded by tumor
viruses have been shown to dysregulate normal cellular processes, such as cell cycle
progression, apoptosis, immune surveillance, and antiviral responses, allowing viral
replication and survival. Many excellent reviews on the impact of tumorigenic
viruses on cellular processes, including cell cycle progression, have been published
(Damania 2007; Dayaram and Marriott 2008; Gatza et al. 2005; Helt and Galloway
2003; Hoppe-Seyler and Butz 1995; McLaughlin-Drubin and Munger 2008; Sears
and Nevins 2002). This review focuses on the biochemical and molecular strategies
used by oncogenic HHVs, including EBV and KSHV, to enhance cell proliferation.
Oncogenic Human Herpesviruses

The Herpesviridae family comprises large, double-stranded DNA viruses with a
genome size of 100200 kb. Members of this family are classified as three subfamilies based on their genomic organization and biological characteristics: alpha (a),
beta (b), and gamma (g). Eight HHVs are known so far. Members of the a-HHV
include herpes simplex viruses (HSV) 1 and 2 (HHV-1 and HHV-2) and varicellar
zoster virus (VZV; HHV-3). Members of the b-HHV include cytomegalovirus
(CMV; HHV-5), HHV-6 variants A and B, and HHV-7. Members of g-hepesviruses
are further classified as g1-herpesviruses (lymphocryptoviruses) and g2-herpesviruses (rhadinoviruses). EBV (HHV-4) and KSHV (HHV-8) belong to g1- and g2HHV, respectively (Damania 2007). Among the members of the HHV family, only
EBV and KSHV have been implicated in a variety of human cancers.
Association of both EBV and KSHV with a number of human cancers derives
from two distinct features of their life cycles, latency and lytic cycle. In a lytic cycle,
viruses replicate extensively and express virtually all viral genes, ultimately leading
to the production of progeny viruses and the death of infected host cells (Ganem
2007). This lytic infection probably occurs either during primary infection or periodically in certain physiologic conditions, causing viral spread between cells and
hosts (Kalt et al. 2009). In latency, on the other hand, the viral genome is maintained
as a circular episome in the nuclei of infected host cells. Only a handful of viral
genes are expressed and progeny viruses are not produced. In addition to these two
life cycles, both EBV and KSHV have other common features: (1) both can infect
B lymphocytes, (2) their latency is associated with human cancers, and (3) it is
difficult to model their lytic replication cycles in vitro (Hume and Kalejta 2009).
EBV (HHV-4). EBV is the first human virus to be directly implicated in carcinogenesis and over 90% of the global adult population is infected with EBV. It is usually
asymptomatic, but a proportion of EBV-infected individuals develop infectious
mononucleosis (IM), a disease characterized by lymphadenopathy and fatigue, later
in life. During acute infection, EBV primarily infects and replicates in the stratified
3 Virus-Mediated Cell Proliferation
47
squamous epithelium of the oropharynx. This is followed by latent infection of

B lymphocytes. While lytic infection is known to be associated with oral hairy leukoplakia, a proliferative disorder in immunocompromised patients, most EBVassociated malignancies are caused by latent infection (Kutok and Wang 2006).
EBV-associated malignancies include Burkitts lymphoma (BL), Hodgkins
lymphoma (HL), nasopharyngeal carcinoma (NPC), T and natural killer (NK) cells
lymphoma, and posttransplant lymphoma (Kutok and Wang 2006). BL is a malignancy principally found in children, especially those who live in regions of Africa
with a high incidence of malaria, although it also occurs more sporadically in other
areas of the world. BL tumor cells are highly linked with EBV as almost all African
BL patients are EBV positive. NPC is a malignancy of the squamous epithelium
situated in the nasopharynx. EBV-associated NPC frequently occurs in Southern
China, Northern Africa, and among Eskimo populations and is thought to arise from
clonal expansion of latently infected cells. HL is the most common EBV-associated
malignancy occurring in the Western world (about 3090% of all HL patients are
EBV positive). EBV is also highly present in immunoblastic lymphomas in HIVinfected individuals (about 70%) as well as in immunosuppressed posttransplant
patients (100%) (Damania et al. 2000).
EBV can infect primary human B lymphocytes in vitro, converting them into
continuously growing, semiactivated, immortalized, and transformed lymphoblastoid cell lines (LCLs). Within LCLs, EBV is latent. Among the >85 genes encoded
by EBV, only 11 viral proteins are expressed in LCLs: six nuclear antigens (EBNAs
1, 2, 3A, 3B, 3C, and 5), three latent membrane proteins (LMPs 1, 2A, and 2B), and
two EBV-encoded small nonpolyadenylated (noncoding) RNAs (EBERs 1 and 2).
Among these, EBNA-2, -3A, -3C and LMP1 are required for the in vitro immortalization of B lymphocytes by EBV (Rickinson and Kieff 2007).
KSHV (HHV-8). KSHV is the second HHV implicated in human malignancies. KSHV
is uncommon in the general population (less than 7%, but some geographical areas
have infection rates as high as 60%). KSHV is primarily transmitted through saliva,
although other transmission routes, including vertical, sexual, blood, and transplantrelated transmission, have also been reported (Pica and Volpi 2007). KSHV can infect
many different types of cells in vitro, including B cells, epithelial cells, endothelial
cells, and cells of the monocyte/macrophage lineage (Brinkmann and Schulz 2006).
In addition, KSHV has been shown to immortalize primary human endothelial cells to
have long-term proliferation and survival and to establish latency in B cells and
endothelial cells in vivo (Brinkmann and Schulz 2006; Damania et al. 2000).
KSHV is the etiologic cause of Kaposis sarcoma, an endothelial neoplasm.
Globally, KS is the fourth most common cancer caused by infection, after gastric
cancer (Helicobacter pylori), cervical cancer (human papillomavirus), and liver
cancer (hepatitis cirrhosis). KS remains a major cause of cancer-related deaths
among immune-suppressed and organ-transplant patients (Kalt et al. 2009).
KSHV infection is also highly associated with two rare atypical B-cell lymphoproliferative diseases: primary effusion lymphomas (PEL)/body cavity B-cell
lymphomas (BCBL) and multicentric Castlemans disease (MCD). These are principally or exclusively of B-cell origin. MCD is a polyclonal B-cell lymphoproliferative
48
S.-H. Lee et al.
disease with dissemination to multiple lymph nodes and other lymphoid tissues.
In AIDS patients, MCD is invariably associated with KSHV infection, whereas
approximately half the cases of HIV-negative individuals are KSHV associated.
PEL is a monoclonal B-cell lymphoproliferative disease marked by rapid proliferation of cells in the pleural, peritoneal, and pericardial cavities. It usually occurs in
AIDS or other immunosuppressed individuals and is often associated with KSHV
and EBV coinfection. Most of what we know about KSHV biology has been
obtained from studying viral gene expression in cultured PEL cells, which grow
readily in culture, stably maintain latent KSHV genome, and express latent transcripts and proteins in virtually all cultured cells (Ganem 2006).
KSHV proteins expressed during latency are believed to contribute to the development of KSHV-associated diseases. Five major latency-associated viral proteins
have been identified in latently infected lymphoma cells: the ORF73-71 locus
encoding latency-associated nuclear antigen (LANA-1, ORF73), viral cyclin
(v-Cyclin, ORF72), viral FLICE inhibitory protein (vFLIP, ORF71, K13), viral
interferon regulatory factor (vIRF) 3 (LANA-2, K10.5), and vIL-6 (K2) (Ganem
2007). All, except for LANA-2 which is exclusively expressed in PEL and MCD,
are also expressed in all KSHV-infected cells and have been shown to affect cellular
proliferation and survival (Ganem 2006).
Viral Strategies for Self-Proliferation

Oncogenic HHVs, including EBV and KSHV, are equipped with strategies that
promote the proliferation of infected host cells for their survival and replication.
Both lytic and latent EBV and KSHV viral proteins demonstrate the ability to activate
growth signaling by functioning as (1) growth factor receptors, (2) growth factor
receptor ligands, (3) signal transduction molecules, (4) cell cycle regulators, or (5)
transcription factors. Interestingly, some viral proteins are of cellular origin.
Viral Proteins Mimicking Growth Receptor

A common feature shared by EBV and KSHV is the presence of membrane-associated
viral proteins located at the left and right ends of the coding regions of their viral
genomes. These viral proteins are LMP1 (EBV), LMP2A (EBV), K1 (KSHV), and
K15 (KSHV). Unlike LMP1 and LMP2A, both K1 and K15 are mainly expressed
during the lytic replication cycle (Brinkmann and Schulz 2006). There is no sequence
homology among the proteins, although there is a limited structural similarity
(Fig. 3.1). Yet, they all function as constitutively active receptors capable of inducing an array of cellular signaling pathways in a ligand-independent manner. In addition,
they all have distinct oncogenic/transforming potentials. Thus, it has been suggested
that these viral proteins act by mimicking normal growth signals required for the
49
NH2
EBV
LMP1
EBV
LMP2A
KSHV
K1
Lyn
NH2
I
T
A
M
COOH
C
T
A
R
1
Lyn
KSHV
K15
Vav
Syk
NH2
TRAFs
PLC
TRAF1,2,3,5
PTKs
I
T
A
M
p85
Syk
COOH
COOH
RIP
C
T
A
R
2
TRAF6
Ca2+
TRADD
COOH
MAPK
PI3K/Akt
MAPK
NH2
JAK
MAPK
PI3K/Akt
2+
Ca
Fig. 3.1 Membrane-associated viral proteins encoded by both ends of EBV and KSHV genomes
proliferation and survival of cells, which ultimately contributes to the transformation

of infected cells (Brinkmann and Schulz 2006; Damania et al. 2000; Schulz 2006).
This section briefly discusses EBV and KSHV viral proteins which enhance selfproliferation by acting as growth factor receptors and their ligands.
EBV LMP1. LMP1, the first open reading frame (ORF) of EBV, is expressed in
all EBV-related malignancies. It is essential for the immortalization of primary B
lymphocytes to LCLs in vitro (Rickinson and Kieff 2007). It is an integral membrane
protein containing a short cytoplasmic N-terminus (23 aa), six transmembrane
(TM)-spanning domains, and a long cytoplasmic C-terminus (200 aa). A substantial
amount of experimental evidence suggests that the six TM domains and the two
cytoplasmic C-terminal domains of LMP1, termed transformation effector sites
(TESs) 1 and 2 or C-terminal NF-kB-activating regions (CTARs)1 and 2, are critical for the conversion of primary B lymphocytes to LCLs by LMP1 (Fig. 3.1,
Soni et al. 2007).
It has been shown that LMP1 markedly mimics an important B-cell activation
receptor CD40, which induces the activation of NF-kB, a key transcription factor
involved in the regulation of cell growth, antiapoptosis, and expression of numerous cytokines upon CD40ligand interaction (Glenn et al. 1999; Hatzivassiliou
et al. 1998; Kilger et al. 1998). Like CD40, LMP1 when expressed in B cells
recruits tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and the
TNF receptor-associated death domain (TRADD) through its CTAR1 and CTAR2,
respectively (Devergne et al. 1998; Eliopoulos et al. 1996; Eliopoulos and Young
1998; Izumi et al. 1997). Unlike CD40, however, LMP1 transduces signals in the
absence of extracellular ligands or cross-linking by self-oligomerizing through its
N-terminal and the TM domains on the plasma membrane. This self-oligomerization mimics the effects of receptor/ligand interactions, giving rise to the constitutive
activation of a number of downstream signaling pathways (Clausse et al. 1997;
50
S.-H. Lee et al.
Eliopoulos and Young 1998; Floettmann et al. 1996; Hatzivassiliou et al. 1998;
Huen et al. 1995; Izumi et al. 1997; Izumi and Kieff 1997; Laherty et al. 1992).
Thus, by mimicking the B-cell activation receptor CD40, LMP1 contributes to the
progression of EBV-associated malignancies. More detailed molecular mechanisms by which LMP1 activates NF-kB signaling are discussed in later in this
review.
In addition to NF-kB signaling, activation of both MAPK and JAK/STAT is also
implicated to be the function of LMP1 (Eliopoulos et al. 1999; Eliopoulos and
Young 1998; Gires et al. 1999; Huen et al. 1995). The MAPK family, a group of
serine/threonine kinases activated in response to extracellular environment signals
such as growth factor, cytokines, and stress signals, is involved in a variety of key
events, including proliferation, differentiation, apoptosis, and migration. The MAPK
family consists of three parallel pathways, namely ERK, JNK, and p38. In epithelial
cells, LMP1-mediated activation of JNK and p38 depends more on its CTAR2
domain (Eliopoulos et al. 1999; Eliopoulos and Young 1998). In lymphocytes,
however, both CTAR1 and CTAR2 are necessary for the activation of JNK and p38
(Soni et al. 2007). LMP1-mediated activation of JNK requires TRAF6, TAK1, and
TAB1, but not TRAF2, TRADD, IRAK4, MyD88, and RIP, indicating that LMP1
selectively utilizes cellular signaling molecules involved in TNFR or IL-1/TLR
receptors that maximize growth and survival signals without inducing apoptosis
(Soni et al. 2007). Meanwhile, JAK/STAT signaling mediated by LMP1 integrates
with the AP-1 transcription factor pathway. The region between CTAR1 and CTAR2
contains proline-rich sequences and is involved in the interaction with members of
the JAK family. Thus, this motif of LMP1 is believed to play a role in JAK3-mediated
activation of STAT3 (Gires et al. 1999). Taken together, LMP1 appears to mediate
constitutive activation of cellular signaling pathways important for controlling
EBV-infected cell survival and proliferation by mimicking activated receptors.
EBV LMP2A. LMP2A is another membrane protein expressed in EBV latently
infected B cells. LMP2A contains 12 TM domains linked by loops, a long cytoplasmic N-terminal (119 aa), and a short C-terminal domain (27 aa). LMP2A aggregates
in the plasma membrane. The N-terminal cytoplasmic domain of LMP2A contains
three tyrosine-based SH2 binding motifs, two of which form a functional immunoreceptor tyrosine-based activation motif (ITAM) (Fig. 3.1, Fruehling and Longnecker
1997). ITAM motifs [(D/E)x7(D/E)x2YxxL/I/Vx68YxxL/I/V, where x is any amino
acid] are composed of a stretch of negatively charged amino acids followed by two
SH2 binding motifs (YxxL/I/V). Found in the cytoplasmic domains of T- or B-cell
receptors, ITAM motifs are tyrosine phosphorylated by Src family protein tyrosine
kinases (SF-PTKs) upon ligand engagement. Signaling molecules containing SH2
domains are then subsequently recruited, leading to the induction of an array of
intracellular signaling pathways. In the case of LMP2A, its ITAM is tyrosine phosphorylated and is required for LMP2A association with the SH2 domain of the
nonreceptor tyrosine kinases, such as Lyn, Fyn, Syk, and Csk (Beaufils et al. 1993;
Burkhardt et al. 1992; Longnecker et al. 1991; Scholle et al. 1999), to induce intracellular calcium mobilization and cytokine production.
51
LMP2A interaction with Lyn and Syk mimics BCR signaling, inducing the
activation of the PI3K/Akt survival pathway in the absence of BCR signals (Caldwell
et al. 1998). The PI3K/Akt signaling pathway plays an important role in mediating
transformation, antiapoptotic effects, invasion, and adhesion. Akt is a serine/threonine
kinase, which phosphorylates and regulates the activities of cell cycle regulatory
proteins, such as GSK-3b and cyclin D. LMP2A is also involved in the activation of
MAPK signaling in various EBV-infected cell lines in vitro (Chen et al. 2002;
Panousis and Rowe 1997). LMP2A binds to and is phosphorylated by ERK, leading
to the activation of MAPK signaling in B-cell lines (Panousis and Rowe 1997).
LMP2A activates JNK, but not p38, in NPC cell lines (Chen et al. 2002). This
in vitro observation is supported by a recent study, in which LMP2A expression in
transgenic mice induced the constitutive activation of the ERK/MAPK and PI3K/Akt
pathways, resulting in cell proliferation and survival (Anderson and Longnecker 2008).
KSHV K1. KSHV K1 is the first ORF located at a position equivalent to that of the
LMP1 of EBV (Lagunoff et al. 1999; Lee et al. 1998b; Zong et al. 1999). K1 is a
46-kDa transmembrane glycoprotein consisting of an N-terminal extracellular
domain, a TM domain, and a short cytoplasmic C-terminal domain (Fig. 3.1). Its
N-terminal extracellular domain contains several N-glycosylation sites and displays
a high degree of genetic variability between different KSHV isolates. A survey of
isolates led to the identification of five major subtypes of K1 (AE), each containing
several distinct variants (Brinkmann and Schulz 2006). In contrast, its C-terminal
cytoplasmic (38 aa) is relatively well-conserved and contains a functional ITAM
similar to the one found in LMP2A (Lee et al. 1998a). The K1 ITAM motif is
phosphorylated and recruits Lyn, Syk, PLCg2, the p85 subunit of PI3K, Vav1, SHP1
and SHP2, RAS-GAP, and growth factor receptor-bound protein 2 (Lagunoff et al.
1999; Lee et al. 2005). Interaction of K1 with these cellular proteins results in the
activation of several transcription factors, including AP-1, NF-AT, and Akt-driven
forkhead box containing proteins, all of which are involved in preventing apoptosis
(Tomlinson and Damania 2004). In addition, K1 interaction with Lyn leads to the
activation of NF-kB in B cells (Prakash et al. 2005). Similar to EBV LMP1 and
LMP2A, K1-mediated signaling occurs constitutively, independent of ligand binding by self-oligomerization through its extracellular domain (Lagunoff et al. 1999).
In addition, K1 induces VEGF and matrix metalloproteinase-9 (MMP-9) in endothelial
cells (Greene et al. 2007), suggesting that K1 may contribute to angiogenesis and
cell division.
KSHV K15. The K15 gene of KSHV is located at a position equivalent to that of
EBV LMP2A (Choi et al. 2000; Glenn et al. 1999; Poole et al. 1999). Sequence
analysis of the K15 gene from different KSHV isolates revealed that the K15 gene
is highly variable, showing as much as 6070% divergence at the amino acid level
(Poole et al. 1999). K15 is a transmembrane protein consisting of 412 TM domains
and a short cytoplasmic C-terminal domain (amino acid 335489) (Fig. 3.1). Like
LMP2A, the cytoplasmic domain of K15 contains multiple signaling motifs, which
have been shown to be highly conserved in most isolates (Poole et al. 1999): a putative SH3 binding motif (P386PPLPP), a potential SH2 binding motif (Y481EEVL), a
52
S.-H. Lee et al.
tyrosine-based signaling motif (Y431ASIL), and a putative TRAF binding motif

(A473TQPTDD) (Glenn et al. 1999; Poole et al. 1999). The cytoplasmic domain of
K15 binds to TRAF1, TRAF2, and TRAF3 in vitro (Glenn et al. 1999). The tyrosine
residue within the Y481EEVL motif of K15 is constitutively tyrosine phosphorylated
by cellular SF-PTKs, including Src, Hck, Lck, Fyn, and Yes (Burkhardt et al. 1992;
Choi et al. 2000; Rajcani and Kudelova 2003). Once phosphorylated, K15 activates the
ERK2 and JNK pathways, which together lead to AP-1 activation (Brinkmann et al.
2003). TRAF2 interaction with the region containing Y481EEVL is involved in K15mediated activation of the MAPK pathway. Tyrosine phosphorylation within the
Y481EEVL motif is required for TRAF2 binding and NF-kB activation (Brinkmann
et al. 2003), indicating the importance of the Y481 residue of K15 in its activation of
diverse signaling pathways. It has been reported that K15 is expressed in KSHVlatently infected PEL and MCD cells (Sharp et al. 2002), leading to the suggestion
that the interaction between K15 and SF-PTKs plays a role in the maintenance of
latency (Cho et al. 2008; Pietrek et al. 2010). Taken together, these findings suggest
that K15 seems to function similarly to LMP1 (e.g., the recruitment of TRAFs and
the activation of NF-kB and JNK) as well as to LMP2A (e.g., the recruitment of
SF-PTKs).
Modulation of Chemokine/Cytokine System

The mammalian chemokine system is composed of chemokines and chemokine receptors. Members of the chemokine superfamily currently consist of at least 46 members
that are structurally related small proteins around 810 kDa in size. Four subfamilies
have been identified so far based on the relative position of their cysteine (C) residues,
which form conserved disulfide bonds: CCL, CXCL, XCL, and CX3L. The majority
are either CCL chemokines (with no intervening amino acids between two cysteine
residues) or CXCL (with a single intervening amino acid (X) between two cysteine
residues) (Mantovani et al. 2010). The main function of chemokines is to attract different cells. For example, the CCL family members are known to attract a variety of cells
from immune system, whereas the CXCL family members mainly attract neutrophils
and lymphocytes. However, chemokines also regulate other biological activities,
such as cell proliferation and differentiation, survival, angiogenesis, and senescence
(Mantovani et al. 2010). Thus, deregulation of chemokine expression has been implicated in tumor growth, angiogenesis, and metastasis (Mantovani et al. 2010).
Biological effects of chemokines are mainly mediated by G protein-coupled
receptors (GPCRs), a diverse family of membrane receptors containing seven TM
domains. While the extracellular domains of GPCRs engage with a variety of
ligands, its cytoplasmic domain couples to heterotrimeric G proteins made up of an
a subunit and a bg heterodimer. Following ligand binding, its TM domains undergo
a series of conformational changes, catalyzing GDP to GTP exchange on a Ga subunit and subsequently generating a free GTP-bound Ga subunit and a free Gbg
heterodimer to activate downstream signaling effector proteins, including PLC and
53
adenylate cyclase (AC), which function as secondary messenger molecules.

Although mutant forms of GPCR or heterotrimeric G proteins are not frequently
found in human cancer, malignant cells often subvert the normal physiological
functions of GPCRs to promote autonomous proliferation, evade immune responses,
increase blood supply, and invade surrounding tissues and disseminate to other
organs (Dorsam and Gutkind 2007). Several members of the HHV family, including
EBV, KSHV, HHV-6, -7, and hCMV-1, encode mimicries of cellular chemokines
and chemokine receptors, indicating that these viruses might have evolved to hijack
host chemokine system for their propagation and replicative advantage (Rosenkilde
et al. 2008).
KSHV Viral Chemokines. KSHV expresses at least three chemokine homologs of
cellular macrophage-inhibitory proteins (MIPs), which are members of the CCL
chemokine family (Moore et al. 1996). For this reason, they were previously named
vMIPI (vCCL1), vMIPII (vCCL2), and vMIPIII (vCCL3) and are encoded by ORFs
K6, K4, and K4.1, respectively. These KSHV-encoded chemokines act on cellular
chemokine receptors expressed on Th2 cells, such as CCR8 (vCCL1 and vCCL2),
CCR3 (vCCL2), and CCR4 (vCCL3), from which their immunomodulatory functions are derived (Boshoff et al. 1997; Dairaghi et al. 1999; Endres et al. 1999; Stine
et al. 2000). Interestingly, vCCLs, unlike their cellular homologs, have been shown
to promote angiogenesis in certain experimental systems (Boshoff et al. 1997;
Simonart et al. 2001; Stine et al. 2000). Since angiogenesis is a key feature of KS,
vCCLs were speculated to be possible angiogenic factors of KS. For instance,
vCCL1 activates the induction of a potent angiogenic factor, VEGF, and its receptor
KDR (Flt-1) in vCCL1-expressing cells. Thus, it has been suggested that increased
signaling mediated by upregulated VEGF and its receptor by vCCL1 may enhance
new blood vessel formation and proliferation of tumor cells within the microenvironment of angiogenic KS lesions (Liu et al. 2001). Since KS lesions are also
characterized by the presence of infiltrating leukocytes and high levels of inflammatory cytokines, the chemotactic properties of vCCLs may further promote infiltration of monocytes/macrophages to KS lesions to facilitate the production of
inflammatory cytokines and proangiogenic factors (Direkze and Laman 2004).
KSHV vGPCR. The KSHV vGPCR, encoded by ORF74, is homologous to the
human chemokine receptors CXCR1 and CXCR2, which are the receptors for the
angiogenic chemokines IL-8 (also known as CXCL8) and growth-related oncogene
a (Gro-a, also known as CXCL1). Similar to cellular GPCRs, KSHV vGPCR is a
seven TM protein with conserved glycosylation sites in its N-terminal and cysteine
residues in its extracellular loops (Fig. 3.2a, Arvanitakis et al. 1997; Bais et al.
1998). Unlike cellular GPCRs, however, vGPCR is ligand-independent and constitutively active due to the presence of several structural changes, including a mutation (Asp142Val) within its highly conserved DRY (Asp-Arg-Tyr) motif (Rosenkilde
et al. 2008). Moreover, compared to other viral and cellular chemokine receptors,
vGPCR shows a higher degree of promiscuity, capable of binding not only cellular
CXCL1 and CXCL8 but also vCCL2, indicating that vGPCR can induce signaling
in a ligand-dependent fashion as well (Geras-Raaka et al. 1998; Gershengorn et al. 1998).
54
S.-H. Lee et al.
KSHV vGPCR
EBV BILF1
Cellular,
Viral ligands
ligands ?
NH2
NH2
COOH
GTP
a
GTP
GTP
MAPK
PI3K/Akt
mTOR
Fig. 3.2 Signaling mediated by viral chemokine receptors
Thus, both ligand-dependent and -independent activation of vGPCR may contribute

to tumorigenesis and/or viral replication uniquely in the microenvironment of
KSHV-associated tumors. The broad signaling capability of vGPCR also encompasses signaling through several G-protein a subunit families (Gas, Gaq, and Gai)
(Shepard et al. 2001; Smit et al. 2002) as well as Rac-1, a member of the Rho family
of monomeric G-proteins (Montaner et al. 2004).
Signaling pathways constitutively activated by KSHV vGPCR include MAPK,
PLC, PI3K, and Akt. Expression of KSHV vGPCR in human umbilical vascular
endothelial cells (HUVECs) results in the induction of PI3K and Akt activity, which
in turn plays a central role in promoting cell survival (Bais et al. 2003; Montaner
et al. 2001; Sodhi et al. 2004). Akt activity is tightly regulated by PI3K. When growth
factor receptors bind to ligands, the catalytic subunit (p110) of PI3K is activated via
recruitment of the regulatory subunit (p85) of PI3K or via Ras activation, both of
which lead to the production of PIP3. Akt in the cytoplasm then binds to PIP3 and
subsequently translocates to the plasma membrane, where its kinase activity is then
fully induced by PI3K-dependent kinases (PDKs). The tumor-suppressor protein
PTEN antagonizes PI3K by dephosphorylating PIP3, reducing Akt translocation to
the cellular membrane, thereby downregulating Akt activity. It has been reported that
treatment of an Akt inhibitor prevents the proliferation of vGPCR-expressing
endothelial cells in vitro and inhibits the tumorigenic potential of vGPCR in mice
(Sodhi et al. 2004). This suggests that the activation of Akt is a significant factor in
the development of vGPCR-induced sarcoma and perhaps KS. Recent studies have
further demonstrated that vGPCR-mediated Akt activity can activate the mTOR
pathway, which also plays a central role in cell proliferation, metabolism, and angiogenesis (Sodhi et al. 2006). Constitutive activation of Akt by vGPCR-mediated PI3K
55
activation results in phosphorylation and subsequent degradation of the tuberous

sclerosis complex (TSC), a negative regulator of mTOR (Sodhi et al. 2006). As a
result, mTOR activity is induced, giving rise to the activation of ribosomal p70 S6
kinase and 4EBP1, the key regulators of the translational machinery, and the promotion of cell proliferation. Treatment of rapamycin, a pharmacological inhibitor of
mTOR, showed a dramatic suppression of vGPCR-expressing cell proliferation
in vitro as well as tumor growth in vivo, indicating that vGPCR-mediated activation
of the PI3k/Akt/mTOR cascade may contribute to vGPCR-mediated sarcomagenesis
(Montaner 2007).
Similar to vCCLs, vGPCR has also been reported to have angiogenic functions.
Endothelial cells ectopically expressing vGPCR have constitutively active VEGF
receptors and increased proliferation (Bais et al. 2003). In addition to VEGF, vGPCR
also upregulates angiogenic chemokines IL-8 and Gro-a in a manner similar to
human CXCR2, a chemokine receptor associated with angiogenesis in a number of
tumors (Montaner et al. 2004). Another signaling pathway induced by vGPCR is the
activation of p38 and ERK and the subsequent phosphorylation of hypoxia-induced
factor 1a (HIF-1a). This, then, acts on the VEGF promoter, resulting in the induction of VEGF expression and secretion (Sodhi et al. 2000). vGPCR can also activate
such transcription factors as NF-AT, AP-1, and NF-kB and promote the expression
of a number of autocrine and paracrine proinflammatory cytokines and growth
factors, such as IL-1b, IL-6, GM-CSF, TNFa, IL-8, and MIP-1, as well as adhesion
molecules, such as VCAM-1, ICAM-1, and E-selectin (Cannon et al. 2003; Couty
et al. 2001; Montaner et al. 2001; Pati et al. 2001, 2003; Schwarz and Murphy 2001;
Shepard et al. 2001; Smit et al. 2002).
As discussed above, vGPCR appears to exert broad effects on cell proliferation,
angiogenesis, and inflammation for a mere single viral gene product. Thus, KSHV
vGPCR, although expressed during the lytic phase of its viral life cycle, clearly
stands out as one of the key factors involved in the proliferation of KSHV-associated
tumor cells.
EBV BILF1. EBV encodes a GPCR homolog called BILF1, which is expressed in
the lytic phase of the viral replication cycle. Although it displays a limited homology to chemokine receptors, sequence analysis revealed that BILF1 has several features
belonging to GPCRs. It contains seven TM domains, conserved cysteine residues in
its N-terminus and in its extracellular loops, seven N-glycosylation sites, and four
intracellular phosphorylation sites (Fig. 3.2b, Paulsen et al. 2005). BILF1 is known
to constitutively activate signaling pathways involved in proliferation (NF-kB)
through Gai (Paulsen et al. 2005). Similar to KSHV vGPCR, the DRY motif in
BILF1 is replaced with an EKT (Glu-Lys-Thr) motif (Paulsen et al. 2005). Alternative
DRY motifs found in KSHV vGPCR have been previously shown to be associated
with constitutive activity as well as transforming activity (Rosenkilde et al. 2008).
Unsurprisingly, a recent study demonstrated that BILF1, similar to KSHV vGPCR,
has both of these abilities in vitro and in vivo (Lyngaa et al. 2010). This study shows
that the EKT motif plays a key role in the constitutive activation of BILF1 and that
constitutive signaling through Gai is associated with BILF1-mediated cell transformation, VEGF secretion, and tumor formation (Lyngaa et al. 2010). Although these
56
S.-H. Lee et al.
studies imply that like KSHV vGPCR BILF1 may play an important role during
EBV infection by mimicking a functional GPCR, additional roles of BILF1 in
immune evasion by targeting PKR and MHC class I (Garcia et al. 2006; Zuo et al.
2009) suggest that BILF1 may function distinctly from KSHV vGPCR. Moreover,
it still remains unknown whether chemokines and other ligands bind to BILF1.
KSHV vIL-6 (ORF K2). Cellular interleukin (IL)-6 is a multifunctional cytokine
involved in the regulation of immune responses, inflammation, oncogenesis, and
angiogenesis (Nishimoto and Kishimoto 2006). Signaling induced by IL-6 requires
the IL-6 receptor (IL-6R) complex composed of gp80 (the a subunit) and gp130
(the b subunit). gp130 is ubiquitously expressed and homodimerizes to transduce
intracellular signaling, whereas gp80 binds to IL-6 but is limited in its availability.
IL-6/IL-6R signaling induces signaling pathways, such as the Ras/MAPK and JAK/
STAT pathways, which are in turn involved in the activation of transcription of IL-6
responsive genes (c-Fos and c-Jun). Many B-cell tumor cell lines depend on IL-6
signaling for growth (Nishimoto and Kishimoto 2006).
KSHV encodes a homolog of cellular IL-6, vIL-6 encoded in (ORF) K2 (Neipel
et al. 1997). It has 24.6% amino acid sequence homology to cellular IL-6. Although
expressed abundantly during lytic replication, various levels of vIL-6 expression have
been detected in latently infected cells (MCD>PEL>>KS; <5% of cells in PEL and in
about 520% of KSHV-infected lymphoid cells in extracavity PEL and MCD, but not
in KS) (Du et al. 2007). Unlike cellular IL-6, however, vIL-6 is selectively glycosylated and signals through only gp130 to activate IL-6 responsive genes and promote
B-cell survival (Chatterjee et al. 2002; Hoischen et al. 2000; Martin and Gutkind 2009;
Molden et al. 1997; Mullberg et al. 2000). Thus, vIL-6 bypasses the normal cellular
checkpoint of gp80 coupling with gp130 for IL-6 signaling (Molden et al. 1997).
Similar to cellular IL-6, vIL-6 has multifunctional roles, targeting various cellular
processes, such as cell proliferation, oncogenesis, and immune responses (Nicholas
2005). Interestingly, it has been reported that vIL-6 can functionally replace cellular
IL-6 in many B-cell tumor cell lines, which are dependent on IL-6 for their growth.
This indicates the substantial functional homology between viral and cellular IL-6
(Burger et al. 1998; Moore et al. 1996). In addition, it has been shown that vIL-6
activates all of the known cellular IL-6-induced signaling pathways, including the
Ras/MAPK and JAK/STAT signaling cascades. Activation of the JAK/STAT
pathway by vIL-6 results in the induction of VEGF (Molden et al. 1997), underscoring the likely importance of the angiogenic property of vIL-6 in the development of
KSHV-associated malignancies. In addition, several lines of evidence suggest that
both cellular and viral IL-6 are important in the proliferation of KSHV-associated
B-cell lymphomas: (1) PEL-derived cell lines depend on vIL-6 and IL-10 for their
growth and the induction of cellular IL-6 expression (Jones et al. 1999); (2) vIL-6
can induce the secretion of cellular IL-6 (Mori et al. 2000); (3) in addition to vIL-6,
LANA-1, vFLIP, and vGPCR can also induce cellular IL-6 expression, most likely
by activating AP-1 and NF-kB (An et al. 2002; Du et al. 2007; Montaner et al. 2004;
Polson et al. 2002); and (4) high serum levels of IL-6 are detected in patients with
PEL and MCD (Ganem 2007). Thus, both cellular IL-6 and vIL-6 signaling may
57
contribute to PEL cell proliferation and to the angiogenesis present in patients with
KSHV-associated lymphoproliferative diseases.
Modulation of Cell Cycle Machinery

Normal tissues maintain cellular quiescence and tissue homeostasis by employing
multiple antiproliferative signals (Hanahan and Weinberg 2000). Similar to growth
stimulatory signals, growth inhibitory signals, including both soluble and immortalized
inhibitors embedded in the ECM and on the surface of nearby cells, are received by
TM receptors and transduced into intracellular antiproliferative signaling circuits,
which eventually block proliferation. Depending on the incoming signal, cells in the
G1 phase of the cell cycle decide whether to initiate proliferation or to prevent proliferation either by forcing themselves out of the active proliferative cycle into a
quiescent (G0) state or by inducing permanent (irreversible) entry into a postmitotic
and differentiated state (Dayaram and Marriott 2008). Much of the antiproliferative
signals induced in normal cells upon receiving growth inhibitory signals are associated
with cell cycle block, in which tumor-suppressor genes, such as p53, and members
of the retinoblastoma (Rb) family (Rb, p107 and p130) play critical roles at the
molecular level. Rb and p53 mutations are observed at high frequencies in a wide
variety of clinically important cancers (Hanahan and Weinberg 2000). Most DNA
tumor viruses encode viral oncoproteins, which can disrupt the function of the p53
and Rb family members at least in part by directly interacting and inactivating them,
which presumably allow infected cells to escape from p53- or Rb-mediated cell
cycle arrest (Hume and Kalejta 2009; OShea 2005). Both EBV and KSHV appear
to encode proteins capable of targeting multiple cellular components of the cell
cycle machinery, including Rb and p53, to promote cell proliferation. Thus, this
section briefly introduces the cellular antiproliferative pathways mediated by Rb
and p53 prior to the discussion of how viral proteins encoded by EBV and KSHV
modulate cellular antiproliferative signals to ultimately contribute to the proliferation
of tumor cells.
Rb-mediated Cell Cycle Arrest. Progression of normal cell cycle transition from G1
through S, and G2 into M, occurs in a sequential and coordinated fashion. For example, the sequential activation of cyclin and cyclin-dependent kinase (Cdk) complexes is a central event in cell cycle transitions: cyclin D and Cdk4, Cdk6 and Cdk2
in G1, cyclin E and Cdk2 in late G1, cyclin A and Cdk2 in S and G2 phase, and
cyclins B and A and Cdk1 in M phase (cyclin B is synthesized in the G2 and M
phase). Activated Cdks then accelerate cell cycle progression (Hume and Kalejta
2009). One of the main substrates of Cdks is the Rb protein, which is known to
contain multiple putative Cdk phosphorylation sites. In the absence of growth stimulatory factors, Rb, constitutively expressed during the G1 phase, exists in a hypophosphorylated form and forms a complex with the E2F transcriptional activator,
resulting in the repression of E2F-mediated transcription of cellular genes required
for a G1 to S phase transition (Rb-mediated G1 cell cycle arrest) (Fig. 3.3, Hume and
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S.-H. Lee et al.

Mitotic
Growth
Factors
DNA Damage
ATM, ATR
p19INK4
p16INK4
Cyclin D
Cdk4/6
Mdm2
p53
p21
E2F
Cdk4/6
Rb
Rb
Cdk6
vCyclin
E2F
Apoptosis
Go
G1
Cyclin D
Cyclin E
Cdk4/6
Cdk2
P15
P16
P18
p19
P21
p27
Cyclin A
DNA
Replication
Cdk2
P21
p27
Fig. 3.3 Rb- and p53-mediated antiproliferative pathways
Kalejta 2009). In the presence of mitogenic growth factors, however, D-type cyclins
(cyclins D1, D2, and D3) are synthesized throughout the G1 phase and activate
Cdk4/6 by forming a complex with either Cdk4 or Cdk6. Active cyclin D-Cdk4/6
complexes then phosphorylate Rb, and hyperphosphorylated Rb is unable to interact
with E2F. The released E2F is then able to stimulate the transcription of cellular
genes essential for subsequent cell cycle progression into the S phase.
Moreover, cell cycle is intricately regulated by a sophisticated interplay of positive
and negative regulatory signals. In the absence of stimulatory growth signals, negative regulatory signals prevent uncontrolled cellular proliferation by maintaining Cdks
in an inactive state. This is achieved by two distinct classes of Cdk inhibitors (CKIs):
the Cip/Kip family includes p21Cip1, p27Kip1, and p57Kip2 and inhibits G1 cyclinCdk
and cyclin BCdk1 complexes, and the other class is the INK4 family including
p16INK4a, p15INK4b, p18INK4c, and p19INK4d, which specifically inhibits G1 cyclinCdk
complexes (cyclin D/Cdk4 or cyclin D/Cdk6). p16INK4a is an inhibitor of cyclin
D-dependent kinases, such as Cdk4 and Cdk6, and blocks Rb function, whereas
p19INK4d blocks the murine double minute 2 (Mdm2)-mediated destruction of p53,
thereby inducing the expression of the Cdk inhibitor p21Cip1 (Vermeulen et al. 2003).
p53-mediated Cell Cycle Arrest. Cellular DNA is continuously exposed to a variety
of endogenous and exogenous genotoxic influences. Thus, cells must have an efficient surveillance system to monitor the integrity of their DNA and eliminate
acquired DNA damage. Upon detecting genomic injuries in the G1 phase, the DNA
damage checkpoint response is induced by activating the DNA damage sensors
59
ATM and ATR; protein kinases capable of phosphorylating a large number of

protein substrates involved in cell cycle control and DNA repair, including the
tumor-suppressor protein p53; and checkpoint kinases (Chk) 1 and 2. Phosphorylation
of p53 by activated ATM/ATR increases its stability and its function as a transcriptional activator. Activation of p53 by DNA damage promotes the transcription of
p21, an inhibitor of G1 cyclinCdk, which consequently causes G1 cell cycle arrest
(p53-mediated G1 cell cycle arrest) (Fig. 3.3). Since genes induced by activated p53
are involved in apoptosis, induction of p53 also leads to apoptosis (p53-mediated
apoptosis) (Fig. 3.3). Under normal conditions, a low level of p53 is maintained by
Mdm2, an E3 ubiquitin ligase that exerts its negative effect on p53 in two ways.
First, the N-terminus of Mdm2 interacts with the transactivation domain of p53,
thus inhibiting the transcriptional activity of p53. Second, the C-terminal ubiquitin
ligase domain of Mdm2 ubiquitinates p53, resulting in p53 degradation through the
ubiquitinproteasome pathway. Additionally, Mdm2 and p53 are components of an
autoregulatory negative feedback loop, wherein p53 induces Mdm2 expression,
while Mdm2 represses p53 activity (Fig. 3.3). This feedback loop confers the tight
regulation necessary for proper p53 function (Lee et al. 2009). Thus, the subversion
of cell cycle components involved in the Rb pathway as well as the p53 pathway
could render tumor cells insensitive to a number of antiproliferative signals.
EBV LMP1. LMP1 has been reported to modestly induce cyclin D2 expression, perhaps
indirectly through the induction of c-Myc and AP-1 transcription factors, and to
maintain Rb in a hyperphosphorylated state in B cells treated with TGF-b (Arvanitakis
et al. 1995; Dirmeier et al. 2005; Hume and Kalejta 2009). In addition, LMP1 downregulates the expression of CKI p16INK4a and inhibits Ras-mediated induction of p16
and p21 (Yang et al. 2000). Recent studies have demonstrated that LMP1 decreases
p27 transcription by inducing the binding of E2F4 and p130, a member of the Rb
family, to the predicted E2F site within the p27 promoter. Furthermore, it increases
the levels of Cdk2 and Rb phosphorylation by inducing the PI3KAkt signaling
pathway (Everly et al. 2009; Mainou et al. 2005, 2007; Mainou and Raab-Traub
2006). Thus, by inducing cyclin D and downregulating both p16 and p27, LMP1
may play a pivotal role in EBV-associated lymphoproliferative diseases.
EBV EBNA-1. EBV EBNA-1 is the only EBV protein consistently expressed in all
proliferating infected cells. It is a multifunctional sequence-specific DNA binding
phosphoprotein required for the replication and maintenance of the EBV genome
(Rickinson and Kieff 2007). The best known function of EBNA-1 is to maintain
latent episomal DNA as an episomal plasmid, which must be distributed into daughter
cells during division (Thompson and Kurzrock 2004). Four functional modules
have been characterized (Rickinson and Kieff 2007): (a) an arginine-rich N-terminal
spanning 89 amino acids; (b) a region encompassing amino acids 90327 of GGA
repeats; (c) amino acids 328386 which are also arginine rich and contain a nuclear
localization signal at amino acids 379386, and (d) the C-terminal comprising
amino acids 451608 that allows sequence-specific DNA binding and dimerization
of EBNA-1 molecules (Rajcani and Kudelova 2003). The two arginine-rich regions
mediate homotypic interactions between DNA-bound EBNA-1 molecules and are
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involved in transcriptional activation, plasmid maintenance, and DNA replication

(Mackey and Sugden 1999). Recently, it has been reported that EBNA-1 increases
cell survival and proliferation by targeting HAUSP, a deubiquitinase that plays a
key role in the regulation of p53 and Mdm2. Overexpression of HAUSP stabilizes
p53, resulting in p53-mediated growth repression and apoptosis, whereas decreased
HAUSP levels destabilize p53. HAUSP also stabilizes Mdm2, a negative regulator
of p53, as described above. Thus, HAUSP plays multiple roles in regulating the
p53Mdm2 pathway. The HAUSP N-terminal TRAF domain (amino acids 53208)
binds not only to the C-terminal regulatory region of p53 (amino acids 357382),
but also EBNA-1 (amino acids 395450) (Holowaty et al. 2003). However, EBNA-1
binds HAUSP much stronger (tenfold) than the p53 regulatory region. The EBNA-1
395450 peptide also displaces the p53 peptide from the p53HAUSP complex.
EBNA-1 binds HAUSP in EBV-infected cells and EBNA-1 interferes with the
UV-induced stabilization of p53 when expressed at levels similar to those in EBVinfected cells. Taken together, several lines of evidence indicate that EBNA-1 can
indirectly destabilize p53 by binding to HAUSP, which could be important for initial cell immortalization by EBV, continued proliferation and survival of latently
infected cells, and/or malignant transformation.
EBV EBNA-3C. The EBNA-3 gene family comprises three genes placed tandemly on
the EBV genome. The products of EBNA-3 genes are EBNA-3A, -3B, and -3C. These
proteins are nuclear phosphoproteins and function mainly as transcriptional regulators. Among these, EBNA-3A and -3C are crucial for B-cell transformation in vitro,
whereas 3B is dispensable (Rickinson and Kieff 2007). In addition to acting as a transcriptional regulator, a number of studies indicate that EBNA-3C can also regulate the
cell cycle by interacting with and modulating several components of the cell cycle
(Kashuba et al. 2003, 2008; Knight and Robertson 2004; Knight et al. 2004, 2005a, b;
Parker et al. 1996, 2000; Saha et al. 2009; Yi et al. 2009). Earlier studies have shown
that EBNA-3C directly interacts with Rb in vitro (Parker et al. 1996, 2000). This interaction conferred rodent fibroblasts resistance to the effects of p16INK4a. Subsequently,
it was reported that EBNA-3C is required for LCLs to proliferate continuously and to
maintain low levels of both p16INK4a protein and mRNA. Although this study suggests
that EBNA-3C may repress p16 expression, evidence of EBNA-3C interaction with
Rb in virus-infected cells was not provided (Parker et al. 2000).
Another mechanism by which EBNA-3C may modulate the Rb pathway is by forming a complex with cyclin A, an activator of S-phase cell cycle (Knight and Robertson
2004; Knight et al. 2004). EBNA-3C was shown to associate with cyclin A in vitro as
well as in LCLs. Interestingly, it was also shown that the expression of ENBA-3C
induces cyclin A/Cdk2 kinase activity and disrupts cyclin A interaction with p27Kip1, a
potent inhibitor of cyclin A/Cdk2 (Knight and Robertson 2004; Knight et al. 2004).
Recent studies further explore the potential roles of EBNA-3C as a modulator of
cell cycle by regulating both Rb and p53 (Kashuba et al. 2008; Knight et al. 2005a,
b; Saha et al. 2009; Yi et al. 2009). It has been reported that EBNA-3C recruits the
SCFSkp2 E3 ubiquitin ligase complex, known to promote the polyubiquitination and
degradation of p27, E2F, and c-Myc, to facilitate the degradation of p27Kip1 and Rb
61
(Knight et al. 2005a, b). One study demonstrated that EBNA-3C can bind to and
translocate MRS18-2, a mitochondrial protein capable of binding to both hypo- and
hyperphosphorylated forms of Rb, into the nucleus, where MRS18-2 competes with
E2F for binding to Rb, thereby allowing free E2F to induce S-phase entry (Kashuba
et al. 2008). In addition to Rb, EBNA-3C has been shown to directly interact with
p53 in vitro (Saha et al. 2009; Yi et al. 2009). Interaction of EBNA-3C and p53
substantially reduces luciferase activity under the control of a p53-responsive
promoter and blocks p53-mediated apoptosis (Yi et al. 2009). Moreover, EBNA-3C
was shown to form a ternary complex with Mdm2 and p53, although EBNA-3C
interaction with Mdm2 was reduced in the presence of p53. EBNA-3C binding to
Mdm2 results in the stabilization of Mdm2, promoting p53 polyubiquitination and
subsequent p53 degradation in various cell lines (Saha et al. 2009).
EBV ENBA-5 (EBNA-LP). EBNA-5 is a nuclear phosphoprotein expressed early
during EBV infection of B cells. Early studies have suggested that EBNA-5 may
dysregulate cell cycle progression by binding to both Rb and p53 (Hoppe-Seyler
and Butz 1995; Szekely et al. 1993). EBNA-5 was reported to bind to Rb in GST
pull-down experiments despite lacking either an LxCxE motif or hydrophobic patch
(Szekely et al. 1993). However, the biological significance of Rb binding remains
unclear since ENBA-5 was unable to counteract the repressive effects of Rb or p107
on a reporter construct under the control of a Gal4E2F1 fusion protein (Inman
and Farrell 1995). Another mechanism by which EBNA-5 may dysregulate cell
cycle progression is by cooperating with ENBA-2. EBNA-5 was shown to interact
with EBNA-2 to drive resting B cells that were stimulated with the EBV gp340
envelope protein into the G1 phase of the cell cycle by upregulating cyclin D2
expression (Sinclair et al. 1994). Moreover, recent studies have hypothesized that
EBNA-5 binds p14ARF and Mdm2, two proteins involved in p53 regulation (Kashuba
et al. 2003, 2010), leading to the inhibition of the transactivating function of p53.
KSHV vCyclin. KSHV vCyclin encoded in ORF72 is a homolog of cellular cyclin
D2, displaying around 53% sequence similarity to cyclin D2 (Li et al. 1997).
Although vCyclin weakly binds to Cdk2, Cdk3, and Cdk4, it strongly forms a complex
with Cdk6 (Chang et al. 1996; Godden-Kent et al. 1997; Li et al. 1997). Tight interaction of vCyclin with Cdk6 results in the robust activation of Cdk6, leading to
enhanced phosphorylation of Rb (Fig. 3.3, Li et al. 1997). Although vCyclin is
structurally and functionally similar to type D cyclins, it has several unique features.
First, unlike cellular type D cyclins, vCyclin exhibits promiscuous substrate specificity,
targeting substrates of type A and E cyclins coupled to Cdk2 kinases. These include
targets of the replication machinery, such as histone H1, p27, Cdc25a (targets of
the cyclin E/Cdk2 complex), ORC1, and Cdc6 (targets of the cyclinA/Cdk2 complex)
(Ellis et al. 1999; Laman et al. 2001; Mann et al. 1999), indicating that vCyclin/
Cdk6 mimics the combined activities of G1- and S-phase cyclin/Cdk complexes
(Verschuren et al. 2004). Second, unlike the cellular cyclin D/Cdk6 complex, which
normally requires Cdk6 phosphorylation through the activation of a Cdk-activating
kinase (CAK), vCyclin is less dependent on host kinases since the vCyclin/
Cdk6 complex does not require phosphorylation by CAK (Child and Mann 2001;
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Kaldis et al. 2001). Third, the vCyclin/Cdk complex can escape from CKI inhibition by targeting p27 and p21, potent inhibitors of cyclin D, A, and E and weak
inhibitors of cyclin B-associated kinases. vCyclin modulates p27 levels by inducing
p27 degradation, which normally occurs in the presence of A/E type cyclins, but not
D type cyclins (Ellis et al. 1999; Mann et al. 1999; Rajcani and Kudelova 2003).
Recently, it has been demonstrated that vCyclin stably associates with p27 in PEL
cells, and that the vCyclin/Cdk6 complex phosphorylates p27 on serine 10, leading
to its sequestration in the cytoplasm and thereby inactivating its antiproliferative
function (Sarek et al. 2006). This may allow PEL cells to proliferate despite their
high levels of p27. Similarly, the vCyclin/Cdk6 complex phosphorylates p21 on
serine 130, bypassing p21-mediated G1 arrest (Jarviluoma and Ojala 2006). Thus,
the vCyclin/Cdk6 protein complex is resistant to many cell cycle checkpoints,
including Rb and CKI, contributing to enhanced rates of cell proliferation.
KSHV LANA-1. KSHV LANA-1 (ORF73) is a major latency-associated nuclear
protein expressed in PEL, MCD, and KS. Thus, it is the most widely used marker
for KSHV latency. There is no known mammalian homolog. It is composed of three
subdomains: (a) a central region containing variable numbers of highly acidic
repeats; (b) a more basic C-terminal region involved in DNA binding and oligomerization; and (c) an N-terminal region implicated in chromatin attachment and
corepressor recruitment (Ganem 2006). LANA-1 is a multifunctional protein. The
best-characterized function of LANA-1 is its involvement in the establishment and
maintenance of the latent viral episome in the nucleus by tethering the viral episome
to host chromatin during mitosis to ensure segregation of viral genomes to daughter
cells (functionally similar to the EBV EBNA-1 protein) (Ballestas et al. 1999; Cotter
and Robertson 1999).
It has been reported that KSHV LANA-1 targets both the Rb pathway and p53
pathway (Friborg et al. 1999; Radkov et al. 2000). The finding that LANA-1 directly
binds Rb through a highly acidic region in vitro and in PEL cells, transactivating
E2F-dependent promoters and the cyclin E promoter (Radkov et al. 2000) indicates
that LANA-1 alleviates Rb-mediated cell cycle arrest. LANA-1 blocks p53-mediated
apoptosis by binding to p53 and inhibiting the transcriptional activity of p53 in PEL
cells (Friborg et al. 1999). This results in the prolonged life span of primary endothelial cells overexpressing LANA-1 (Watanabe et al. 2003), suggesting a potential
role of LANA-1 in promoting cell survival.
KSHV vIRFs. The KSHV genome encodes four potential homologs of cellular IRFs
and they are thus named vIRF 1 (K9), 2 (K11), 3 (K10.5; LANA-2), and 4 (K10).
KSHV vIRFs are numbered based on the order of their discovery and characterization, not on their homology to any particular cellular IRF. For example, vIRF3 is not
the viral homolog of cellular IRF3; it is more closely related to IRF4 (Ganem 2006).
Most of vIRFs are lytic proteins, but vIRF3 is a latent gene primarily expressed in
latently infected B cells, not in KS spindle cells (Ganem 2006). In addition to the
downregulation of IFN-mediated host innate immune responses, KSHV vIRFs share
one more common feature, which is the targeting of p53-mediated cell growth
control (Lee et al. 2009). vIRF1 interacts with p53 to suppress its acetylation, inhibiting
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the transcriptional activation of p53 and efficiently preventing p53-mediated apoptosis

(Nakamura et al. 2001; Seo et al. 2001). Moreover, vIRF1 interacts with ATM to
block its activity, thereby reducing p53 phosphorylation at its serine 15 residue and
increasing p53 ubiquitination (Shin et al. 2006). In conjunction with vIRF1, the B
cell-specific, latently expressed vIRF3 also interacts with p53, thereby inhibiting
p53-mediated transcription and apoptosis (Rivas et al. 2001). Silencing it by siRNA/
shRNA in PEL cell lines results in increased apoptosis and caspase3/7 activity, suggesting that this protein contributes to the survival of PEL cells (Carbone and
Gloghini 2008). Recently, it was shown that vIRF4 interacts with Mdm2, leading to
a reduction of p53 via proteosomal degradation, thereby effectively suppressing p53mediated apoptosis and establishing favorable circumstances for viral replication
(Lee et al. 2009). Collectively, it is clear that the downregulation of p53-mediated
cell growth control is a common characteristic of the vIRFs (Lee et al. 2009).
Subversion of Cellular Signaling Pathways

Associated with Cell Proliferation
Constitutive Activation of NF-kB Signaling Pathway. One of the unique mechanisms
that both EBV and KSHV utilize to promote lymphoma induction is the constitutive
activation of the NF-kB pathway, which regulates the expression of a large number
of genes involved in cell proliferation (de Oliveira et al. 2010). NF-kB, a transcription factor, is composed of homo- and heterodimers of five subunits, including c-Rel,
NF-kB1 (p50), NF-kB2 (p52), p65 (RelA), and RelB. NF-kB1 and NF-kB2 are produced as p105 and p100 precursors, respectively. In nonstimulated cells, NF-kB is
sequestered in the cytoplasm by forming a complex with inhibitory kB (IkB). Various
biological stimuli, including TNFa, interleukin-1, the lipopolysaccharide (LPS),
viral infection, and ligands for antigen receptors, however, lead to the phosphorylation and subsequent ubiquitination of IkB for proteosomal degradation. NF-kB is
then released and translocated to the nucleus to induce the transcription of target
genes. Two protein kinases, IkB kinase a (IKKa) and IKKb, mediate the phosphorylation of IkB and represent a convergence point for signal transduction pathways
leading to NF-kB activation. Most of the IKKa and IKKb molecules in the cell are
in IKK complexes that also contain a regulatory molecule, IKKg/Nemo. The interaction of IKKg/Nemo with IKKa and IKKb is critical for the assembly of the IKK
complex and for increasing IKK activity (Hacker and Karin 2006). Two distinct
NF-kB pathways, termed canonical (classical) and noncanonical (alternative), have
been defined. IKKb and IKKg/Nemo play a major role in IkB phosphorylation in
canonical NF-kB activation upon TNFa, IL-1, and LPS stimulation. Noncanonical
NF-kB activation involves proteasome-mediated processing of p100 into p52 and
this requires IKKa and NF-kB-inducing kinase (NIK), a member of the MAP3K
superfamily responsible for the phosphorylation and activation of IKKa, to respond
to a subset of TNFa family members, such as B-cell activating factor (BAFF), CD40
ligand, and lymphotoxin (LT)aLTb heterodimers (Hacker and Karin 2006).
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K1
LMP1
Lyn
Syk
Lyn
TRAF1,2,3,5
TRAFs
Syk
PTK
TRAF6
RIP
TRADD
GTP
vFLIP
g
g
IKK
complex
IKK
complex
DED DED
b
NF-kB IkB
NF-kB IkB
IkB
NF-kB
Ub
NF-kB
Ik B
NF-kB
Ub
NF-kB
Fig. 3.4 Activation of NF-kB signaling by EBV and KSHV
Several viral proteins expressed during lytic and latent infection of EBV and
KSHV have been shown to modulate NF-kB signaling pathways in favor of viral
replication and survival (Fig. 3.4, de Oliveira et al. 2010). Among them, EBV LMP1
and KSHV vFLIP, expressed during latency, are largely responsible for the constitutive activation of NF-kB in infected cells (Fig. 3.4, de Oliveira et al. 2010). In addition, these two viral proteins can similarly activate both canonical and noncanonical
NF-kB (de Oliveira et al. 2010).
EBV LMP1. LMP1 activates NF-kB via its CTAR1 and CTAR2 regions by recruiting TRAFs and TRADD, respectively. The TRAF family consists of seven members, all of which contain a RING finger domain associated with E3 ligase activity
and several zinc finger motifs at their N-terminus with the exception of TRAF1.
The C-terminal domain of all TRAF proteins have a novel TRAF domain composed of a coiled-coil domain (TRAF-N) and a conserved TRAF-C domain at the
C-terminus, which is responsible for homo- and heterodimerization of the TRAF
proteins as well as for their direct and indirect interactions with cognate surface
receptors. TRAF proteins interact with members of the TNFR superfamily, which
directly or indirectly recruits specific TRAFs to their intracellular domain to mediate various downstream signaling pathways involved in survival, proliferation, differentiation, activation, and migration. Both TNFR2 and CD40 directly recruit
TRAFs via a consensus TRAF-binding motif (PxQxT, where x is any amino acid)
in their intracellular domain. Members of TNFR superfamily that contain a
death domain (DD) in their intracellular domain, such as TNFR1, first recruit a
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DD-containing adaptor protein, TRADD, via a DDDD homotypic interaction.

TRADD then recruits TRAF2 and RIP for survival signaling, and FADD and
caspase-8 for the induction of apoptosis (Soni et al. 2007).
LMP1 CTAR1 has a PxQxT TRAF binding motif and is therefore able to recruit
DD-containing domain proteins, such as TRADD and RIP1. LMP1 CTAR1 can activate
the noncanonical NF-kB pathway in fibroblast cell lines and epithelial and B cells by
recruiting TRAFs (1, 2, 3, and 5), whereas LMP1 CTAR2 has a YYD motif and can
activate the canonical NF-kB pathway by directly interacting with RIP and TRADD,
followed by the recruitment of TRAF6. Although TRAF6 does not bind to LMP1
directly, it is essential for LMP1-mediated NF-kB activation in MEFs (Soni et al. 2007).
Thus, LMP1 can activate NF-kB signaling by acting as a constitutively active TNFR,
producing effects similar to those induced by B-cell activation receptor CD40 or a combination of TNFR1 and TNFR2 (Figs. 3.1 and 3.4a, Rickinson and Kieff 2007).
KSHV K13 (vFLIP). The KSHV K13, encoded in ORF71 of the viral genome, is a
homolog of cellular FLIP [FLICE (FADD-like IL-1b converting enzyme)-inhibitory protein], thus named vFLIP. Similar to cellular FLIPs (cFLIPs) and several
other vFLIPs encoded by herpesvirus saimiri (HVS), equine herpesvirus, and
Molluscum contagiosum poxvirus, KSHV vFLIP contains two tandem death-effector
domains (DEDs) and can block Fas-mediated apoptosis (Belanger et al. 2001; Bertin
et al. 1997; Hu et al. 1997; Thome et al. 1997). Among the known vFLIPs, only
KSHV vFLIP shares functional homology to cFLIPs, with the ability to activate the
NF-kB pathway but in a constitutively active manner (Chaudhary et al. 1999). This
may indirectly contribute to antiapoptotic function of vFLIP by enhancing the
transcription of a number of antiapoptotic proteins. In PEL cells, vFLIP interacts
with components of the IKK complex and RIP, a protein kinase that plays an essential role in TNFR1-mediated NF-kB activation (Liu et al. 2002). Yeast two-hybrid
screening identified IKKg/Nemo as a binding partner of vFLIP (Fig. 3.4b, Field
et al. 2003). In addition, KSHV vFLIP associates with Hsp90, an important component of IKK complexes (Field et al. 2003). Inhibition of Hsp90 in PEL cells results in
a significant reduction of cell death and IKK activity induced by vFLIP, suggesting
that the activity of the vFLIPIKK complex depends on Hsp90 (Field et al. 2003).
Despite its weak detection in PEL cells and endothelial cells carrying KSHV, vFLIP
is the major factor promoting tumor cell survival. In PEL cells, siRNA-mediated
knockdown of vFLIP expression significantly reduced NF-kB activity by approximately 80% and increased the sensitivity of PEL cells to external apoptotic signals,
subsequently enhancing apoptotic levels, suggesting that vFLIP is essential for PEL
cell survival (Guasparri et al. 2004).
KSHV vFLIP also constitutively activates the noncanonical NF-kB pathway by
upregulating the expression of NF-kB precursor protein p100 and enhancing its
processing to p52 subunit in MEF and 293T cells stably expressing KSHV vFLIP
(Matta and Chaudhary 2004). Knockdown of KSHV vFLIP by siRNA in PEL cells
leads to a marked reduction of p100 processing and a significant decrease in cell
proliferation (Matta and Chaudhary 2004), indicating that activation of the noncanonical NF-kB pathway induced by KSHV vFLIP is also required for the growth
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and proliferation of PEL cells. One of the unique features of KS is the involvement
of cytokines and growth factors in the pathogenesis of this disease. The NF-kB
pathway is well-known for its role in the transcriptional activation of several
cytokines and chemokines. In fact, vFLIP mediates the induction of IL-6 and IL-8
expression through its activation of NF-kB (An et al. 2002; Sun et al. 2006). Both
KSHV vIL-6 and KSHV vGPCR are proposed to contribute to the production of
cytokines involved in KS development as discussed above. Unlike KSHV vFLIP,
however, none of these genes are major latency-associated genes. Thus, vFLIP
expressed in latently infected cells is a likely candidate for viral genes responsible
for the promotion of tumor cell proliferation.
Modulation of Notch Signaling Pathway. Notch signaling is involved in diverse cellular processes, including cell fate decision, differentiation, and proliferation.
Deregulation of Notch signaling has been implicated in a number of human malignancies (Bolos et al. 2007; Leong and Karsan 2006). In mammals, the Notch receptor family consists of four members, Notch1, 2, 3, and 4, and the Notch ligand
family consists of five members, Jagged-1 and 2 and Delta-like-1, -3, and 4. Both
Notch receptors and Notch ligands are type I single-pass TM proteins. Notch
receptors have a large extracellular domain and a cytoplasmic domain. The extracellular domain of Notch receptors consists of 29 and 36 EGF-like repeats involved
in ligand binding, followed by three cysteine-rich LIN12/Notch repeats (LNRs)
involved in the prevention of receptor activation in the absence of ligands. The
Notch intracellular domain (NotchIC or NICD) consists of a RAM23 domain, six
ankyrin/cdc10 repeats involved in proteinprotein interactions, two nuclear localization signals (NLSs), a transcriptional activation domain (TAD), and a PEST
sequence involved in degradation of Notch. Two consecutive proteolytic cleavages
of these receptors occur upon ligand binding: TNF-a converting enzyme (TACE)mediated cleavage on the extracellular side near the TM domain, followed by
g-secretase complex-mediated intramembrane cleavage within the TM domain. As
a result, NotchIC is released and translocated into the nucleus to induce target gene
transcription. In the absence of nuclear NotchIC, the transcription-repressor protein
C protein-binding factor 1 (CBF1, also known as RBP-Jk or CSL) binds to CBF1
binding sites of Notch target gene promoters and represses transcription of Notch
target genes (Fig. 3.5a). When Notch signaling is activated, NotchIC enters the
nucleus, binds CBF1, and recruits mastermind-like (MAML) transcription activator.
MAML then recruits the histone acetyltransferase p300/CBP, resulting in the activation of Notch target gene transcription (Fig. 3.5b). Activated Notch signaling has
been linked to the development of hematologic malignancies and solid tumors in
which a number of cellular signaling pathways are modulated to result in increased
cell proliferation and inhibition of apoptosis (Leong and Karsan 2006). Both EBV
and KSHV exploit components of the Notch signaling pathways to promote
proliferation and viral gene expression (Hayward 2004; Hayward et al. 2006).
EBV and Notch Signaling. EBV EBNA-2, one of the first viral proteins expressed
after EBV infection in vitro, is essential for the proliferation of EBV-infected cells.
It is a transcriptional activator that upregulates almost all viral genes and many cellular
67
Ligand
Notch
Receptor
NotchIC
Coactivator
Complex
b
EBNA-2
Coactivator
Complex
RBP-J
RBP-J
d
a
Corepressor
Complex
RTA
RBP-J k
RBP-J k
Fig. 3.5 Subversion of Notch signaling pathway by EBV and KSHV
genes by interacting with other transcription factors, not by directly binding to DNA
(Hayward 2004; Hayward et al. 2006). Similar to NotchIC, EBNA-2 interacts with
the repression domain of CBF1/RBP-Jk, which is the same binding domain of
NotchIC, resulting in the activation of CBF1/RBP-Jk-responsive reporter gene
promoters, EBV latency promoters (with the exception of LMP1), and affecting the
expression of the same cellular genes targeted by NotchIC, including c-Myc
(Fig. 3.5c, Hayward 2004). Interaction of EBNA-2 with CBF1/RBP-Jk is thought
to be necessary for the proliferation of EBV-infected lymphoblastoid cells since
blockage of EBNA-2CBF1/RBP-Jk interaction with a cell-permeable EBNA-2TAT peptide caused growth arrest and concurrent induction of p21 (Farrell et al.
2004). Unlike Notch receptor, however, EBNA-2 functions as a constitutively active
Notch receptor because it activates target genes in the absence of ligands (Kohlhof
et al. 2009). In addition, unlike NotchIC, which is mainly regulated by processes,
such as differential expression of Notch ligands, receptor recycling, and protein
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turnover, the activity of EBNA-2 is either negatively or positively regulated at the

level of the CBF1/RBP-Jk complex by other viral proteins; the EBNA-3 family
competitively binds CBF1/RBP-Jk, resulting in the loss of CBF1/RBP-Jk binding
to its target promoters and subsequent downregulation of EBNA-2-mediated transcriptional activation, while EBNA-5 (EBAN-LP) acts as a coactivator of EBNA-2mediated transcriptional activation (Hayward 2004). Nonetheless, Notch1-IC, only
if expressed at extremely high levels or in combination with LMP1, has been shown
to rescue proliferation of immortalized B cells to some extent in the absence of
EBNA-2 (Gordadze et al. 2001; Hofelmayr et al. 2001). A more recent genomewide expression analysis utilizing EBV-infected B cells, however, has shown that
neither Notch1-IC nor Notch2-IC can functionally replace EBNA-2 function and
that Notch1 is more potent in regulating genes associated with differentiation/
development while EBNA-2 is more potent in inducing viral and cellular genes
involved in proliferation, survival, and chemotaxis (Kohlhof et al. 2009). Thus, this
study suggests that Notch1 and EBNA-2 have profoundly different effects on
cellular processes.
KSHV and Notch Signaling. Unlike EBV, subversion of Notch signaling pathway by
KSHV appears to be critical for a productive lytic infection rather than latency. The
expression of KSHV replication and transcription activator (RTA) protein is necessary and sufficient for KSHV reactivation from latency. KSHV RTA activates target
gene expression not only by directly binding to recognition sequences in the KSHV
genome, but also by indirectly interacting with cellular DNA-binding proteins on
responsive promoters. One of the cellular DNA-binding proteins targeted by RTA is
the CBF1/RBP-Jk protein, notable because RTA target genes are less responsive in
fibroblasts derived from RBP-Jk null mice (Liang and Ganem 2003). RTA also
binds the same repression domain of CBF1/RBP-Jk targeted by NotchIC and EBV
EBNA-2, resulting in the activation of both cellular (CD21 and CD23) and viral
(vIL-6 and vGPCR) gene transcription (Fig. 3.5d, Chang et al. 2005; Hayward
2004; Hayward et al. 2006; Liang et al. 2002; Liang and Ganem 2003). In tetracycline-inducible BCBL-1 PEL cells, NotchIC was capable of inducing the expression
of 24 KSHV genes, including the vIL-6, but did not induce the full spectrum of viral
lytic gene expression and replication (Chang et al. 2005), indicating that the transcriptional responses to NotchIC overlap with but differ from those of KSHV RTA.
Although KSHV latent genes expressed in KSHV-associated tumor cells play significant roles in the development of KSHV-associated diseases, lytic KSHV genes
expressed from a small percentage of cells also contribute to KSHV-associated
pathogenesis. Two lytic genes, such as vIL-6 and vGPCR, are now recognized to
have NotchIC-responsive promoters (Liang and Ganem 2003). Thus, the induction
of vIL-6 and vGPCR by NotchIC may contribute to the proliferation of KSHVinfected tumor cells.
Modulation of Wingless-Type (Wnt) Signaling Pathway. The Wnt-mediated signaling pathway is a highly conserved pathway between species and has been implicated
in embryonic development, cell polarity and adhesion, apoptosis, and tumorigenesis
(Akiyama 2000; Polakis 2000). It is activated when Wnts, secreted glycoproteins,
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b
b
b
b
Fig. 3.6 Wnt signaling pathway
bind and activate the Frizzled (Fz) receptors, seven-pass TM receptors with an extracellular N-terminal cysteine-rich domain, on target cells in a paracrine manner. Fzs
then cooperate with LRP5/6 coreceptor, a single-pass TM protein, to activate
b-catenin, a key transcriptional activator of the Wnt signaling pathway involved in
the transcription of more than 30 different genes in the nucleus by forming a complex with the T-cell factor (Tcf)/lymphoid-enhancer factor (Lef) family of transcription factors. In the absence of Wnt signaling, the cytosolic pool of b-catenin is
held in a destruction complex comprising casein kinase 1 (CK1), glycogen synthase
kinase 3-b (GSK-3b), axin, and adenomatous polyposis coli (APC) (Fig. 3.6a).
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Both axin and APC function as scaffold proteins, binding both GSK-3b and
b-catenin. GSK-3b, a serine/threonine kinase, phosphorylates both axin and APC,
strengthening the formation of the complex. CK1 and GSK-3b sequentially phosphorylate N-terminal Ser/Thr residues of b-catenin. Phosphorylated b-catenin is then
recognized by b-TrCP, a component of an E3 ubiquitin ligase complex, resulting in
the rapid degradation of b-catenin by the 26S proteasome. In the presence of Wnt,
the activated receptor Fz binds dishevelled (Dvl) proteins, which recruit the destruction complex through its interaction with axin. Coreceptors LPR5/6 are then phosphorylated by GSK-3b and CK1g and subsequently recruit axin, which leads to
dissociation of destruction complex and stabilization of b-catenin in the cytoplasm.
Stabilized b-catenin then enters the nucleus, where it associates with Tcf/Lef transcription factors to activate many target genes involved in cell cycle progression,
cell growth, and cell proliferation. Notable b-catenin target genes include c-Myc,
c-Jun, and cyclin D1. Both c-Myc and Cyclin D1 can promote G1 to S phase cell
cycle transition (Fig. 3.6b, Clevers 2006; Karim et al. 2004). Dysregulation of Wnt
signaling pathway and its components has been implicated in different stages of
tumorigenesis ranging from initiation, proliferation, and progression to the accumulation of mutations (Karim et al. 2004). Both EBV and KSHV are capable of constitutively activating Wnt signaling pathways by directly or indirectly inactivating
GSK-3b, resulting in the enhanced proliferation of virus-infected cells.
EBV and Wnt Signaling. EBV LMP2A expression in epithelial cells activates the
PI3K/Akt pathway, resulting in the inactivation of GSK-3b, increased cytoplasmic accumulation of b-catenin, enhanced nuclear translocation of b-catenin, and
subsequently upregulation of a reporter responsive to Tcf (Morrison et al. 2003).
EBV infection in B cells constitutively induces the accumulation of b-catenin in
the cytoplasm and the translocation of phosphorylated and inactivated GSK-3b
into the nucleus. Unlike LMP2A-mediated regulation of b-catenin in epithelial
cells, however, there was no nuclear localization of b-catenin in B cells nor
increased transcription of b-catenin-dependent target genes (Everly et al. 2004).
EBV LMP1 has been shown to upregulate b-catenin in B-lymphoma cells by
increasing its stability through the inhibition of Siah-1, an E3 ubiquitin ligase that
binds APC and promotes b-catenin degradation in a GSK-3b-independent fashion
(Jang et al. 2005). Thus, EBV proteins expressed during latency target GSK-3b,
an inhibitor of Wnt signaling, resulting in the constitutive activation of the Wnt
signaling pathway.
KSHV and Wnt Signaling. It has been reported that KSHV LANA-1 dysregulates
the Wnt pathway by binding and sequestrating GSK-3b in the nucleus, which leads
to b-catenin stabilization, nuclear entry, and subsequent activation of cyclin D1, a
protein involved in cell proliferation (An et al. 2005; Fujimuro and Hayward 2003;
Fujimuro et al. 2005, 2003; Hayward et al. 2006). Consistent with this, transient
LANA-1 expression stimulates S-phase entry (Fujimuro and Hayward 2003). These
studies indicate that tumor cells expressing LANA-1 are more prone to survive and
proliferate in the absence of Fz receptor ligands.
71
Summary
Although the pathogenesis of EBV and KSHV-induced malignancies is still not
completely understood, similar to the accumulative mutational process of cancer
induction, these viruses affect a variety of host cell processes, including cell cycle
progression, DNA damage response, and numerous intracellular signaling pathways
to promote cellular proliferation, and ultimately facilitate the development of malignancies. This review summarizes the recent progress made in our understanding of
the biochemical and molecular mechanisms, whereby EBV and KSHV, two oncogenic, large DNA HHV, induce cell proliferation, with a particular focus on the
contribution of viral proteins to this process. Both EBV and KSHV can induce
uncontrolled cell proliferation, resulting in lymphomas, Hodgkins diseases, and
Kaposis sarcoma. Overall tumor cell number is determined by the rate of not only
cell proliferation (birth), but also apoptosis (death). Although this review does not
discuss EBV- or KSHV-mediated antiapoptosis mechanisms, most of viral proteins
discussed in this review, in fact, have demonstrated the ability to inhibit apoptosis as
well. Moreover, both EBV and KSHV are equipped with many different strategies
to evade cellular apoptosis machineries and immune surveillance systems, suggesting that the promotion of cellular proliferation may represent one of the several
mechanisms employed by these viruses for enhancing their survival and replication
and to contribute to tumorigenesis. Thus, understanding the functional interplay
between viral proteins with cellular proteins involved in cellular proliferation
may provide a basis for the development of future therapeutic strategies to tackle
virus-mediated tumorigenesis.
Acknowledgment This work was partly supported by the U.S. Public Health Service grants
CA082057, CA31363, CA115284, Hasting Foundation, and Fletcher Jones Foundation.
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Chapter 4
Viral-Encoded Genes and Cancer

Blossom Damania
Tumor Viruses and Cancer

Cancer is a multistage process. Most cancers do not arise from mutation of a single
gene, but rather from cumulative accumulation of multiple gene mutations.
Mutations that contribute to tumorigenesis generally occur in one of three types of
genes a proto-oncogene; a tumor suppressor gene; or genes involved in DNA replication and repair, chromosome segregation, or cytokinesis. The definition of a
proto-oncogene is a wild-type gene, which when mutated can give rise to an oncogene. Proto-oncogenes include genes that encode for proteins involved in cell
growth, cell survival, or cell signaling. The accumulation of multiple genetic insults
in a single cell leads to the loss of cell cycle checkpoints and the loss of programmed
cell death (or apoptosis), which consequently results in continual cell proliferation.
In contrast, tumor suppressors are proteins that normally function to inhibit cell
growth and enhance cell death pathways. When mutated, tumor suppressors lose the
ability to block cell growth or activate apoptosis; thereby, contributing to the proliferative phenotype of a cancer cell.
The combination of oncogenes and tumor suppressor mutations occurring in a
multistage process leads to transformation. Several studies have supported this
multistage mutational model of cancer. Both breast and colorectal tumor models
reveal many gene mutations (Wood et al. 2007).
Generally speaking, the road that a normal cell traverses to become a cancer
cell includes (1) hyperplasia or an abnormal increase in cell number; (2) dysplasia, which results in disorganized tissue architecture; (3) benign tumor, which is a
hyperproliferative tissue mass that is not invasive; (4) invasive tumor, where the
tumor begins to penetrate into adjacent tissues; and (5) metastasis, where the
B. Damania (*)
Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
e-mail: damania@med.unc.edu
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B. Damania
tumor cells move to distal sites. Each of these stages is driven by genetic mutation,
with the end result being a combination of gene mutations that give rise to cancer.
Thus, each type of cancer that arises has a specific genetic signature comprised
of a particular combination of genetic mutations (Nowell 2002). Although the
above descriptors seem primarily related to cell growth, the prevention of cell
death, a contributing factor to the development of cancer, is as important as cell
proliferation. Apoptosis or programmed cell death is a tightly orchestrated process in which cells die in response to particular stimuli. Apoptosis is an important
cellular defense mechanism against transformation. The p53 tumor suppressor is
one of the major regulators of apoptosis in the cell. Thus, cellular gene mutations
that prevent a damage-induced apoptotic response in the cell contribute to the
tumorigenesis process.
Oncogenic viruses are comprised of both RNA and DNA tumor viruses. These
tumor viruses are associated with cancer in their natural hosts or in non-native hosts.
Viruses have contributed to, and revolutionized, the field of cancer biology. The
study of tumor viruses has been the key to understanding the genetic basis of transformation. In addition, the study of these viruses has also contributed to our understanding of virus-induced cancers. The study of tumor viruses initiated the concept
of oncogenes and tumor suppressors in modern cancer biology.
Retroviruses and the Concept of Oncogenes

Retroviruses are RNA viruses that undergo reverse transcription as part of their
lifecycle. Following infection, the viral RNA genome is transcribed by the reverse
transcriptase enzyme that is packaged in the virion. This produces a double-stranded
DNA copy of the RNA genome with the help of the viral integrase can be integrated
into the host chromosomal DNA.
The avian sarcoma virus, Rous sarcoma virus (RSV), was the first known tumor
virus and also a retrovirus. The study of RSV led to the discovery of oncogenes
(Vogt 1997). RSV was shown to be capable of transforming cells in culture assays
(Temin and Rubin 1958). The RSV-encoded gene named src or viral src (v-src) was
essential for the transformation of this virus. Additionally, src was also found to be
present in normal cells. This discovery was seminal in linking the concept of cancer
development to a transforming gene like src. It also revealed that normal cells
contained genes that were homologous to v-src and led to the concept of protooncogenes that were present in normal cells.
Since the discovery of RSV and src, many other oncogenic retroviruses of chickens and mice, which contained transforming genes, were identified. These included
the Abelson murine leukemia virus (Ab-MLV) containing the abl oncogene, the
Harvey murine sarcoma virus (Ha-MSV) containing the ras oncogene, the MC29
avian myelocytomatosis virus containing the myc oncogene and the Moloney
83
Table 4.1 A subset of retroviruses encoding for viral oncogenes that are homologous to cellular
proto-oncogenes
Virus
Viral oncogene
Viral oncoprotein
Rous sarcoma virus
src
Tyrosine kinase
Abelson murine leukemia virus
abl
Tyrosine kinase
Moloney murine sarcoma virus
mos
Serinethreonine kinase
Murine sarcoma virus 3611
raf
Serinethreonine kinase
HardyZuckerman-4 feline sarcoma
kit
Receptor tyrosine kinase
virus
Avian erythroblastosis virus
erbB
Epidermal growth factor receptor
Harvey murine sarcoma virus
H-ras
GDP/GTP binding
Kirsten murine sarcoma virus
K-ras
GDP/GTP binding
FBJ osteosarcoma virus
fos
Transcription factor
Avian sarcoma virus-17
jun
MC29 myelocytoma virus
myc
Reticuloendotheliosis virus
rel
murine sarcoma virus (Mo-MSV) containing the mos oncogene (Rosenberg and
Jolicoeur 1997).
Similar to the situation with src, all the oncogenes carried by these tumor retroviruses were subsequently shown to be homologous to genes present in the normal
cells. The cellular proto-oncogenes were of many different types including kinases,
G proteins, growth factors, and growth factor receptors, all of which were involved
in signal transduction and cell proliferation (Table 4.1). This led to the concept of
molecular piracy by the virus. However, the viral gene and the cellular counterpart
are not usually identical. Retroviral oncogenes are generally mutated and expressed
constitutively from the control of the viral long terminal repeat (LTR), which is
thought to lead to transformation of the host cell or animal. Moreover, certain retroviruses contain two oncogenes, e.g., an isolate of avian erythroblastosis virus
encodes for v-erbA and v-erbB oncogenes. These observations gave rise to the idea
that oncogenes can synergize with each other since the isolate containing two oncogenes was more transforming than an isolate containing the v-erbB oncogene
(Rosenberg and Jolicoeur 1997).
The retroviruses described earlier are oncogenic retroviruses. However, a second
class of non-oncogenic retroviruses exist that do not contain viral oncogenes, e.g.,
avian leukosis virus (ALV). Rather, these viruses induce transformation because
their viral DNA genome is integrated upstream of a cellular proto-oncogene resulting in dysregulated expression of the cellular gene. This allows the cellular protooncogene to be expressed constitutively and at very high levels (Kung et al. 1991).
Such expression of a growth regulatory proto-oncogene allows for cellular transformation by giving the cell a proliferative and survival advantage over normal cells.
This initiates the process of tumorigenesis and additional mutations acquired after
this event may give rise to bonafide tumors.
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Small DNA Tumor Viruses and the Concept of Tumor

Suppressors
Small DNA tumor viruses include the adenoviruses, polyomaviruses, and papillomavirus families. Unlike retroviruses, these DNA viruses do not encode homologs
of cellular proto-oncogenes, but contain unique viral oncogenes that are important
for cell transformation (Cole 1996; Howley 1996; Shenk 1996). Studies of these
small tumor DNA viruses led to the identification of tumor suppressor genes. Due
to their small genome size, these DNA tumor viruses are dependent on the host cell
machinery to replicate their viral genomes. Viral proteins stimulate quiescent cells
to enter the cell cycle to replicate both the cellular and the viral genomes. Simian
virus 40 (SV40) was the first small DNA tumor virus to be studied extensively due
to its presence as a contaminant in the Sabin polio vaccine.
Polyomaviruses are small DNA tumor viruses that are widely prevalent throughout the animal kingdom. SV40, the prototype polyomavirus, encodes for a protein
named large T antigen (T ag), which is required for viral DNA replication and for
pushing the cell into the cell cycle. Homologs of large T antigen are found in all
human polyomaviruses, including JCV, BKV, and Merkel cell polyomavirus (MCV)
(White et al. 2009). SV40 large T is a potent transforming protein in many different
cell and animal model systems (Butel and Lednicky 1999).
SV40 large T antigen was found to coimmunoprecipitate with p53, which was
later coined a tumor suppressor gene due to its ability to inhibit cell proliferation
(Lane and Crawford 1979; Linzer and Levine 1979). Hence, the p53 protein was
discovered due to its interaction with SV40 large T antigen.
Adenoviruses are a different small DNA tumor virus family that encode for two
different viral oncoproteins, E1A and E1B (Shenk 1996). E1B was also found to
bind p53 while a different tumor suppressor protein, Rb, was identified as a cell
protein that interacted with the E1A oncoprotein in adenovirus-transformed cells
(Whyte et al. 1988). SV40 Tag was later found to interact with Rb as well.
The papillomaviruses are a third group of small DNA tumor viruses, which
encode their own unique transforming proteins, E6 and E7. Key to E6s and E7s ability
to immortalize or transform cells is their ability to bind p53 and Rb, respectively
(Howley 1996).
Common Properties Shared by the Small DNA Tumor Viruses

Small DNA tumor viruses share the common property of inducing DNA synthesis
in resting cells. This is an essential function of small DNA tumor viruses since
they need to activate the DNA replication machinery of the host cell to replicate
their own viral genomes. The binding of different viral oncoproteins with the
cellular tumor suppressor proteins, Rb and p53, is key for allowing the small DNA
tumor viruses to induce cell proliferation, and as a result, transform these cells.
85
Large T
E1A
E7
Large T
E1B
E6
Rb
p53
Rb
E2F
Apoptosis
Growth Arrest
Large T
E1A
E7
E2F
Cell Cycle Progression
Fig. 4.1 Common targets of small DNA tumor viruses. (a) p53 negatively regulates cell cycle
progression and positively regulates the induction of apoptosis in response to aberrant proliferation
signals, DNA damage or cellular stress. Binding of p53 by the small DNA tumor viral proteins,
Large T, E6 and E1B allows cells to survive and escape p53 checkpoints in the face of DNA damage stress signals. This allows for cell cycle progression and replication of the viral genome.
(b) E2F is a transcription factor that under normal conditions is bound to Rb and upon phosphorylation by cyclin-dependent kinases is released from Rb enabling its binding to cellular promoters
of genes involved in cell cycle progression. Small DNA tumor viral proteins, large T, E1A and E7
dissociate Rb from E2F
Each small DNA tumor virus encodes unique proteins that bind and inactivate the
function of these tumor suppressor proteins, which are common targets of these
viruses.
Figure 4.1 depicts how binding p53 and Rb by proteins encoded by the small
DNA tumor viruses may lead to cell cycle progression and cellular transformation.
SV40 large T antigen, HPV E6, and adenovirus E1B-55K bind p53 and prevent its
DNA binding activity, induce its degradation, or inhibits its transactivation, respectively; thereby, inactivating p53 function. Large T antigen, HPV E7 and adenovirus
E1B all possess a common LXCXE motif, which bind Rb, release E2F, and initiate
cell cycle progression (Butel 2000).
Although SV40 encodes for one protein (large T antigen) that inactivates both Rb
and p53, the adenoviral proteins E1A and E1B-55K as well as the HPV proteins E6
and E7 show functional cooperativity in inactivating the function of both p53 and RB.
This synergistic inactivation of the tumor suppressor genes results in transformation.
Since the discovery of these critical proteins, the cancer biology field has also
determined the transforming potential of viral oncogenes in combination with cellular
oncogenes. For example, transfection of fibroblasts by a cellular ras oncogene did not
induce transformation by itself. However, the fibroblasts did get transformed if a
second oncogene such as a viral gene (large Tag) or cellular gene (myc) was introduced together with the cellular ras gene (Land et al. 1983). Just as oncogenes cooperate to induce transformation, it has also been shown that expression of an oncogene
together with the loss of a tumor suppressor gene or expression of a mutant tumor
suppressor gene can cooperate to induce tumorigenesis (Zambetti et al. 1992).
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B. Damania
Viruses Associated with Human Cancer

Several types of neoplastic disease in humans are linked to viral infection. Viruses
from diverse families have been etiologically linked to human malignancy
(Table 4.2). Two RNA viruses hepatitis C virus (HCV) and human T-cell leukemia
virus 1 and 2 (HTLV-I, 2) are linked to human cancer. HTLV-1, 2 is associated with
adult T-cell leukemia (ATL) (Yoshida 1999) and HCV is the etiological agent of
hepatocellular carcinoma (HCC) (zur Hausen 1999).
Among the small DNA tumor viruses, the aforementioned human papillomavirus is linked to cervical cancer, a subset of head and neck cancers, skin cancers in
patients with epidermodysplasia verruciformis (EV), and anogenital cancers (zur
Hausen 1996). Additionally, MCV polyomavirus has been linked to cases of Merkel
cell carcinoma (Feng et al. 2008). There is also a question as to whether SV40 and
the human polyomaviruses, JCV and BKV, are linked to different types of brain
tumors (Pagano et al. 2004). Another virus, HBV, is associated with HCC in addition to HCV (Robinson 1999).
Two DNA herpesviruses are also linked with human malignancies. EBV is the
etiological agent of nasopharyngeal carcinoma (NPC), African Burkitts lymphoma,
posttransplant lymphomas (PTLD), Hodgkins disease, and some gastric cancers
(Damania 2004; Pagano et al. 2004). Kaposis sarcoma-associated herpesvirus
(KSHV) is linked to Kaposi sarcoma (KS) and two lymphoproliferative diseases,
primary effusion lymphoma and multicentric Castlemans disease (Damania 2004;
Pagano et al. 2004). In this chapter, we discuss the details of a subset of genes
encoded by these oncogenic viruses that appear to play a contributing role in the
development of human cancer. These genes modulate diverse cellular pathways
including transcription, cell cycle progression, signal transduction, and apoptosis
(Table 4.3).
Table 4.2 Viruses associated with human malignancies

Virus family
Virus
Type of human cancer
Retroviridae
HTLV-1, 2
T-cell leukemia
Flaviviridae
HCV
Hepatocellular carcinoma
Hepadnaviridae
HBV
Polyomaviridae
MCV
Merkel cell carcinoma
JCV, BKV, SV40
Brain and other tumors?
Papillomaviridae
HPV
Cervical cancer
Herpesviridae
EBV
Burkitts lymphoma, Hodgkins lymphoma,
nasopharyngeal carcinoma, post-transplant
lymphoproliferative disease, gastric cancer
KSHV
Kaposis sarcoma, primary effusion lymphoma,
multicentric Castlemans disease
87
Table 4.3 Viral oncogenes encoded by human tumor viruses

Virus
Oncogene(s)
Mode of action
HTLV-1, 2
Tax
Transactivator of viral and cellular genes
HCV
Core proteins, NS3,
Inhibition of apoptosis, induction of oxidative
NS4B and NS5A
stress, activation of signal transduction
proteins
pathways, immune evasion
HBV
HBx
MCV
Large T antigen
Inactivation of tumor suppressor proteins,
promotion of cell cycle progression
JCV, BKV, SV40
Large T antigen
Brain tumors?
HPV
E6, E7
Inactivation of tumor suppressor proteins;
induction of genomic instability
EBV
LMP1
Activation of NF-kB and signal transduction
pathways
LMP2A
Signal transduction; Inhibition of B cell
differentiation
EBNA-1
Inhibition of apoptosis, promotion of
tumorigenesis, viral latency
EBNA-2
EBNA-3A, B, C
Inactivation and degradation of p53 and Rb;
promotes transformation and metastasis
KSHV
K1
Activation of signal transduction pathways;
angiogenesis; promotes transformation
LANA
Inactivation of tumor suppressors, viral latency
vFLIP
Inhibition of apoptosis
vGPCR
angiogenesis; promotes transformation
Kaposin
promotes transformation
vIL-6
cell proliferation
vIRF-1
Evasion of host immune responses, promotes
transformation
HTLV-1-Encoded Oncogenes
Unlike the aforementioned murine and avian retroviruses, HTLV-1 does not contain
a viral oncogene derived from a cellular proto-oncogene. It encodes a unique protein, Tax, which is a transcription factor that is essential for cellular transformation.
Tax has been shown to potently activate the NF-kB pathway and turn on expression
of NF-kB responsive genes such as the IL-2 receptor and several NF-kB responsive
cytokines involved in T-cell proliferation. Tax upregulates the expression of Bcl-XL;
thereby, conferring resistance of Tax expressing cells to apoptosis (Matsuoka and
Jeang 2007). Furthermore, Tax interacts with chromatin remodeling proteins
including SWI/SNF and HDAC-1 and inhibits DNA repair mechanisms, which leads
to genomic instability and transformation (Matsuoka and Jeang 2007).
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HCV-Encoded Oncogenes
Transgenic mice that contain the entire HCV genome or just the HCV core protein
have been shown to develop liver steatosis and hepatocellular carcinogenesis (Lerat
et al. 2002; Moriya et al. 1998). It is thought that the HCV core protein is a major
factor in the induction of oxidative stress leading to the induction of transformation.
Additionally, the HCV NS5A protein can sequester p53 in the cytoplasm, activate
STAT3 signaling, and inhibit TNF alpha-mediated apoptosis (Levrero 2006). HCV
core, NS3, NS4B, and NS5A proteins, have been shown to transform cells in vitro
and alter oncogenic signaling pathways (Levrero 2006). HCV NS3/4A also inhibits
innate immune responses through cleavage of the mitochondrial antiviral signaling
(MAVS) protein (Li et al. 2005).
HPV-Encoded Oncogenes
Almost 100% of cervical cancers contains HPV genomic DNA. Additional cancers
associated with HPV include vulvar, vaginal, penile, oropharyngeal, and some skin
cancers. Of the more than hundred HPV strains known thus far the high-risk
viruses HPV type 16 and 18 are associated with cervical and anal cancer. HPV
strains 31 and 45 are also found in these cancers and together with 16 and 18 account
for 80% of them. Low-risk viruses (strains 6 and 11) are associated with benign
lesions such as condyloma accuminata.
The E6 and E7 genes encode for the major oncoproteins of HPV. In general, E6
and E7 proteins of high-risk strains of HPV are more efficient at inactivating tumor
suppressor proteins than those of the low risk types. In high grade cervical dysplasias,
HPV 16 and 18 E6 and E7 oncoproteins are consistently over-expressed as a consequence of deletion of the E2 repressor gene upon integration of the HPV genome
into the host chromosome. HPV E6 induces the ubiquitination-mediated degradation
of p53 through the ubiquitin ligase, E6-AP, which prolongs survival of the infected
cell (Scheffner et al. 1993). HPV E7 induces the proteosomal degradation of Rb and
related proteins so that cell growth is dysregulated (Dyson et al. 1989). E7 also
induces genomic instability (Duensing et al. 2000).
HBV-Encoded Oncogenes
The HBx protein encoded by HBV is considered to be a viral oncogene since transgenic mice expressing HBV X develop hepatocarcinogenesis (Zeichner et al. 1991).
The HBx protein is a transcription factor that transactivates cellular growth factors,
cytokines, and proto-oncogenes. HBx binds to p53, thereby inactivating it. HBx also
activates several cell signaling molecules including src kinase through activation of
Pyk-2 kinase and induces cytosolic calcium (Bouchard et al. 2001).
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EBV-Encoded Oncogenes
EBV encodes for many different viral proteins that control various cellular pathways.
The primary EBV oncogene, LMP1, is essential for EBV transformation of B cells
and can transform rodent fibroblasts (Rickinson and Kieff 2001). LMP1 interacts with
TNF receptor-associated factors (TRAFs) (Mosialos et al. 1995). LMP1 is a potent
activator of the NF-kB signaling pathway in both lymphocytes and epithelial cells
(Miller et al. 1998). Transgenic mice expressing LMP1 under the control of the
immunoglobulin promoter have an increased frequency of B-cell lymphomas
(Uchida et al. 1999). LMP1 activates the expression of cellular genes involved in
cell signaling and proliferation including Bcl-2, ICAM-1, epidermal growth factor
receptor (EGFR), matrix metalloproteinase 9, vascular endothelial growth factor
(VEGF), and hypoxia-inducible factor HIF-1alpha (Pagano et al. 2004). Additionally,
LMP1 activates the phosphatidylinositol 3-kinase (PI3K) and mitogen activated
kinase pathways (MAPK) (Dawson et al. 2003; Mainou et al. 2005; Mainou and
Raab-Traub 2006).
Although LMP2A is dispensable for EBV-immortalization of B lymphocytes
(Longnecker et al. 1993), it induces a hyperproliferative response and prevents
differentiation in epithelial cells and is likely to contribute to epithelial transformation (Scholle et al. 2000). The cytoplasmic domain of LMP2A contains several
tyrosine-based SH2 binding motifs including a functional ITAM (Fruehling and
Longnecker 1997). In keratinocytes, LMP2A activates both the PI3K and betacatenin signaling pathways (Morrison et al. 2003). Additionally, LMP2A transgenic
mice do not progress to full B cell development, causing immunoglobulin-negative
B cells to home to peripheral lymphoid organs (Caldwell et al. 1998). It is therefore
thought that LMP2A mediates survival of immature EBV-infected B cells in the
absence of functional BCR signaling.
EBV nuclear antigen 2 (EBNA-2) is a promiscuous transcriptional activator, of
both viral and cellular genes (Grossman et al. 1994; Kaiser et al. 1999; Wang et al.
1991, 2000). Deletion of the EBNA-2 gene from wild-type EBV renders the mutant
virus incapable of immortalizing B lymphocytes (Cohen et al. 1989; Hammerschmidt
and Sugden 1989; Rabson et al. 1982). It is believed that the transactivation ability
of EBNA-2 contributes to the transforming mechanism of EBV.
The genes encoding EBNA 3A, 3B, and 3C lie in a tandem array in the viral
genome. EBNA 3A and 3C are essential for B-cell transformation (Sample and
Parker 1994; Tomkinson et al. 1993), while 3B has been shown to be dispensable
(Tomkinson and Kieff 1992). All three EBNA-3 proteins can prevent EBNA-2
transactivation function by hindering its ability to bind RBP-Jk (Le Roux et al.
1994; Marshall and Sample 1995; Robertson et al. 1996). EBNA 3C has been shown
to cooperate with the proto-oncogene, Ras, to immortalize and transform rodent
fibroblasts (Parker et al. 1996) and promote metastasis (Kaul et al. 2007). It can
directly interact with the retinoblastoma (Rb) tumor suppressor protein, rendering it
inactive, promoting its degradation, and thereby contributing to tumorigenesis
(Knight et al. 2005; Parker et al. 1996). EBNA 3C also induces ubiquitin-mediated
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degradation of p53 through stabilization of Mdm2 (Saha et al. 2009). In sum, EBNA
3C promotes cellular proliferation by overcoming cell cycle control checkpoints. It
also cooperates with EBNA-2 and 3A to modulate cellular gene expression in EBVinfected lymphocytes.
EBV EBNA-1 might also contribute to EBV transformation since an EBNA-1deleted virus showed greatly reduced ability to immortalize B cells (Humme et al.
2003). Additionally, EBNA-1 transgenic mice develop B-cell lymphomas (Wilson
et al. 1996) and expression of EBNA-1 can enhance the tumorigenicity of EBVnegative NPC epithelial cells (Sheu et al. 1996). This property of EBNA-1 may be
linked to its ability to induce Bcl-xL (Tsimbouri et al. 2002). Similar to EBNA 3C,
EBNA-1 can also promote metastasis (Kaul et al. 2007).
KSHV-Encoded Oncogenes
Similar to EBV, KSHV encodes for a number of different viral genes, some of
which have been shown to be transforming in different model systems.
Located in a similar position as EBV LMP1, the first open reading frame of
KSHV encodes for K1, a 46-kDa type I transmembrane protein (Lagunoff and
Ganem 1997). K1 is expressed at low levels during latency and is highly upregulated during early lytic replication (Chandriani and Ganem 2010; Wang et al. 2006).
Its expression has been detected in KS, PEL, and MCD (Jenner et al. 2001; Lagunoff
and Ganem 1997; Lee et al. 2003; Wang et al. 2006). The K1 protein possesses an
immunoreceptor tyrosine-based activation motif (ITAM) that is important for lymphocyte activation signaling (Lee et al. 1998) and has been shown to transform
Rat-1 rodent fibroblasts by inducing morphological changes and foci formation
(Lee et al. 1998). Transgenic mice expressing the K1 gene develop tumors with
features resembling spindle-cell sarcomatoid tumors and malignant plasmablastic
lymphomas (Prakash et al. 2002). K1 activates PI3K (p85 subunit), Akt, Vav, and
Syk kinases in B cells (Lee et al. 1998; Tomlinson and Damania 2004). In addition,
K1 can prevent death receptor-mediated apoptosis of B lymphocytes by inhibiting
the induction of FasL expression and activating the PI3K/Akt pathway (Tomlinson
and Damania 2004), and requires heat shock proteins (Hsp) 40 and 90 for its
antiapoptotic function (Wen and Damania 2010). In epithelial and endothelial cells,
K1 induces the expression and secretion of angiogenic factors, including VEGF and
matrix metalloproteinase-9 (Wang et al. 2004) and activates the PI3K/Akt/mTOR
pathway (Wang et al. 2006). Furthermore, K1 can immortalize and extend the life
span of primary endothelial cells (Wang et al. 2006). Cumulatively, these data suggest a paracrine model in which K1-mediated secretion of cytokines is involved in
the development of KSHV-associated diseases (Wang et al. 2004). Thus K1 appears
to be important in KSHV-associated tumorigenesis and angiogenesis. Another
transforming protein of KSHV is the viral G-protein-coupled receptor encoded by
Orf74 (Arvanitakis et al. 1997; Guo et al. 1997). vGPCR can transform murine
NIH3T3 cells (Bais et al. 1998) and a subset of vGPCR transgenic mice developed
91
KS-like angioproliferative lesions with surface markers and cytokine profiles resembling those of KS (Guo et al. 2003; Montaner et al. 2003; Yang et al. 2000). The
expression of vGPCR can be found in a small fraction of KS, PEL, and MCD samples
(Sodhi et al. 2000). Like K1, vGPCR can immortalize endothelial cells (Bais et al.
2003; Montaner et al. 2001). In contrast to its cellular homologs, vGPCR is constitutively active (Rosenkilde et al. 1999). Several signaling pathways including the
mitogen-activated protein kinases (MAPKs) (Sodhi et al. 2000), PLC (Bais et al.
2003), PI3K (Bais et al. 2003), and Akt (Montaner et al. 2001) pathways have been
shown to be activated by vGPCR.
In addition to its role in the establishment and maintenance of KSHV latency
(reviewed in Dittmer 2008; Rainbow et al. 1997; Verma et al. 2007), the latencyassociated nuclear antigen (LANA) may also be involved in oncogenesis due to its
ability to bind p53 and inhibit p53-driven transactivation and apoptosis (Friborg
et al. 1999; Si and Robertson 2006). LANA can also inactivate the tumor suppressor
retinoblastoma (Rb) gene, allowing the E2F transcription factor to induce G1/S cell
cycle progression (Radkov et al. 2000). LANA also interacts with the tumor
suppressor, Sel10, and suppresses ubiquitination and degradation of intracellular
Notch (Lan et al. 2007). LANA induces expression of human telomerase reverse
transcriptase (hTERT) and survivin (Lu et al. 2009; Verma et al. 2004; Watanabe
et al. 2003). Finally, transgenic mice expressing LANA under the endogenous
LANA promoter developed splenic follicular hyperplasia with increased germinal
centers as well as lymphomas (Fakhari et al. 2006). Another latent gene product,
viral Fas-associated death-domain like IL-1 beta-convertase enzyme (FLICE) inhibitory protein (vFLIP) is encoded by Orf71 (Dittmer et al. 1998; Fakhari and Dittmer
2002; Jenner et al. 2001). Similar to cellular FLIPs, vFLIP may inhibit death receptor signaling by preventing the association between caspase-8 and Fas-associated
death domain (FADD) (Belanger et al. 2001; Djerbi et al. 1999). vFLIP is also a
strong activator of the NF-kB pathway (Chaudhary et al. 1999; Field et al. 2003;
Guasparri et al. 2004, 2006; Liu et al. 2002). The enhanced NF-kB signaling appears
to contribute to the transforming potential of vFLIP in Rat-1 fibroblast assays and
tumors in nude mice (An et al. 2003).
Kaposin A, B, and C are three proteins encoded by the K12 gene of KSHV. The
smallest isoform, Kaposin A, demonstrates oncogenic potential in different assays
(Kliche et al. 2001; Muralidhar et al. 1998). Viral interleukin-6 encoded by OrfK2 is
a homolog of cellular IL-6 (Neipel et al. 1997) and is detected in KS, PEL, and MCD
samples (Parravicini et al. 2000; Staskus et al. 1999). Similar to cellular IL-6, vIL-6
activates MAPK and the Janus tyrosine kinases signal transducers and activators of
transcription (JAK/STAT) (Molden et al. 1997). This results in increased VEGF
expression and signaling in an autocrine/paracrine fashion (Liu et al. 2001). However,
in contrast to cellular IL-6, vIL-6 signaling occurs through gp130 and does not
require the gp80 subunit (IL-6Ra) (Molden et al. 1997). Additionally, vIL-6 also
transforms murine fibroblasts and when these cells were injected into immunocompromised mice, they formed highly vascularized tumors (Aoki et al. 1999). Another
viral homolog, the viral interferon regulatory factor 1 (vIRF-1) suppresses both type
I and type II interferon responses (Burysek et al. 1999; Gao et al. 1997; Li et al. 1998).
92
B. Damania
In addition to suppressing the host antiviral response, vIRF-1 is a potential oncogene

since NIH3T3 cells stably expressing vIRF-1 are transformed and form tumors in
nude mice (Gao et al. 1997).
Common Properties Shared by the Large DNA Tumor Viruses

A common property shared by the large DNA tumor viruses EBV and KSHV is that
they are both members of the gammaherpesvirus family and induce tumors in their
hosts. Additionally, both EBV and KSHV contain a distinct ORF at the left end of
their respective genomes (LMP1 and K1, respectively) that have characteristic
transforming potential. Although LMP1 and K1 do not share sequence similarity,
the ability to transform cells and activate critical signaling pathways to augment cell
survival and proliferation is conserved. Additionally, these viruses also encode for
similarly functioning proteins at the right end of the viral genome, EBV LMP2A
and KSHV K15. These two proteins interact with kinases associated with the B-cell
receptor (BCR) and alter signaling pathways in the cell (Beaufils et al. 1993;
Brinkmann et al. 2003; Brinkmann and Schulz 2006; Burkhardt et al. 1992; Glenn
et al. 1999; Longnecker et al. 1991; Scholle et al. 1999). Cross-linking of the B-cell
antigen receptor (BCR) triggers a signal transduction cascade that leads to the activation of B cells. Both EBV LMP2A and KSHV K15 antagonize BCR-induced
signaling events (Beaufils et al. 1993; Burkhardt et al. 1992; Glenn et al. 1999;
Longnecker et al. 1991; Scholle et al. 1999). It is thought that this helps maintain
viral latency in B lymphocytes.
The viral proteins encoded close to the terminal repeats of EBV (LMP1 and
LMP2A) and KSHV (K1 and K15) also exhibit the highest sequence divergence
among individual viral isolates (Nicholas et al. 1997, 1998; Palefsky et al.
1996; Zong et al. 1999, 2002). This is likelya result of their close proximity to
the terminal repeats of the viral genome, which is a region of high
mutagenicity.
Summary
There is a lot to be learned about cancer biology by studying the common and
distinct mechanisms by which tumor viruses modulate cell pathways. As described
above, all tumor viruses encode proteins that interact intimately with host cell pathways to mediate cell proliferation and cell survival. Most of these viruses target
common cellular proteins and cell signaling pathways that appear to be essential for
the survival of all tumor viruses in the host cell. These include activation of multiple
oncogenes and inhibition of tumors suppressors, evasion of the host immune
system, immune evasion, and the induction of cell proliferation and cell survival
programs.
93
Acknowledgments BD is a Leukemia & Lymphoma Society Scholar and a Burroughs Welcome

Fund Investigator in Infectious Disease. Her work is supported by grants CA096500, DE18281,
and HL083469 from the NIH.
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Chapter 5
Oncogenic Viruses and Cancer Transmission

Robin A. Weiss
Infection and Human Cancer

Physicians are able to reassure patients and those who care for them that cancer is
not catching in the sense that you do not develop cancer as a result of close contact
with someone who has the disease. Veterinarians might be more cautious, as there
is a malignancy of dogs and another of the Tasmanian devil that are transmitted
directly through contact, as described at the end of this chapter. However, while
human cancer is not directly contagious, there is compelling evidence that nearly
20% of the global burden of human cancer has an infectious etiology amounting to
almost 2 million new cases each year (Parkin 2006). Approximately 5.5% of tumors
are attributable to bacteria, such as Helicobacter pylori (Polk and Peek 2010) causing
stomach cancer and mucosal-associated lymphoid tumors (MALTs), and to helminths
associated with cancers of the urinary bladder and the gall bladder. The remaining
infective cancers are due to virus infection representing some 13.5% of all human
cancer. This is good news for cancer prevention because it opens the path to cancer
control through screening and immunization.
The notion that some types of cancer might be transmissible was first promulgated
in 1842 by Domenico Rigoni-Stern (Scotto and Bailar 1969), who was a surgeon
with epidemiological and statistical interests in Verona in Northern Italy. He observed
that nuns in the citys convents tended to develop breast cancer more frequently than
other women, but suffered less cancer of the uterine cervix. He suggested that the
constriction of tight corsets might promote breast tumors but that cervical cancer
was associated with sexual activity. With modern eyes, we can say that the higher
incidence of breast cancer had an endocrine explanation and the lower rate of cervical
cancer owed its benefit from less exposure to human papillomaviruses (HPVs).
R.A. Weiss (*)

Division of Infection and Immunity, University College London, London, UK
e-mail: r.weiss@ucl.ac.uk
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R.A. Weiss
Nulliparous women in general develop breast cancer more frequently than those
who have children; moreover, nuns were usually recruited from relatively wealthy
families, and there is a positive correlation between body mass index and breast
cancer. On the other hand, most of the women who joined religious orders (other
than widows) would not have encountered infection by a sexually transmitted virus.
Rigoni-Stern conducted his analysis before the germ theory of transmissible diseases
was accepted (although he was an active promoter of vaccination) and before the elaboration of the cell theory of cancer, yet his observations remain pertinent today.
The infectious agents known to be associated with human cancers have been
identified during the last 50 years (Table 5.1). A vintage year for discovery was
1983, when HPV-16, HIV-1, and H. pylori were first reported (Weiss 2008). Where
there was clear epidemiological evidence of a transmissible agent causing the cancer,
the discovery of the virus often resulted from a specific search for it, e.g., cervical
carcinoma, Burkitts lymphoma, and Kaposis sarcoma. On the other hand, some
cancers were not identified as having an infectious etiology until well after the agent
had been characterized, e.g., hepatocellular carcinoma (HCC), nasopharyngeal
carcinoma, and stomach cancer.
Virus discovery was usually followed by meticulous epidemiological analyses
using serological or molecular markers and established the links between virus
infection and malignant disease. For example, it took more than 12 years following
the discovery of hepatitis B virus (HBV) by Baruch Blumberg (Chap. 2) as a cause
of serum hepatitis and cirrhosis for R. Palmer Beasley to demonstrate unequivocally through a study of a cohort of 22,707 Taiwanese men that, contrary to the
prevailing view, HBV was also a major cause of liver cancer (Beasley et al. 1981).
The discovery of human T-cell leukemia virus type 1 (HTLV-1) in the USA (Poiesz
et al. 1980) in a case of cutaneous CD4-positive lymphoma was soon followed by
its link to adult T-cell leukemia (ATL) in Japan (Miyoshi et al. 1981; Hinuma et al.
1981), and later to tropical spastic paraparesis in the West Indies (Gessain et al.
1985). The identification of the Caribbean islands as an endemic region of HTLV-1
transmission (Blattner et al. 1982) followed on from an observation in London
(Catovsky et al. 1982) that immigrants from Jamaica presented with a type of leukemia indistinguishable from Japanese ATL and were seropositive for HTLV-1.
The public health importance of different oncogenic viruses varies greatly, ranging
from major global causes of mortality, such as liver and cervical cancer, to relative
rarities, like ATL and Merkel cell skin cancer (Table 5.1). Given that the Merkel cell
polyomavirus (PyV) was discovered as recently as 2008 (Feng et al. 2008; Becker,
Chap. 18), further cancer associated viruses may be revealed. That seems possible
for rare cancers which are increased in immunodeficiency, for example conjunctival
squamous carcinoma in African AIDS (Waddell et al. 2010), although a sensitive
subtractive transcriptome analysis of this tumor did not reveal any viral sequences
(Feng et al. 2007). Further investigation of EpsteinBarr virus (EBV)-negative
AIDS lymphomas might also lead to the discovery of a new agent.
Acute lymphocytic leukemia (ALL) in children is thought to be linked to delayed
infection among children living in relative isolation (Kinlen et al. 1990; Greaves
2006). However, a specific oncogenic virus is not necessarily involved as several
1967
1979
1980
1983
1983
1989
1994
HBV
HPV-5
HTLV-1
HIV
HPV-16, -18, etc.
HCV
KSHV
Skin Ca
ATL
KS
Lymphoma
Cervical Ca
Other anogenital Ca
HCC
Tumor
BL
NPC
HCC
490,000
65,000
195,000
<1,000
3,340
Estimated annual new

cancer cases globally
6,700
78,000
340,000
~3.0
~3.0
~5.0
<0.1
0.5
~20
Carriers who
develop cancer (%)
<0.1
<1.0
~15
Hepatitis
Cirrhosis
None
Genital warts
Nonmalignant disease
Infectious
mononucleosis
Hepatitis
Cirrhosis
Wart
TSP/HAM
AIDS
Blood
Blood
Sexual
Contact
Milk, blood
Sexual
Blood
Sexual
Transmission route
Saliva
KS
66,000
<1.0
Saliva
PEL, CD
<0.1
Merkel PyV
2008
Skin Ca
~1,000
<0.1
None
Contact
EBV EpsteinBarr virus, HBV hepatitis B virus, HPV human papillomavirus, HTLV human T-cell leukemia virus, HIV human immunodeficiency virus, KSHV
Kaposis sarcoma herpesvirus, PyV polyomavirus, BL Burkitts lymphoma, NPC nasopharyngeal carcinoma, HCC haptocellular carcinoma, Ca cancer, ATL
adult T-cell leukemia, KS Kaposis sarcoma, PEL primary effusion lymphoma, CD Castlemans disease, TSP/HM tropical spastic paraparesis/HTLV-associated
myelopathy, AIDS acquired immune deficiency syndrome
First reported
1964
Virus
EBV
Table 5.1 Viruses causing cancer in humans
5 Oncogenic Viruses and Cancer Transmission

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R.A. Weiss
kinds of infection promoting lymphocyte activation could be triggers for the appearance
of ALL if infection is delayed (Greaves 2006). The infection hypothesis is consistent with the higher incidence of childrens ALL in more affluent social classes, as
was seen for paralytic poliomyelitis in the 1950s, because poorer children tended to
acquire asymptomatic poliovirus infection and subsequent immunity early in life. It
represents a situation analogous to the hygiene hypothesis for the increasing prevalence of allergies (Rook 2007).
Certain common human viruses can transform cells in culture and can induce
tumors in experimentally inoculated animals, yet are not known to cause tumors in
their natural host. Thus, the human polyomaviruses, BK, and JC (Hirsch, Chap. 16;
Khalili, Chap. 17) and human adenovirus types 2 and 12 (Ricciardi, Chap. 20) can
transform rodent cells in culture, and are potently oncogenic in newborn rats and
hamsters. However, there is no firm epidemiological evidence to suggest that these
commonplace human infections are linked to any human malignancy. One possible
explanation for this paradox is that human cells are permissive for lytic replication
of these viruses which would kill the host cell, whereas rodent cells permit the
expression of only the early, transforming genes, such as polyoma T antigens and
adenovirus E1A and E1B. But these viruses also spawn defective variants during the
multiple rounds of replication in the human body, so cell transformation by viruses
lacking late, lytic viral genes might be expected to occur. One could argue that the
human immune system would nonetheless control or eliminate the development of
viral tumors, yet even in immunocompromised people (Wood, Chap. 32), tumors
caused by polyomaviruses or adenoviruses have not been documented. Perhaps this
question merits reinvestigation. Conversely, Zur Hausen (2001) has suggested that
zoonotic animal viruses that can induce cell proliferation might be tumorigenic in
humans if human cells were nonpermissive for lytic replication.
Contribution of Tumor Viruses of Animals to Molecular Biology

Veterinary malignant diseases were first shown to be transmissible in the nineteenth
century which we now know are caused by oncogenic viruses. Enzootic bovine
leukosis is caused by a deltaretrovirus related to the HTLV and Jaagsiekte in sheep,
caused by the betaretrovirus, JRSV (Fan, Chap. 30). The first cell-free filtrate
hence a virus shown to cause malignancy was the erythroleukemia virus of chickens reported by Ellerman and Bang in 1908 (Coffin et al. 1997). And 100 years
ago, Peyton Rous described his eponymous sarcoma virus (Rous 1911; Parent,
Chap. 28). In 1932, Richard Shope discovered the first oncogenic papillomaviruses
in rabbits (Tooze 1980) while in 1936, Bittners milk factor (mouse mammary tumor
virus (MMTV)) was the first oncogenic virus to be defined in laboratory mice (Weiss
et al. 1984). It is curious that in some animal species the proportion of tumors with
a viral etiology is higher than that of humans (e.g., cats and chickens), whereas
other well-studied species do not appear to harbor any oncogenic viruses (e.g., dogs
and horses), although cross-species infection of horses by bovine papillomavirus
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type 1 induces the benign tumor, equine sarcoid. Cancer in wild animals is more
difficult to study (McAloose and Newton 2009) and therefore few oncogenic viruses
of wild species are known. One that has recently emerged is the retrovirus-causing
acute leukemia in koalas (Tarlinton et al. 2006).
In addition to their importance as pathogens, tumor viruses have played a pivotal
role in gaining insight into basic molecular and cell biology and into mechanisms of
nonviral oncogenesis. This benefit to our understanding resulted from the intensity
of studies of animal tumor viruses as models from the 1960s onward (Tooze 1980;
Weiss et al. 1984; Coffin et al. 1997). Thus, splicing of eukaryotic mRNA transcripts
was first discovered in polyomaviruses and adenoviruses. The first nuclear location
signal in a protein was identified in SV40 large T antigen, and origins of DNA
replication and control in mammalian systems were first analyzed with these
DNA tumor viruses. The tumor-suppressor gene, p53, was first identified as a protein
to which large T antigen bound and inhibited or sequested its function (Howley
and Livingston 2009). RNA tumor viruses led to the identification of oncogenes
before cellular oncogenes were discovered, reverse transcriptase which allowed the
synthesis of cDNA, and retroviral vectors which became standard tools in gene
transfer and gene therapy.
Control and Prevention of Viral Cancers

We live in an age of optimism that we can prevent millions of potential cancers through
screening and immunization with tumor virus vaccines. Cervical cancer screening
by cytologists essentially entails the recognition of abnormal, HPV-infected cells.
In Japan, the screening of blood donations for HTLV-1 has led to a drastic diminution of horizontal virus transmission, and antenatal screening helps to prevent the
next generation acquiring HTLV-1 vertically via mothers milk, which is the main
route of transmission.
The first really successful anticancer vaccine was the Mareks disease virus
(MDV) vaccine against this alpha herpes virus causing T-cell lymphomas in the
domestic fowl (Parcells and Morgan, Chap. 13). However, as this vaccine protects
against tumor development rather than virus infection, it has led to increased MDV
virulence later. Immunization against HBV (Blumberg, Chap. 2; Mason, Chap. 22)
prevents HBV infection and hence hepatitis. It was initially prepared by purification
of noninfectious 22-nm particles present at high titer in the plasma of viremic
patients, but then the subunit vaccine was produced which represents the first vaccine
of any kind to be developed through recombinant DNA technology. There have now
been sufficient years of follow-up to discern a fall in hepatocellular cancer incidence in immunized populations. The recent development of two commercially
available, efficacious vaccines against genital HPV transmission promises to greatly
reduce cervical cancer (Roden and Wu 2006).
The delivery and coverage of HBV and HPV immunization programs in resourcepoor countries, where these viruses are most prevalent, presents a challenge in
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R.A. Weiss
public health economics and disease prevention. Protective vaccines against EBV
and HTLV-1 could be developed and produced if they were deemed to be of public
health benefit, but to date they have not been regarded as a major market opportunity
for vaccine companies. HCV and HIV vaccines would be of obvious and immediate
benefit, but alas, we still await safe and efficacious vaccines for these viruses.
Transmission Dynamics of Oncogenic Viruses

DNA Tumor Viruses
Many DNA viruses with oncogenic potential are acquired early in life, but the tumors,
if any, tend to present much later. For example, the major route of transmission of
EBV and Kaposis sarcoma-associated herpesvirus (KSHV or HHV-8) is from
mother to child via saliva (Boshoff and Weiss 2006; Schulz, Chap. 10), but Kaposis
sarcoma occurs mainly in elderly men, although KSHV-associated lymphoma may
occur in children and young adults. Evidence from seroconversion also suggests
that polyomavirus infections, such as BK and JC, occur in childhood. A recent study
indicates that Merkel cell polyomavirus and novel human polyomaviruses are
chronically shed from the skin and may, therefore, be transmitted by contact
(Schowalter et al. 2010). HPVs infect the dividing, basal cells of epithelia but its late
proteins for viral maturation are only expressed in differentiated squamous cells.
Thus, carcinoma in situ and eventually invasive cancer develop in cells that do not
achieve sufficient differentiation to complete lytic viral replication. HPV infections
of the skin (e.g., HPV-1, HPV-5) are similarly shed in squames, and some genital
HPV types are vertically transmitted, as HPV-11 and HPV-16 have been found in
pediatric laryngeal warts. Transmission probably occurs when the baby passes
through the dilated cervix. Of course, the main route of transmission of these genital
HPV strains is sexual (You and Wells, Chap. 19).
Oncogenic Retroviruses
The transmission dynamics and pathogenesis of retroviruses vary according to the
type of tumor virus and its mode of transmission. Let us take the classical example
of avian leukosis viruses in chickens (Weiss et al. 1984). Horizontal transmission
between birds (except to the newly hatched) usually results in a transient viremia
followed by an immune response that controls the virus load in the tumor-target
organ (the bursa of Fabricius producing B cells) so that leukemia rarely ensues.
However, if the same virus infects the chick embryo through secretion into egg
albumen in the hens oviduct, then chronic viremia without an immune response
often results many weeks after hatching in B-cell leukemia. The mechanism of
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oncogenesis is through the integration of the proviral long terminal repeat (LTR)
adjacent to a cellular oncogene (Beemon and Rosenberg, Chap. 27), so the higher
the rate of replication, the greater the chance of integration proximal to an oncogene.
A similar mechanism applies to MMTV acquired through suckling colostrums
immediately after birth, except that lymphocytes provide an intermediate cellular
reservoir until the offsprings mammary glands become infected upon maturity
and pregnancy (Ross, Chap. 29). On the other hand, Jaagsiekte virus of sheep
(Fan, Chap. 30) has a strict cell tropism for type II pneumocytes which require lungto-lung transmission via aerosols.
Murine and feline leukemia viruses present a rather more complex series of
events leading to cancer (Beemon and Rosenberg, Chap. 27). An ecotropic virus is
the primary agent, replicating from cell to cell and increasing in titer. It has an
endogenous, Mendelian origin in mice, such as the AKR strain, but is horizontally,
exogenously acquired among cats (FeLV-A). However, a recombinant virus between
these transmissible, ecotropic viruses and endogenous retroviral envelopes with
xenotropic or polytropic properties eventually emerges and it is this recombinant
genome that has oncogenic properties. Thus, the oncogenic agent itself is generated
anew in each infected host, and the transmissible virus is a precursor.
Retroviruses have also been transmitted across species barriers, sometimes
between large taxa, to infect new hosts. For instance, the gibbon ape leukemia virus
and the koala virus are both closely related to gammaretroviruses of rodents in
Southeast Asia. Whether retroviruses and polyomaviruses of animal origin are
circulating in humans is discussed later.
Retroviruses which bear oncogenes of cellular origin appear to be severely
restricted in transmission. Most are defective, requiring a helper leukemia virus to
complement missing or defective replicative genes (Beemon and Rosenberg, Chap.
27; Parent, Chap. 28). Despite the immense interest in these viruses for unraveling
molecular mechanisms of oncogenesis, I am not aware of any cases of oncogenebearing viruses which are naturally transmitted between hosts, with the possible
exception of the cyclin-bearing fish retroviruses (Kurth and Bannert 2010). Each
one of the sarcoma viruses or acutely transforming leukemia viruses of chickens,
mice, cats, and primates appears to be transmissible only by experimental inoculation. We can regard them as one-off, dead-end transductions whose tumors came to
the notice of pathologists like Peyton Rous, but which play no role in the natural
history of virus transmission.
Iatrogenic Infections
Hepatitis viruses are transmitted parenterally (Lindenbach, Chap. 23; Mason, Chap.
22). HBV is often naturally acquired early in life, perinatally in Asia and during
infancy in Africa. But iatrogenic (medically induced) transmissions also play a
significant role in tumor virus transmission. Where nonsterile injection equipment
was used, the dynamics and prevalence of infection by HBV, HCV, and HIV may
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have been greatly increased in Africa during the twentieth century, as argued by
Drucker et al. 2001. The campaign to eradicate bilharzia (Schistosomiasis) in Egypt
from the 1950s to 1980s by using an intravenous drug, tatar emetic, greatly exacerbated the spread of HCV because syringes and needles were not replaced for
each individual when entire villages were treated (Frank et al. 2000; Strickland
2006). A related veterinary example was the spread of bovine leukemia virus (BLV)
infection when whole herds were dehorned using common equipment, although
injection for brucellosis vaccination did not markedly increase the risk of BLV
transmission (Lassauzet et al. 1990).
Regarding zoonotic infection by tumor viruses, millions of humans were exposed
to SV40 virus because it was a containment of poliovirus vaccine propagated in
primary kidney cultures derived from rhesus macaques (Shah and Nathanson 1976).
Whether or not SV40 has taken off as a circulating virus in the human population
remains controversial (Butel, Chap. 15, and Sect. 4.7). The discovery of SV40 in
rhesus kidney cultures (Sweet and Hilleman 1960) soon led to the replacement by
African green monkey kidneys as the preferred substrate for the propagation of
polio vaccines. It was not known at the time that African green monkeys frequently
harbor a strain of simian immunodeficiency virus (SIVagm), but fortunately SIVagm
does not transfer to humans as it is sensitive to human host cell restriction factors.
A claim that HIV-1 came into humans as a result of a batch of polio vaccine being
prepared in chimpanzee kidney cultures (Hooper 2001) has not been upheld for two
major reasons: first, a vial from the incriminated lot of polio vaccine was analyzed
and found to have been prepared in rhesus cells and was not contaminated by SIVcpz
(Berry et al. 2001); second, estimates of the time since the most recent common
ancestor of HIV-1 Group M (the pandemic group) reliably show that HIV started to
circulate in humans in Cameroon and the Congo several decades before the polio
vaccine trials were conducted there (Sharp et al. 2001).
These tales of contamination and of near misses emphasize how important it
remains to ensure that biologicals and the injecting equipment used to deliver them
are free from contaminating viruses. Novel therapies, such as xenotransplantation,
need to consider the potential hazards of virus transmission (Weiss 1998).
Transmission and Virulence

Cancer is a multistep process of progressive somatic mutations, and oncogenic
viruses may push the infected cell past one or more of those steps, but not all of
them. Cancer-associated viruses are persistent infections in the hosts in which the
cancer develops. Indeed, the virus is usually present in the cancer cell clone itself:
EBV genomes are found in the nasopharyngeal carcinoma and lymphoma cells with
which it is associated, HTLV-1 in ATL cells, HBV in HCC cells (often integrated
into chromosomal DNA), but HCV-linked liver tumors do not invariably contain the
virus. However, HIV is a cancer-associated virus without infecting the tumor cells.
It acts indirectly by allowing opportunistic oncogenic viruses to exert their effect via
109
immune deficiency (Wood, Chap. 32), although some investigators have proposed
an additional role for the Tat protein in oncogenesis (Aoki and Tosato 2007).
One of the reasons that certain DNA viruses are potentially oncogenic is that they
need to kick-start the cell-replicative machinery the S phase of the cell division
cycle in order to replicate themselves. The small DNA tumor viruses, such as
HPV, polyomaviruses, and adenovirses, do not encode all the enzymes for DNA
replication in the viral genome, and require cellular enzymes to achieve replication.
Oncogenic herpesviruses may be similar in this regard, but pox viruses, which
replicate in the cytoplasm, are less dependent upon the cell cycle. This reasoning
may explain the convergent evolution of disparate DNA tumor viruses to target the
inactivation of host tumor suppressors, such as retinoblastoma protein and p53
(Jung, Chap. 3). Oncogenic retroviruses in general need to infect dividing cells. An
interesting exception is JSRV (Fan, Chap. 30) which infects terminally differentiated type II pneumocytes, and its envelope glycoprotein serves as an oncogene
which returns these cells to a dividing state.
It is not clear how early after an infection an incipient precancerous cell lineage
occurs. The tumors tend to present late in the life span of the host. Moreover, tumors
develop in only a minority of infected individuals, perhaps up to 20% in HBV
infection, around 1% in HTLV-1 and in genital HPV infection, and <0.01% in EBV
and cutaneous HPV infection (Table 5.1). Thus, the rare malignant state of an
infected cell is not directly related to virus transmission; rather, it should be regarded
as collateral damage, an occasional side effect of infections that require the host
cells to be in a proliferative state.
As oncogenic viruses are necessary if not sufficient for the tumors to develop,
immunization against infection can prevent the oncogenic process commencing.
Mareks disease (MD) (Parcells and Morgan, Chap. 13) is an interesting exception
in that the vaccines protect mainly against tumorigenesis rather than against infection. MD vaccines protect with great efficacy against the development of the disease
but do not prevent MDV transmission. Nonetheless, the MD vaccines dampen down
virus transmission and, therefore, elicit selective pressure on the virus to circumvent
this restriction. Over the 40 years since the first MDV vaccine was introduced, virus
escape has resulted in the emergence of progressively more virulent MDV strains as
novel, more potent vaccines were developed (Gimeno 2008). MD represents the
best example for tumor viruses of a positive correlation between virulence and
onward transmission. A corollary of this phenomenon is that vaccines which provide
sterilizing immunity against primary infection (as for HBV and genital HPV) are
less likely to exert selective pressure for the evolution of virulence.
Multifactorial Facets of Viral Cancers

As oncogenic viruses only cause cancer in a minority of infected individuals,
many cofactors influence the likelihood of malignancies developing. Further
somatic mutations in the cell lineage leading to cancer are necessary, and several
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Mendelian-inherited predispositions both to viral infection and cancer emergence

affect cancer incidence. These genetic predispositions are best understood for retroviral oncogenesis in mice, but they are also well-recognized in humans. In addition,
environmental agents, including other infections, also play a role in certain types of
viral cancer. Three human examples of the multifactorial elements in human cancer
illustrate the complexities of viral oncogenesis.
Hepatitis B Virus and Hepatocellular Carcinoma

HCC is the third most common cause of cancer incidence (Parkin 2006), but the
substantial proportion of the burden due to HBV should be preventable by vaccination
(Goldstein et al. 2005). Liver mycotoxins in the diet, such as aflatoxin B, act synergistically with HBV infection to increase the incidence of HCC three- to sixfold
over than seen for exposure to chemical or virus alone (Kirk et al. 2006; Wild and
Montesano 2009). Aflatoxin B is a product of Aspergillus mold species which
grow on maize and groundnuts, especially in tropical humid conditions. Aflatoxin
B is often present in the diet in regions, such as China and West Africa, where HBV
infection is also prevalent. It is a genotoxic agent making mutagenic adducts to
DNA and it induces mutations in key genes, such as the tumor suppressor p53.
Aflatoxin B becomes concentrated and metabolized in the liver which is its main
site of mutagenic action. Regenerating hepatocellular cells in HBV infection, therefore, become targets for the chemical carcinogen in the diet.
It is not yet known precisely to what extent dietary aflatoxin B interacts with
HCV (Wild and Montesano 2009). Because primary HCV infection usually occurs
at a later age than HBV, the synergy for HCC may be lower, although aflatoxin B
can act as an initiating carcinogen. Clearly, it is of health benefit to reduce the levels
of mycotoxins in the diet through dry storage and other means, as well as controlling
hepatitis infections.
EpsteinBarr Virus-Associated Tumors

EBV infects the vast majority of people in all populations, and is linked to several
types of lymphoma, nasopharyngeal carcinoma, and gastric cancer (Robertson,
Chap. 8), yet few infected individuals develop EBV-associated diseases. The virus
is usually acquired via saliva in infancy to no ill effect unless the child inherits the
rare genetic disorder, Duncans syndrome. Delaying infection until adolescence
(and probably receiving a higher infecting dose through kissing) is likely to result
in infectious mononucleosis, a polyclonal lymphoproliferative but nonmalignant
disease. As discussed earlier, it is not uncommon to see an increase with age in the
virulence of primary infections, e.g., polio virus and varicella-zoster virus, as maternal
antibodies partially protect the infant from disease.
111
EBV-linked tumors require cofactors in addition to EBV. Burkitts lymphoma

(BL) in children only occurs in areas of endemic malaria infections, and the chronic
activation of B cells due to malaria may allow EBV-infected premalignant clones to
expand. Moreover, in every case of BL, including the EBV-negative adult cases,
the lymphoma cells have chromosome translocations linking one of the highly
active promoters of immunoglobulin genes to c-myc on chromosome 8. Thus, of the
oncogenic virus, malaria infection and somatic rearrangement of chromosomes
act in combination to promote the malignancy. A further cofactor might be local
inflammation because BL in children frequently presents in the upper or lower jaw
during the time of secondary tooth development.
Undifferentiated nasopharyngeal carcinoma (NPC) causes considerably more
mortality than BL (Table 5.1), although the tumor has been studied less intensively.
Like BL, NPC prevalence tends to be geographically restricted and it occurs most
commonly among people in Southern China and in people of Chinese origin. It is
thought that predisposing host genetic factors must be determinants of tumor development. Certain major histocompatibility complex genes are associated with risk of
NPC (Hildesheim et al. 2002), although the association does not appear to be strong
enough to wholly explain the geographic clustering. There is also a theory that an
environmental factor, such as nitrosamines in the diet, may cause somatic mutations
that trigger NPC several decades later (Zheng et al. 1994).
Other tumors which are sometimes associated with EBV infection, such as
Hodgkins lymphoma, gastric cancer, and immunoblastic large cell lymphoma
(LCL), have other risk factors, e.g., immune deficiency for LCL.
Epidermodysplasia Verruciformis
Epidermodysplasia verruciformis (EV) is a very rare genetic disorder affecting
DNA repair and immune functions in the skin. This leads to multiple warts developing
all over the body, and these warts are initiated by papillomaviruses of the HPV-5
group (Kremsdorf et al. 1982). If the skin of EV patients is exposed to sunlight or
other sources of ultraviolet radiation, malignant skin tumors appear. Thus, two distinct
environmental factors HPV and sunlight generate a malignancy which is vastly
exacerbated in patients carrying defective EV alleles.
Skin tumors of the type seen in EV patients are rarely seen in people not carrying
EV-defective genes. But HPV-5-related viruses are thought to be near-universal
infections because all EV-affected individuals develop warts if not malignancies.
Moreover, using polymerase chain reactions (PCRs), the viruses can be detected in
normal skin and are expressed in psoriasis (Orth et al. 2001). However, other
HPV types are more commonly associated with squamous skin cancer and can be
frequently detected in normal skin and hair follicles (Plasmeijer et al, 2010). Like
EBV, usually nonpathogenic, ubiquitous cutaneous HPV strains elicit a lethal
malignant disease in rare individuals and occur more frequently in immunocompromised individuals.
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Distinguishing Tumor Viruses from Rumor Viruses

Distinguishing genuine infection from artifact is not as easy as it might at first sight
appear. Voisset et al. (2008) reviewed this topic in detail concerning the claims of
retroviruses in humans, and similar problems arise with certain DNA tumor viruses.
Our interest was aroused when we imagined that we had discovered a novel human
retrovirus linked to rheumatoid arthritis (Griffiths et al. 1997) but later found that
it was actually an endogenous retroviral genome of rabbits (Griffiths et al. 2002).
The virus could be found at extremely low virus load usually requiring nested PCR
for detection. The error only became evident when we managed to clone host DNA
sequences at the integration sites flanking the LTRs of the virus, and a BLAST
search showed that they were rabbit sequences. Strangely, the false-positive detection of this retrovirus was also detected by independent groups in Europe and the
USA (Voisset et al. 2008), and occurred significantly more frequently in pathological
tissue samples than in controls. The only reason we could suggest for this discrepancy was that the pathology samples might have been opened more often than the
control blood samples and hence have had more opportunity for contamination by
extraneous DNA molecules in the laboratory.
There is a long history of reports of DNA sequences and antigens related to animal
tumor viruses in human tumors (Voisset et al. 2008). The presence or not of MMTV
in human breast cancer biopsies remains controversial, but definitive evidence, to
my mind, has never quite materialized. PCR detection of MMTV sequences is genuine
enough (Pogo et al. 2010) but could well result from contamination like we found
with the rabbit virus. Antigens cross-reacting with antibodies to MMTV envelope
glycoproteins gp52 are not quite convincing, and the in situ hybridization of MMTV
on human chromosomes only lights up one chromatid of a mitotic pair (Liu et al.
2001), again leading one to doubt the interpretation that this represents reliable
evidence of MMTV infection in human breast cancer.
A virus sequence similar to xenotropic mouse leukemia virus is called xenotropic murine-related retrovirus (XMRV) (Singh, this volume). XMRV was detected in
stromal cells of a subset of human prostate cancer biopsies in patients with a defective RNase L gene, which affects the interferon response pathway (Urisman et al.
2006). Such a defect might predispose people to zoonotic infection with a mouse
retrovirus or aid its transmission from human to human. But the link to RNase L
alleles has not been upheld in another study which detected XMRV in the prostatic
epithelium rather than in the stroma (Schlaberg et al. 2009). Another report (Hohn
et al. 2009) failed to detect XMRV in prostate cancer.
XMRV has also been reported in a majority of patients with chronic fatigue
syndrome (CFS) (Lombardi et al. 2009) but could not be confirmed in other cohorts
with similar symptoms (Erlwein et al. 2010; Groom et al. 2010; Switzer et al. 2010).
XMRV has been isolated in infectious form as well as by PCR (Lombardi et al.
2009) from prostate tumor cell lines and CFS biopsies. However, it should be born
in mind that any human tumor that has been grown as a xenograft in mice or has
been propagated in culture in the same facility as cell lines producing xenotropic
113
murine retroviruses is at risk of contamination. Many murine monoclonal antibody

preparations contain xenotropic MLV (Weiss 1980), and therefore the viral genes
are present in most research laboratories. XMRV has a signature in the gag sequence
that distinguishes it from classical MLV-X from NZB mice, but different mouse
strains harbor many variants of this retrovirus. Thus, the link between XMRV and
prostate cancer or CFS and whether the virus is a genuine human transmissible
agent remains to my mind unresolved.
The current XMRV controversy is reminiscent of the SV40 issue in the mid
1990s (Brown and Lewis 1998). As already mentioned, this polyomavirus of rhesus
macaques did contaminate certain early batches of polio vaccine to which millions
of people were exposed (Shah and Nathanson 1976) and hence it might have
established a foothold in the human population. Thus, when Carbone et al. (1994)
reported the presence of SV40-like DNA in human mesothelioma biopsies, many
investigators turned their attention to it. Indeed, Bergsagel et al. (1992) had previously detected SV40-related sequences in pediatric central nervous system tumors,
such as ependymoma and choroid plexus tumors. Assuming that these tumors
contained SV40 rather than a human polyomavirus, such as JC on BK, the data
suggest that SV40 has now become a transmissible human virus (rather like HIV-1)
because these cancer cases occurred in children born one or two generations after
polio vaccines were cleaned up. In the years since this controversy was actively
debated, the evidence does not appear to have hardened for a role of SV40 in human
tumors (Shah 2007). So I veer toward the skeptical view, whereas Butel (Chap. 15)
has a more positive opinion.
An impediment to sorting tumor virus from rumor virus is that investigators tend
to become drawn into taking entrenched positions. Those who obtain positive results
may declare that those who cannot confirm such data do not know how to do PCR
detection diligently! On the other hand, the doubters like me point out that the
laboratories with positive results are often those that work on that type of virus in
any case, and therefore would be more prone to contamination by virus or PCR.
As detection techniques become increasingly sophisticated, we must be careful to
match sensitivity with specificity, including the purity of tissue sources.
Transmissible Cancer Cells

Around the time that HIV-1 was discovered, I read a paper by Hayes et al. (1983)
postulating that Kaposis sarcoma (KS) in AIDS may have a similar mode of transmission to canine-transmissible venereal tumor (CTVT) of dogs, namely, that the
sexually transmissible agent could be the tumor cell itself. This hypothesis was
disproved for KS when the Kaposis sarcoma-associated herpesvirus was discovered
(Chang et al. 1994; Schulz, Chap. 10), but the question whether it remained true for
the canine tumor intrigued me.
CTVT was first experimentally transplanted in 1876. It was extensively used in
cancer research before inbred rodents became available, and it was cited (as Stickers
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sarcoma) by Rous (1911). The cellular theory of CTVT transmission was based on
its ease of experimental transmission and on the presence of marker chromosomes
in tumors arising in different dogs (Murgia et al. 2006; Murchison 2008). Further
suggestive evidence for cellular transmission came from the detection in all CTVT
biopsies of a unique LINE-1 retrotransposon insertion site (Amariglio et al. 1991).
Many veterinarians seemed to accept the cellular theory of CTVT transmission,
but when I discussed the hypothesis with oncologists and immunologists, it was
usually greeted with disbelief. We, therefore, investigated the phenomenon employing
modern forensic DNA markers in order to test whether CTVT differed genetically
from the dogs bearing the tumors. Indeed, CTVT specimens collected from five
continents differed from the host dogs, but belonged to a single clonal lineage of
cancer cells. Thus, a canine somatic cell had evolved to become a transmissible
oncogenic parasite (Murgia et al. 2006).
A second example of a parasitic cancer has been found in the marsupial Tasmanian
devil known as Tasmanian devil facial disease (TDFD) (Pearse and Swift 2006;
Murchison 2008; Murchison et al. 2010). While CTVT is mainly spread through
sexual intercourse, TDFD is transmitted through biting and fighting. TDFD was
first seen in 1996 and has recently emerged in a population which is partially
inbred and shows restricted MHC polymorphism (Jones et al. 2004). CTVT probably
originated in a gray wolf some thousands of years ago and can spread to all dog
breeds and mongrels (Murgia et al. 2006; Rebbeck et al. 2009).
These two tumors in animals represent transmissible cancers that are not caused
by viruses. The question arises whether similar tumors might be found in humans.
To date, only iatrogenic cases are known, when immunosuppressed transplant
recipients have been unwittingly transplanted with apparently healthy tissue or
organs that contained occult cancer cells (Barozzi et al. 2003; MacKie et al. 2003).
The world of transmissible tumor viruses and tumor cells is fascinating.
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Chapter 6
DNA Viruses and Cancer: Taking

a Broader Look
James C. Alwine
Introduction
Increasingly evidence suggests that a variety of infectious agents, including bacteria,
parasites, and viruses, contribute to oncogenesis. At present, more than 20% of
worldwide cancers are believed to be caused or associated with infection by these
agents (zur Hausen 2009). Mechanistically, these agents may contribute to oncogenesis directly or indirectly. For example, many of the viruses encode oncoproteins, which can directly transform cells by affecting the function of major cellular
growth control proteins such as p53 and the retinoblastoma proteins (Rb) (OShea
2005); or transform using modified host gene, which have integrated into the viral
genomes (Chen and Barker 1985). Other viruses, as well as the bacteria and parasites,
may function more indirectly through immunosuppression, chronic inflammation,
suppression of apoptosis, and induction of genetic instability (zur Hausen 2009). It
is highly likely that we have only begun to discover the extent to which infectious
agents contribute to oncogenesis especially via an indirect route.
In this regard, it is worthwhile to take a broader look at the potential means by
which those DNA viruses not known to frankly transform cells may mediate more
subtle means of manipulating cells toward oncogenesis (oncopotentiation), or tumor
cells toward more serious malignancy (oncomodulation) (Michaelis et al. 2009).
The successful replication of mammalian DNA viruses such as polyomaviruses,
adenoviruses, and herpesviruses requires viral adaptation of the host cell to establish
an environment that can accommodate the increased demands for nutrients, energy,
and macromolecular synthesis that accompany viral infection. In order to do this
DNA viruses must control key cellular processes, for example, signaling pathways,
stress responses signaling, and metabolism, that affect broad aspects of cellular
J.C. Alwine (*)

Department of Cancer Biology, Abramson Family Cancer Research Institute,
School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
e-mail: alwine@upenn.edu
119
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J.C. Alwine
Fig. 6.1 The phosphotidylinositol-3 kinase (PI3K)Akttuberous sclerosis complex (TSC)

mammalian target of rapamycin (mTOR) signaling pathway. The diagram shows the control of
mTOR complex 1 (mTORC1) in the phosphorylation of p70S6 kinase (S6K) and the eIF4Ebinding protein (4E-BP), which control cap-dependent translation by controlling the formation of
the eIF4F translation initiation complex
synthesis, growth and survival. Thus we examine how DNA viral-mediated alterations
in these processes might contribute to the progression toward transformation when
the virus infects preoncogenic cells; or how these viral effects could increase malignancy if the virus infected a tumor cell.
We consider these effects for DNA viruses in general, but also focus more specifically on the b-herpesvirus, human cytomegalovirus (HCMV). HCMV is not believed
to be capable of frankly transforming human cells; although, there are reports in the
literature suggesting a hit and run transformation mechanism (Shen et al. 1997).
However, the recent development of more sensitive methods for the detection of
HCMV has implicated the virus in the development of several cancers, including
glioblastoma (Cobbs et al. 2008; Mitchell et al. 2008), colorectal cancer (Harkins
et al. 2002), and prostate cancer (Samanta et al. 2003). The role of HCMV in these
cancers has been proposed to by oncomodulation (reviewed in Michaelis et al. 2009;
Soderberg-Naucler 2008). It is proposed that by this process, HCMV in tumor cells
increases their malignancy by deregulating vital cellular processes (Bentz and
Yurochko 2008; Castillo and Kowalik 2004; Lukac and Alwine 1999; Sanchez and
Spector 2008; Streblow et al. 2008; Yu and Alwine 2002; Zhu et al. 1995). In the following section, we will examine how HCMV and other DNA viruses can alter the
phosphotidylinositol-3 kinase (PI3K)Akttuberous sclerosis complex (TSC)
mammalian target of rapamycin (mTOR) pathway (Fig. 6.1) in ways that mimic
effects seen in oncogenesis. We will also discuss viral alterations of cellular metabolism that also mimic what happens in many tumor cells. The data suggest that while
some DNA viruses, like HCMV, may not frankly transform cells, they can certainly
alter signaling and metabolism in ways that could potentiate transformation or increase
the malignancy of a cancer cell.
DNA Viruses and Cancer: Taking a Broader Look
121
The PI3KAktTSCmTOR Signaling Pathway

A good example of a major cellular signaling pathway targeted by many DNA
viruses is the PI3KAktTSCmTOR pathway (Fig. 6.1). As discussed below,
mammalian DNA viruses interact with one or a few components of this pathway at
some point in their life cycle. This is done in order to activate or inhibit the growth,
metabolic, antiapoptotic, and translational functions the pathway controls (Cooray
2004; Datta et al. 1999). The significance of this is that each component of the
PI3KAktTSCmTOR pathway is known to be an oncoprotein. Thus the DNA
viruses are altering the pathway in a manner that could contribute to transformation
or the progression of oncogenesis.
Akt (also called PKB) is the cellular homolog of the oncoprotein encoded by the
AKT8 retrovirus (Bellacosa et al. 1991). Akt can be activated by several mechanisms (Datta et al. 1999; Plas and Thompson 2005; Sarbassov et al. 2005b), the best
understood involves PI3K in response to insulin and other tropic factors. Akt activation depends on activation of PI3K to phosphorylate PIP2 (phosphotidylinositol
(PI)-3,4-bisphosphate), creating PIP3 (PI-3,4,5-triphosphate) at the plasma membrane. For example, insulin binding to the insulin receptor leads to the activation of
PI3K and converts PIP2 to PIP3. PIP3 binds both Akt and PDK1 (phosphoinositidedependent protein kinase-1), recruiting both to the plasma membrane allowing
PDK1 to phosphorylate Akt on threonine 308 (T308). Activated Akt affects multiple
cellular targets, which increase metabolism, growth, synthetic processes, and proliferation while suppressing apoptosis (Cass et al. 1999; Datta et al. 1999; Hill et al.
1999; Summers et al. 1998; Ueki et al. 1998). Since these processes benefit viral
replication, it is not surprising that many DNA viruses have developed means to
activate the Akt pathway (Cooray 2004).
However, these viruses can affect more aspects of the pathway than just Akt. An
important downstream effect of Akt is the activation of mTOR kinase (also known
as RAFT1 or FRAP), which exists in two complexes, mTOR complex 1 (mTORC1)
and complex 2 (mTORC2) (Fig. 6.1). The major difference between the two is the
substrate specificity factors, raptor in mTORC1 and rictor in mTORC2 (Kim et al.
2002; Sarbassov et al. 2004). Functionally, the two complexes have very different
substrates in uninfected cells. The substrates and functions of mTORC2 are less
well characterized than mTORC1; however, the data suggests that mTORC2 functions in (1) organizing actin cytoskeleton (Jacinto et al. 2004; Sarbassov et al. 2004),
(2) regulating cell growth and proliferation, (3) promoting the activation of the
serum glucocorticoid-induced protein kinase (SGK), and (4) promoting the phosphorylation of proline-directed serine or threonine sites in the turn motif of Akt and
protein kinase C isoforms (reviewed in Alessi et al. 2009). A primary function of
mTORC1 is to control cap-dependent translation (Mamane et al. 2006; Reiling and
Sabatini 2006; Sarbassov et al. 2005a). When mTORC1 is active, it phosphorylates
p70S6 kinase (S6K) and the eukaryotic initiation factor 4E (eIF4E) binding protein
(4E-BP1) (Fig. 6.1). Phosphorylation of S6K activates it, promoting the formation of
translation initiation complexes (Mamane et al. 2006); this includes the phosphorylation
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J.C. Alwine
of ribosomal protein S6. The phosphorylation of 4E-BP1 is a major point of control

in cap-dependent translation and regulates the function of the eIF4F translationinitiation complex. eIF4F binds to the 5-cap of an mRNA, which is the first step in
the initiation of cap-dependent translation. The eIF4F complex (Fig. 6.1) contains
eIF4E, which is the subunit that actually binds to the 5-cap, eIF4G, eIF4A, and the
Mnk1 kinase. To form the eIF4F complex eIF4E must bind to eIF4G, the scaffolding protein of the eIF4F complex. However, eIF4G binding and cap-dependent
translation, can be inhibited by 4E-BP1 binding to eIF4E, which displaces eIF4G
and inhibits eIF4F complex formation. mTORC1 regulates the binding of 4E-BP1
to eIF4E by controlling 4E-BP1 phosphorylation. Under positive growth conditions, mTORC1 is active and maintains 4E-BP1 in its hyperphosphorylated state
where it is incapable of binding eIF4E. This allows eIF4E to remain active in the
eIF4F complex and promote cap-dependent translation. However, under negative
growth conditions, for example, during stress or inhibition of mTOR kinase activity,
mTORC1 is inactive; 4E-BP1 becomes hypophosphorylated, binds efficiently to
eIF4E, displaces eIF4G and inhibits cap-dependent translation (reviewed in
Buchkovich et al. 2008b).
The signaling between Akt and mTORC1 involves the TSC, which comprises
TSC1 and TSC2 (also known as hamartin and tuberin, respectively) (Jozwiak 2006;
Krymskaya 2003), and Rheb-GTP (Astrinidis and Henske 2005; Long et al. 2005a, b).
Rheb-GTP function is mediated by the TSC, which stimulates the intrinsic GTPase
activity of Rheb, converting it from Rheb-GTP to Rheb-GDP, which cannot activate
mTOR kinase. Akt phosphorylation of the TSC inactivates the TSC, allowing
Rheb-GTP levels to remain high and maintain mTOR kinase activity in mTORC1
(Long et al. 2005a). A number of cellular stress responses activate the TCS to inhibit
mTORC1 and cap-dependent translation. Viral infections activate many of these
stress responses and many viruses have developed means to circumvent them to
maintain cellular functions like translation. As discussed below, a number of viruses
circumvent the effects of stress responses by targeting the TSC for inhibition or
degradation, thus maintaining mTORC1 activity. Hence, viruses that want to maintain mTORC1 activity and cap-dependent translation will want to either (1) activate
Akt to maintain the TSC in an inactive state, (2) directly block TSC function, or (3)
in some other way maintain mTORC1 activity.
The Effects of Mammalian DNA Viruses on the Activation

of PI3KAktTSCmTOR Pathway and the Maintenance
of Cap-Dependent Translation
Nearly every family of mammalian DNA viruses can be shown to affect some
aspect of the PI3K-Akt-TSC-mTOR pathway. This has been previously reviewed
in Buchkovich et al. (2008b). In the following discussion, we will consider the
past and more present data regarding the polyoma, papilloma, adeno, and herpes
viruses, i.e., the mammalian DNA viruses, which replicate in the nucleus and have
123
double-stranded DNA genomes. Thus we will not discuss the single-stranded

DNA parvoviruses and the cytoplasmic-replicating Pox viruses.
The polyoma viruses, for example, mouse polyoma (Py) virus and simian virus 40
(SV40) and the papilloma viruses contain covalently closed circular double stranded
(ds) DNA genomes of 58,000 base pairs encoding 58 temporally expressed early
and late proteins. Differential splicing of the early gene transcript of both viruses
produces mRNAs for several early proteins called large and small tumor (T) antigens
(LT and ST, respectively) for both Py and SV40, and middle T antigen (MT), which
is specific to polyoma (Ichaso and Dilworth 2001). The plasma membrane-bound
PyMT is a potent oncogene that alters the activities of important signaling proteins
(Gottlieb and Villarreal 2001; Utermark et al. 2007), including PI3K, which is important for transformation mediated by PyMT (Utermark et al. 2007). PI3K activation
by PyMT leads to the phosphorylation of a number of cellular targets, including Akt.
Presumably, the activation of Akt leads to the activation of mTORC1.
SV40 large T (SVLT) transforms mouse and human cells primarily by binding
and inactivating p53 and the Rb family proteins (Simmons 2000). Additionally, it
has been reported that SVLT transformation requires insulin receptor substrate-1
(IRS-1) (Rundell and Parakati 2001), which is key in the activation of PI3K by
insulin. SV40 small t (SVSt) binds to protein phosphatase 2A (PP2A), a major
phosphatase controlling many cellular functions. This interaction is required for
SV40-mediated transformation of human cells (Rundell and Parakati 2001). Akt
and mTOR are activated early during SV40 infection. Both SVLT (Yu and Alwine
2002; Yu et al. 2005) and SVSt (Yuan et al. 2002) have been shown to activate Akt;
however, the mechanisms involved remain unclear. Activation by SVLT can be
inhibited by PI3K inhibitors, indicating that SVLT activates PI3K. The interaction
between SVSt and PP2A is required for the SVSt-mediated activation of Akt (Yuan
et al. 2002), thus inhibition of PP2A appears to be needed to maintain Akt activity.
The papillomaviruses cause warts and several members of this virus family cause
cancer, including the high-risk human papillomaviruses (HPVs), which cause cervical cancer (Mirzamani et al. 2006; Schiffman et al. 2007). HPVs express several
early proteins, two of which, E6 and E7, are required for maintenance of HPVrelated oncogenesis through interactions with p53 and the retinoblastoma protein
(Rb), respectively. However, it has been shown that HPV E7 can interact with PP2A,
interfering with its interaction with Akt and, similar to SVSt, inhibiting Akt dephosphorylation (Pim et al. 2005). It has also been reported that the HPV 16 E5 protein
may be required for activation of PI3K and Akt (Kim et al. 2006). As the papillomavirus infection progresses, the need to activate Akt specifically to maintain mTOR
activity can be circumvented by the E6 protein, which binds to tuberin in the TSC;
this directs tuberin to proteasome-mediated degradation (Lu et al. 2004). As a result,
mTORC1 remains active.
The adenoviruses possess linear dsDNA genomes of 3038 kb encoding 3040
proteins. Early adenovirus proteins are responsible for preparing the cell to execute
viral replication, adenovirus proteins, E1A and E1B-55K, target Rb and p53, respectively (Helt and Galloway 2003; Querido et al. 1997, 2001). It has been suggested
that E1A mediates increased phosphorylation of 4E-BP and S6K (de Groot et al. 1995;
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J.C. Alwine
Gingras and Sonenburg 1997); other studies suggest that the early adenoviral
proteins E4-ORF1 and E4-ORF4 are involved in the activation of PI3K, resulting in
the activation of Akt and mTORC1 (OShea et al. 2005).
The human herpesviruses (HHV) include herpes simplex virus (HSV) 1 and 2,
varicella zoster virus (VZV), HCMV, Epstein-Barr virus (EBV), HHV-6, HHV-7,
and Kaposis sarcoma herpesvirus (KSHV, also known as HHV-8). These are large
DNA viruses with linear dsDNA genomes up to 230 kb, capable of encoding up to
200 proteins, as is the case with HCMV, the largest HHV. The host ranges and the
rates of the replicative cycles of the various HHVs differ greatly. For example,
HSV-1 has a broad host range and replicates very rapidly. Conversely, HCMV is
human specific and has a very slow replicative cycle, thus it must keep host cells
relatively functional for an extended period despite the stress of the infection on the
cells. Data suggest that HCMV does this very well by affecting many cellular signaling pathways in a way that inhibits stress signals or circumvents their inhibitory
effects (Alwine 2008; Buchkovich et al. 2008a, b; Child et al. 2004; Chuluunbaatar
et al. 2010; Hakki et al. 2006; Isler et al. 2005a, b; Kudchodkar et al. 2004, 2006,
2007; Mohr 2006; Moorman et al. 2008; Walsh et al. 2005; Xuan et al. 2009).
HCMV infection can be sustained under stress conditions that would kill uninfected
cells (Buchkovich et al. 2008a; Isler et al. 2005a, b). Regarding the effects of HHVs
on the PI3KAktTSCmTOR pathway, much is known about HCMV and HSV.
HCMV infection activates Akt through stimulation of T308 phosphorylation via
activation of PI3K (Johnson et al. 2001; Yu and Alwine 2002) and stimulation of
S473 phosphorylation via activation of mTORC2 (Kudchodkar et al. 2006).
However, the need for AKT activation during lytic infection for both HCMV and
HSV infections has come under question by the observations that Akt becomes
highly hypophosphorylated after 24 hpi in HCMV infected cells (Kudchodkar et al.
2006) suggesting that it is only needed during the early stage of infection.
Additionally, the HCMV early protein UL38 binds to and inactivates the TSC to
eliminate TSC-mediated inhibition of mTOR (Moorman et al. 2008), thus the activity of Akt as an inhibitor of the TSC is moot.
HSV appears to eliminate the need for Akt altogether, recent data has shown that
it encodes an Akt surrogate (HSV Us3), which allows mTORC1 to be constitutively
active (Chuluunbaatar et al. 2010).
HCMV infection also appears to targets the mTOR complexes and functionally
alters them to maintain 4E-BP phosphorylation and cap-dependent translation
(Kudchodkar et al. 2006). Additionally, it can act downstream of mTOR by inducing the activity of the Mnk1 kinase to phosphorylate eIF4E in the eIF4F complex
(Walsh et al. 2005) (Fig. 6.1).
Figure 6.2 summarizes this discussion showing all of the intersections between
DNA viruses and the PI3KAktTSCmTOR pathway. We see events that activate
PI3K, Akt, mTORC1, and inhibit the TSC. Each one of these activation or inhibition
events is known to be associated with oncogenesis (Averous and Proud 2006;
Engelman 2009; Henske 2004; Lu et al. 2010; Zhou and Huang 2010). Thus the
PI3KAktTSCmTOR pathway provides a vivid example of how DNA viruses can
affect cellular signaling in way that can be oncopotentiating and oncomodulatory.
125
Fig. 6.2 The points in the

PI3KAktTSCmTOR
signaling pathway that are
activated or inhibited by
polyoma, adeno, and herpes
viruses. See text for details
Viruses and the Warburg Effect: Viral Effects on Glucose

and Glutamine Metabolism
The effect of viruses on metabolism has recently begun to be examined in detail.
The findings, bases primarily on studies of HCMV, suggest that some DNA viruses
can alter metabolism in a manner similar to that seen in many tumor cells, thus suggesting another way viruses may promote the progression of oncogenesis. In 1924,
Otto Warburg observed that cancer cells metabolize glucose differently than normal
cells (Warburg et al. 1924). Like normal cell, cancer cells use glycolysis to convert
glucose to pyruvate (Fig. 6.3); however, normal cells and cancer cells differ in their
utilization of pyruvate. Pyruvate enters the tricarboxylic acid (TCA) cycle in normal
cells where it is catabolized to CO2 and promotes oxidative phosphorylation. In
cancer cells, pyruvate is converted to lactate (Fig. 6.3), and does not enter the TCA
cycle, even if sufficient oxygen is present to support mitochondrial oxidative phosphorylation (Vander Heiden et al. 2009). Such utilization of glucose is classically
known as the Warburg effect and it has several important implications. Specifically,
when pyruvate does not enter the TCA cycle only two ATP molecules are produced
per molecule of glucose; however, by completing the TCA cycle and oxidative
phosphorylation, an additional 36 ATP molecules are produced per molecule of
glucose. While this appears inefficient, the shift from the complete catabolism of
glucose in cancer cells, and other rapidly proliferating cells, results in the utilization
of glucose carbon in biosynthetic pathways for the production of macromolecules
needed to construct progeny cells (Vander Heiden et al. 2009, 2010). Another implication of this utilization of glucose is that glucose-derived carbon does not enter the
TCA cycle; this not only limits ATP production, but also limits the production of
TCA cycle intermediates, which are important biosynthetic precursors.
126
J.C. Alwine
Fig. 6.3 The metabolism of glucose and glutamine in normal, tumor, and HCMV-infected cells.
See text for details
Depriving the TCA cycle of glucose-derived carbon must be compensated and

recent studies show that in cancer cells exogenous glutamine is used as a carbon
source (DeBerardinis et al. 2007, 2008; Wise et al. 2008). Glutamine is converted to
a-ketoglutarate via glutaminase (Glnase) and glutamate dehydrogenase (GDH)
(Fig. 6.3). Thus, in a process called anaplerosis glutamine provides TCA cycle
intermediates to maintain the cycle (Fig. 6.3) and NADH for oxidative phosphorylation (DeBerardinis et al. 2007, 2008). Most normal, quiescent cells use only a small
amount of consumed glutamine in this way. Thus, both glucose and glutamine
metabolism are dramatically altered in tumor cells (DeBerardinis et al. 2008;
Newsholme et al. 1985; Wise et al. 2008).
Interestingly, HCMV-infected cells appear to induce metabolic changes similar
to those observed in tumor cells. Recent studies have shown that glucose metabolism is dramatically altered in HCMV-infected cells (Munger et al. 2006, 2008). In
these studies, it was shown that glucose carbon does not complete the TCA cycle.
Instead, much of the glucose derived pyruvate into the TCA cycle as far as citrate,
which is formed via the conversion of pyruvate to acetyl CoA (AcCoA) and the
subsequent reaction of AcCoA with oxaloacetate (OAA) (Fig. 6.3). Citrate in the
mitochondrion can be shuttling from the mitochondria to the cytoplasm where it is
converted back to AcCoA and OAA by ATP-citrate lyase (ACL). This provides
AcCoA in the cytoplasm for use in synthetic processes such as fatty acid synthesis
(Fig. 6.3) (Munger et al. 2008). The utilization of glucose carbon for fatty acid synthesis is essential for the success of the HCMV infection (Munger et al. 2008). This
is presumably due to the large amounts of membranes required during infection
(Buchkovich et al. 2010; Das et al. 2007; Homman-Loudiyi et al. 2003; Sanchez
et al. 2000; Seo and Britt 2006).
127
The diversion of citrate from the TCA cycle can be considered a modified
Warburg effect since glucose-derived carbon is being removed from the TCA cycle
for synthetic purposes. HCMV-infected cells, like tumor cells, must find other ways
to maintain the TCA cycle and produce ATP. Several recent studies have shown that
HCMV induces a program for the anaplerotic use of glutamine. Metabolomic data
charting metabolic flux suggested that glutamine utilization in the TCA cycle was
increased in HCMV-infected cells (Munger et al. 2006, 2008). The critical nature of
glutamine for the success of HCMV infection was demonstrated by direct measurement of glutamine metabolism in infected cells (Chambers et al. 2010). These data
showed that glutamine was necessary for ATP production in infected human fibroblasts, but not in uninfected cells. HCMV-induced glutamine utilization was also
indicated by increased glutamine uptake and increased ammonia production resulting from glutaminolysis. Correspondingly, there were HCMV-induced increases in
the levels and enzymatic activities of glutaminase and GDH, the two enzymes that
must be activated to convert glutamine to a-ketoglutarate for entry into the TCA
cycle (Fig. 6.3). That glutamine is used to maintain the TCA cycle in infected cells
was confirmed by the observation that TCA cycle intermediates such as a-ketoglutarate (Fig. 6.3) could rescue both ATP production and viral growth under conditions of glutamine deprivation (Chambers et al. 2010). Hence, HCMV-infected
cells, like many tumor cells, activate a program, whereby, glutamine utilization
increases specifically to maintain the TCA cycle allowing glucose to be used
biosynthetically.
There remains much to be determined about the effects of HCMV and other
viruses on metabolism and how this relates to pathogenesis. However, it is important to understand since it may provide new insights into viral therapy and new
drug targets for viral diseases. However, there is more to consider. As described
above, the alterations in cellular metabolism during HCMV infection are comparable to that seen in many tumor cells. As we noted in the case of viral alterations
in cell signaling what HCMV is doing could potentiate oncogenesis by setting up
a metabolic condition that favors the needs of the pre-cancerous or cancerous cell.
Indeed, the viral-mediated alterations in cellular signaling, stress responses, and
metabolism could result in not only oncopotentiation and oncomodulation, but also
unexpected pathogenesis, potentially implicating HCMV and other viruses as
agents or subtle cofactor in many maladies.
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Chapter 7
Herpesviruses and Cancer

David Everly, Neelam Sharma-Walia, Sathish Sadagopan,
and Bala Chandran
Introduction
Over 200 herpesviruses that are known to infect humans and a spectrum of animal
species including oysters are classified under Herpesviridae (order: Herpesvirales)
due to common characteristics such as large double-stranded, linear DNA genomes
encoding 100200 genes encased within an icosahedral capsid, which is itself
wrapped in the tegument protein layer containing both viral proteins and viral
mRNAs and a lipid bilayer envelope bearing many viral glycoproteins. Most importantly, they establish lifelong latent infection in the infected host and periodically
reactivate to reinfect. It is obvious that for the successful persistence in the host
these viruses must have evolved to utilize several avenues such as evading the host
intrinsic, innate, and adaptive immune responses, and modulation of apoptosis,
transcription, host cell metabolism, transport, and cell cycle. Though transformation leading to cancer and death of the host is not an advantage for the maintenance
of species, events such as reduction in immune and other surveillance mechanism
can lead to tumor formation by some of the members of herpesviruses.
The notion that herpesviruses play roles as the etiological agents of human and
animal malignancies came from the initial electron-micrographic discovery of
Epstein-Barr virus (g-1 herpesvirus) particles in Burkitts lymphoma (BL) cells in
1966 by Epstein, Achong, and Barr (Epstein et al. 1966a, b). This was further
substantiated by the identification of Mareks disease virus (MDV), an a-herpesvirus,
as the causative agent of Mareks disease (MD). Subsequent studies discovered that
Kaposis sarcoma-associated herpesvirus (KSHV), a g-2 herpesvirus, is etiologically
associated with Kaposis sarcoma (KS).
B. Chandran (*) D. Everly N. Sharma-Walia S. Sadagopan

H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology,
Chicago Medical School, Rosalind Franklin University of Medicine and Science,
North Chicago, IL 60064, USA
e-mail: bala.chandran@rosalindfranklin.edu
133
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D. Everly et al.
EBV is a g-1 herpesvirus and the type species of the genus Lymphocryptovirus
(LCV). Thirty different LCV, 26 of them novel, were detected in primates by a
panherpesvirus PCR assay. Nineteen LCV from chimpanzees, bonobos, gorillas,
and other Old-World primates were closely related to EBV. Seven LCV originating
from New-World primates were related to callitrichine herpesvirus 3 (CalHV-3), the
first recognized New-World LCV. Another LCV from gorillas and three LCV from
orangutans and gibbons were only distantly related to EBV and CalHV-3, which
were tentatively assigned to a novel genogroup of Old-World primate LCV. Another
transforming, EBV-related virus from spontaneous B cell lymphomas of common
marmosets (Callithrix jacchus) has been isolated.
KSHV is classified as a member of the rhadinovirus subgroup of gammaherpesviruses. Rhadinoviruses have been found in many species, including cattle,
mice, and both Old-World and New-World primates such as herpesvirus saimiri
(HVS), Rhesus rhadinovirus (RRV), mouse herpesvirus 68 (MHV-68), alcelaphine
herpesvirus 1 (AHV1), bovine herpesvirus 4 (BHV-4), equine herpesvirus 2 (EHV2),
and ovine herpesvirus 2 (OvHV-2).
Viruses that infect New-World monkeys include HVS, infecting the squirrel
monkey, and herpesvirus ateles (HVA), infecting the spider monkey. The macaques
of Asia are the only Old-World primate species documented thus far to harbor rhadinoviruses. RRV is widespread among rhesus macaques (Macaca mulatta). Its genome
organization is very similar to that of KSHV, with homologues of several potentially
pathogenic KSHV genes. Another rhadinovirus sequences have been identified in
macaques suffering from retroperitoneal fibromatosis (RF), a mesenchymal neoplasm
with vascular components. Both the rhesus macaque and the pigtailed macaque
(Macaca nemestrina) harbor RF herpesviruses (RFHVMm and RFHVMn). RF
herpesviruses are more closely related to KSHV than KSHV is to RRV.
Individual excellent chapters of this book discuss separately the role of LCV,
RRV, RFHV, MHV-68, and MDV in cancer. The focus of this chapter is primarily on
the general biology of the two human oncogenic herpesviruses: EBV and KSHV.
EBV Biology
After the initial identification of EBV in a BL biopsy, seroepidemiological studies
demonstrating increased antibody titers to EBV antigens solidified the connection
between the virus and BL (Burkitt 1958; Epstein et al. 1964; Levy and Henle 1966).
Subsequently, serological analyses of EBV antigens made the connection to infectious
mononucleosis and undifferentiated nasopharyngeal carcinoma (NPC) (Klein et al.
1968; Henle et al. 1970; zur Hausen et al. 1970). Since then, EBV has been associated with an increasing number of human malignancies. Although EBV is an
extremely successful pathogen that has infected greater than 95% of the individuals
by adulthood worldwide, only a subset of infected individuals develops malignancies
containing EBV. Understanding the viruss contribution to malignant transformation
has revealed a complex relationship between the virus with host factors, genetic
events, environmental influences, and other pathogens. The contribution of EBV to
135
Gastric and Nasopharyngeal

Carcinoma Latent infection of
mucosal epithelial cells in the
presence of other mutations
(Latency II)
Hodgkins disease EBV

infection in the presence of
other mutations (Latency II)
EBV
EBV
EBV
EBV EBV
T
EBV EBV
Germinal Center
B
EBV
EBV
EBV
EBV
EBV
Immunosuppressed lymphomas
Proliferation of latently infected Bcells (Latency III)
Memory
B-cell
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
Burkitts lymphoma
C-myc translocation
in the presence of
EBV (Latency I)
Fig. 7.1 Model of EBV life cycle and cancer contribution. EBV normal infectious cycle (solid
arrows) is initiated when the virus infects and replicated in the oral epithelial cells leading to the
latent infection of B-cells. Infected B-cells can enter lytic cycle leading to the infection of epithelial cells and possible transmission of the virus or can remain latent and go through the germinal
center reaction. Eventually the latently infected cell becomes a memory B-cell. EBV can also
contribute to the oncogenic process (dotted arrows). In the presence of immunosuppression EBV
can drive the proliferation of latently infected cells (immunosuppressed lymphomas). Alternatively,
germinal center B-cells latently infected with EBV that undergo mutation, e.g. c-myc translocation
(Burkitts lymphoma) or other mutations (Hodgkins disease), can lead to lymphoma development.
Latent infection of epithelial cells in the presence of mutations and/or environmental or genetic
predispositions can lead to carcinoma (gastric or nasopharyngeal carcinoma). Viral gene expression profile is indicated
cancer is intimately related to the natural life cycle of the virus (Fig. 7.1).
Understanding the mechanisms of cancer development have yielded insights into
the viral life cycle, and conversely, understanding the viral life cycle has led to
insights into the processes of malignant transformation.
EBV primarily infects two cell types: B-lymphocytes and epithelial cells. Like all
herpesviruses, infection with EBV can result in either lytic or latent infection.
Infection of epithelial cells primarily results in lytic infection. Lytic infection results
in production of infectious viral particles and the death of the infected cell. Infection
of B-lymphocytes normally results in latent infection. In latent infection, there is
limited viral gene expression and the virus seeks to maintain the viral DNA within a
cellular reservoir, i.e., memory B-cells for EBV. The malignancies of EBV are associated with the latent cycle of the virus. The viral proteins that ensure the survival of
the latently infected cell and maintenance of viral DNA are hypothesized to play
roles in the development of the malignancies associated with EBV (Table 7.1).
Latency 3
Latency 3
Latency 2, 3
Latency 2, 3
Latency 2, 3
Latency 1, 2, 3
Variable
EBNA-LP
BHRF1
LMP1
LMP2A
LMP2B
EBERs
miRNAs
Augment EBNA2 activity

Bcl2 homolog
TRAF-binding; NF-kB, PI3K,
MAPK, and JNK signaling
Syk and Lyn-binding; PI3K
signaling
Unknown
PKR-binding
Unknown targets
Table 7.1 EBV proteins with potential involvement in oncogenesis

Gene
Expression
Function
EBNA1
Latency 1, 2, 3
Episomal origin binding
EBNA2
Latency 3
RBP-Jk binding; transcription
activator
EBNA3A
Latency 3
RBP-Jk and CtBP binding
EBNA3B
Latency 3
RBP-Jk binding
EBNA3C
Latency 3
RBP-Jk and CtBP binding,
Deubiquitination
Regulate LMP2A function

Inhibit innate immunity
Cellular and viral gene expression
B-cell receptor mimic
Unknown
Increased tumorigenicity
Unknown
BIM and p16INK4A downregulation

Unknown
BIM and p16INK4A downregulation,
Rb and p27KIP1 regulation, Mdm2
regulation
Unknown
Antiapoptotic
Growth, antiapoptotic, and increased
motility and invasion
Cell growth and survival
Transcriptional and epigenetic regulation

Unknown
Transcriptional and epigenetic regulation
Transcriptional regulator
Antiapoptotic
CD40 mimic, antiapoptotic
Possible cancer contribution

Genomic instability
Myc upregulation
Proposed latency function

Episome replication and partitioning
LMP and cellular gene transcription
136
D. Everly et al.
137
During latent infection, a variety of latent proteins and RNAs can be expressed
depending upon promoter usage. EBV nuclear antigens (EBNAs) are encoded from
several alternatively spliced primary transcripts. The transcripts are named for the
BamH I library fragment from which the transcript arises. EBNA transcripts arise
from promoters in the W, C, and Q fragments named Wp, Cp, and Qp, respectively.
Wp and Cp transcripts are alternatively spliced to encode EBNA1, EBNA2,
EBNA3A, EBNA3B, EBNA3C, and EBNA-LP, while Qp transcripts express only
EBNA1. One of the viral Bcl2 homologues, BHRF1, can also be expressed from
Wp and Cp. The latent membrane proteins (LMPs), LMP1, LMP2A, and LMP2B,
are expressed from individual promoters near the terminal repeats of the genome.
EBV also expresses noncoding RNAs. EBER1 and EBER2 are abundant nonpolyadenylated of 167 and 173 base pairs, respectively. A number of viral microRNAs
(miRNAs) are also expressed during viral latency from the BamH I A and BHRF1
RNAs. Increased antibody titers to EBV latent proteins and in some cases EBV lytic
proteins are associated with the development of EBV-associated malignancies.
The correlation between the increased EBV-specific antibodies and the development of malignancies suggested a role of the virus in contributing to cancer.
At least three different, increasingly restrictive latent gene expression patterns
have been observed, termed latency III, latency II, and latency I. Alternatively, these
patterns have been described as the growth program, default program, and latency
program, respectively. The later nomenclature accounts for what is hypothesized to
occur during B-cell infection in a healthy individual. Following initial infection of a
nave B-cell, all of the latent proteins are expressed in the growth program to induce
the proliferation of the latently infected cell. As the latently infected cells move
through the germinal center reaction from centroblasts to centrocytes and in the face
of increased immune selection, only the LMPs and EBNA1, the default program,
are expressed. Finally, as the infected cell is differentiated into a memory B-cell,
only EBNA1 of the latency program is expressed. It is hypothesized that if the
latently infected memory B-cell stops dividing altogether, no EBV proteins may be
expressed, also called latency 0. These gene expression profiles approximate the
gene expression profiles observed in different EBV-associated cancers. However,
there can be significant variability in the EBV genes expressed in specific cancers
that are not accounted for by the latency nomenclature. The different EBV latent
proteins that play roles in the establishment of latency undoubtedly play roles in
cancer development under specific circumstances.
EBV and Burkitts Lymphoma

Although the BL was the first cancer to be associated with EBV, understanding the
contribution of EBV to the development of the tumor has been a longstanding mystery.
BL is endemic to equatorial Africa and is nearly always associated with EBV; by
contrast, spontaneous BL is also observed but it is rarely associated with EBV.
The primary oncogenic event of BL is a reciprocal chromosomal translocation
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D. Everly et al.
between the cellular oncogene, c-myc, and the genes of the immunoglobulin loci,
7585% t(8;14)(q24;q32) Ig heavy-chain (m), 5% t(2;8)(p12;q24) Ig light-chain (k),
and 10% t(8;22)(q24;q11) Ig light-chain (l) (Dalla-Favera et al. 1982; Lenoir et al.
1982; Magrath 1990). This translocation results in the uncontrolled expression of
c-myc, which is both necessary and sufficient to induce the proliferation of the tumor
cells (Lombardi et al. 1987; Polack et al. 1996). However, overexpression of c-myc
can also activate p53-dependent antioncogenic pathways (Cherney et al. 1997).
The Em-myc mouse model, which mimics the translocation, demonstrates that genetic
or epigenetic changes in the ARF/MDM2/p53 pathways are required for tumor
development (Eischen et al. 1999). Two nonmutually exclusive hypotheses for the
contribution of EBV to BL are (1) EBV may increase the chance of the c-myc translocation or (2) EBV may antagonize the cellular antioncogenic defenses. Interestingly,
EBV-associated BL development is also associated with infections of the malarial
parasite Plasmodium falciparum (Kafuko and Burkitt 1970) or HIV infection in the
context of AIDS. The association with malaria and AIDS is suggestive of the contribution of immunosuppression and thus higher levels of virus to the development of
BL. P. falciparum can induce reactivation of lytic EBV replication in EBV-infected
BL cell lines and infected primary B-cells that may increase the pool of latently
infected B-cells in which the c-myc translocation might occur (Chene et al. 2007).
The contribution of the viral proteins and RNAs of EBV to the development of BL
has been difficult to decipher. Paradoxically, the transforming proteins of EBV are not
expressed in BL, reinforcing the fact that the primary oncogenic event of BL is the
overexpression of c-myc. The presence of EBV within BL cell lines increases their
resistance to cytotoxic agents and leads to increased tumorigenicity. Most BL express
only EBNA1 and the EBERs. EBNA1, which is the expressed in almost all EBVpositive BL, is required for the maintenance of the viral episome within proliferating
cells. Expression of EBNA1 in EBV-negative BL and expression of dominant-negative
versions of EBNA1 in EBV-positive BL suggest that EBNA1 may also increase cell
survival and inhibit p53-dependent apoptosis (Kennedy et al. 2003; Saridakis et al.
2005). A role for EBNA1 in inducing genetic instability has also been proposed
(Gruhne et al. 2009). The expression of the EBERs has been reported to increase the
tumorigenicity of BL cell lines (Komano et al. 1999; Ruf et al. 2000, 2005).
Examining the viral genomes within BL has yielded interesting clues as to how
the virus might contribute to the survival of the tumor cells. A series of deletions
have been identified in up to fifteen percent of BL samples examined (Kelly et al.
2002, 2005). Although the exact coordinates of the deletion vary, nearly all remove
the EBNA2 coding sequences. Such deletions produce viruses are defective in
transformation and establishment of latency, suggesting that the selection for these
deletions in BL is specific for the development of cancer rather than selection for
increased fitness of the resulting viruses. Expression of RNAs capable of encoding
BHRF1 and EBNA3 proteins from the Wp promoter has been detected in the deleted
BL (Kelly et al. 2006). EBNA2-deleted episomes have increased expression of one
of the viral Bcl2 homologues, BHRF1, that was previously only observed to be
expressed during lytic replication. BHRF1, which has been found to be expressed
139
during latency as well as during lytic replication, is a potent inhibitor of apoptosis

(Kelly et al. 2009). This could be a critical inhibitor of myc-induced apoptosis
following the chromosomal translocation. Both EBNA3A and 3C have been reported
to downregulate the proapoptotic protein Bim, and deletion of EBNA3A and 3C
results in increased sensitivity to cytotoxic agents (Anderton et al. 2008). EBNA3C
also regulates MDM2/p53 and Rb function (Knight et al. 2005; Saha et al. 2009; Yi
et al. 2009). Expression or roles of EBV miRNAs in BL development have not been
established.
In summary, it is clear that the primary event of BL development is the c-myc
translocation. EBV may play a role in compensating for the translocation by inhibiting
apoptosis. A number of functions of the viral proteins which may help in inhibiting
apoptosis have been identified. EBV may also increase directly or indirectly increase
the probability of translocation. The discrete roles of these EBV proteins as well as
the interaction between EBV and other infectious agents will continue to be elucidated to determine the pathogenesis of EBV in BL.
EBV and Nasopharyngeal Carcinoma

EBV was associated with NPC shortly after BL and is the cancer that is most highly
associated with EBV. Like BL, NPC has a specific geographical distribution suggestive of specific genetic and/or environmental influences to the development of cancer.
NPC is endemic to regions of China and South-East Asia. Incidence is particularly
high in Cantonese males (Yu and Yuan 2002). Computation approaches have suggested that EBV latent proteins may be poorly recognized by the MHC haplotypes
of the affected ethnicities (Edwards et al. 2004). In addition, the environmental
cofactor of nitrosamines consumed in salted fish at an early age is thought to be an
important contributing factor to the development of cancer (Huang et al. 1978; Yu
et al. 1986). Increased IgA antibodies to the EBV viral capsid antigen (VCA) are
useful in the diagnosis of the disease (Henle and Henle 1976).
Unlike most of the other cancers associated with EBV, NPC results from latent
infection of epithelial cells rather than B-lymphocytes. Although infection of epithelial
cells by EBV was initially controversial, lytic infection of the epithelial cells,
particularly in the oral mucosa, is now assumed to be important for the amplification
of the virus in primary infection to establish a sufficient latent B-cell reservoir.
Inefficient in vitro epithelial infection models have made it difficult to understand
the factors that influence how or why latent infection is induced in epithelial cells.
Genetic and epigenetic analyses have clearly defined several events that are required
for the development of cancer. Loss or silencing of genes on chromosomes 3p, 9p,
and 14q are responsible for increased susceptibility presumably through the loss of
tumor suppressors p16IN4A, p14ARF, RASSF1A, and most recently a novel tumor
suppressor, Mirror-Image POLydactyly 1 (MIPOL1) (Lo and Huang 2002; Cheung
et al. 2009). Current models of NPC development suggest that following loss of
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these tumor suppressors EBV latently infects the epithelial cells and induces precancerous lesions. Following subsequent genetic and epigenetic events, the precancerous
lesions develop into invasive and metastatic NPC (Raab-Traub 2002).
Several EBV genes are expressed in NPC, which is the prototypical latency II
profile. EBNA1, EBERs, LMP1, and LMP2A are consistently detected in EBVpositive NPC, although the LMPs are detected less often, especially at the protein
level (Tsao et al. 2002a). LMP1 is assumed to drive the proliferation and induce
some of the transformed phenotypes of the tumor cells. Expression of LMP1 in
epithelial cells and EBV-negative NPC cell lines induces increased motility and
invasion (Tsao et al. 2002b; Shair et al. 2008). LMP1-specific therapies are being
developed and tested. Interestingly, the EBV miRNAs arising from the BART RNAs
are highly expressed in NPC, although the function of these miRNAs and their role
in NPC pathogenesis are unknown currently (Cai et al. 2006).
EBV and Hodgkins Disease

Like BL and NPC, an infectious agent was suspected as a contributor to Hodgkins
Disease (HD) and increased antibodies to EBV-specific antigens initially solidified
this connection (MacMahon 1966; Levine et al. 1971). In situ hybridization established the presence of EBV DNA and EBER RNA in the tumors as well as the fact
that the HD tumors were clonal for the EBV episome (Anagnostopoulos et al. 1989;
Wu et al. 1990; Weiss et al. 1991). This suggests that the tumor arises from the
expansion of a single transformed clone. Increased risk of HD is the only EBVassociated cancer correlated with a history of infectious mononucleosis (Gutensohn
and Cole 1980). Association between EBV and HD varies by ethnic group, sex, and
age (Armstrong et al. 1998). Generally, HD in children and older adults is associated
with EBV, while HD in young adults is not associated as often with EBV. Although
HD is only slightly increased in AIDS, the majority of them contain EBV (Uccini
et al. 1990).
HD has a latency II EBV gene expression pattern (Deacon et al. 1993). The malignant cells of HD, Hodgkin ReedSternberg cells, express LMP2A and/or the viral
oncoprotein LMP1 in the absence of EBNA2 (Weiss et al. 1987, 1989; Pallesen
et al. 1991; Murray et al. 1992). There is some correlation between the pathways
and proteins activated by LMP1 in vitro and the EBV-positive HD, suggesting that
LMP1 signaling is active in the tumor cells (Herbst et al. 1996; Durkop et al. 1999;
Murray et al. 2001; Hinz et al. 2002). However, EBV-negative HD often has similar
profiles, suggesting that the tumors may arise by alteration of the cellular environment by either cellular or viral methods (Knecht et al. 1999). In particular, NF-kB
and possibly Akt appear to be activated and critical to the growth of both EBVpositive and EBV-negative tumors (Bargou et al. 1997; Morrison et al. 2004).
Comparison of EBV-positive and -negative tumors has highlighted the importance
of deregulation of specific pathways critical to the development of HD whether by
viral or other means.
141
EBV and Lymphomas in Immunosuppression

A number of EBV-associated malignancies occur in the context immunosuppression. Lymphoproliferations that occur during immunosuppression as a result of
therapies after organ transplantation are known as posttransplant lymphoproliferative
disorders (PTLD) (Gulley et al. 1993; Capello et al. 2005; Gottschalk et al. 2005;
Taylor et al. 2005). Similar tumors are observed in inherited immunodeficiency and
in the context of AIDS (Ambinder 2001). The majority of the lymphomas in the
context of immunosuppression has gene expression of Latency III/growth program
and is analogous to cell lines formed as the result of immortalization of B-cells
in vitro by infection with EBV. PTLD will often regress following removal of immunosuppression but aggressive non-Hodgkins lymphoma, which does not resolve,
also can result.
Like latency III, generally all of the proteins of EBV are expressed in immunosuppressed lymphomas. The latent membrane proteins, LMP1, LMP2A, and LMP2B,
and EBV nuclear antigens, EBNA1, EBNA2, EBNA3A-C, and EBNA-LP, are all
expressed and thought to drive the proliferation of the tumor cells in the absence of
immune surveillance. The noncoding RNAs, EBERs and miRNAs, are also expressed,
but their role in tumor growth and development has not been determined. As the viral
proteins are hypothesized to induce the growth of these tumors, they likely would be
the most sensitive to therapies that specifically target viral proteins.
EBV and Gastric Carcinoma

Gastric carcinoma is the second most common carcinoma worldwide and 10% of the
cancers contain EBV making the new cases of EBV-gastric cancer nearly 90,000
worldwide (Takada 2000). Like HD and NPC, gene expression in gastric carcinoma
is latency II. EBNA1 is the only EBNA detected in gastric carcinoma. The EBERs and
EBV lytic protein BZLF1 is also detected in the tumors. Detection of the LMPs in
gastric carcinoma is variable and LMP2A is most often detected while LMP1 expression has also been reported (Imai et al. 1994; Ryan et al. 2009). As with BL and HD,
there may be other factors that influence the association of EBV with the tumor.
EBV and Other Cancers

EBV is also associated with other cancers. It is associated with some T-cell and
NK-cell lymphomas (Jones et al. 1988; Kikuta et al. 1988; Harabuchi et al.
1990; Pallesen et al. 1993; Nagata et al. 2001; Lan et al. 2008). EBV is also
associated with leiomyosarcoma (McClain et al. 1995). There have been reports
of EBV in breast cancer and hepatocellular carcinoma (Bonnet et al. 1999;
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Sugawara et al. 1999). However, some of these associations are controversial

and the necessary immunohistochemical and gene expression profiles have not
been rigorously demonstrated.
EBV Animal Models

Because of the clinical importance of EBV and the importance of developing EBVtargeted therapies, a number of EBV animal models have been developed. Nonhuman
primates have yielded limited effectiveness. Old-World monkeys are not infected by
EBV possibly due to presence of related cross-reacting antibodies from endogenous
herpesviruses as detailed above. Several species of New-World monkeys can be
infected with EBV; however, they cannot be infected via the natural, oropharyngeal
route and fail to develop most EBV-related pathologies (Niedobitek et al. 1994).
Although EBV does not infect mouse cells, a number of mouse models have been
developed that mimic aspects of EBV disease. Tumor xenografts can be grown in
nude or SCID mice. SCID mice injected with lymphocytes from seropositive donors
can develop lymphoproliferative diseases similar to PTLD (Mosier et al. 1988).
A number of knock-in mouse models have been developed in which the expression
of latent viral proteins is targeted to specific cell types, including LMP1 (epithelial
targeted (Wilson et al. 1990; Curran et al. 2001), LMP1 (B-cell targeted (Kulwichit
et al. 1998; Uchida et al. 1999), LMP2A (Caldwell et al. 2000), and EBNA1 (Kang
et al. 2005). Recently, humanized mice in which immunocompromised or immunodeficient mice, typically nude, SCID, or NOG mice, are populated with human
immune stem cells and then used to study human adaptive and innate immune
responses to EBV infection (Melkus et al. 2006). Each model system has offered
unique insights into EBV biology and/or the pathogenesis of EBV-related malignancies but also fails to fully explain the complex interconnection between EBV
and its host leading cancer development.
Understanding the contribution of EBV to the development of human cancer
helps scientists understand the nature of cancer and the pathogenesis of the disease.
It also offers insights into the viral life cycle and how the virus uses the host cell to
its advantage. The presence of EBV within the tumor also offers a unique pharmacological target in which the virus and thus the tumor cells may be subject of a
therapy that does not affect normal cells and tissues.
KSHV or Human Herpesvirus-8 (HHV-8) and Kaposis Sarcoma

KS was recognized as early as 1872 by the Hungarian dermatologist Moritz
Kaposi as a rare multiple idiopathic pigmented hemangiosarcoma seen as purple blotches on the skin of extremities with some visceral involvement. This form
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of KS known as classical KS occurs most commonly in Eastern Europe and North

America among older men of Italian or Jewish ancestry. However, in the 1950s
and 1960s, another aggressive form known as endemic KS was recognized among
younger individuals in central Africa and KS was reported to represent 9% of all
neoplasms in Uganda (Tornesello et al. 2010 ; Buonaguro et al. 2003). Clinically,
the African aggressive forms have been described as nodular, florid, infiltrative
and lymphoadenopathic. The lymphoadenopathic form is usually seen in African
children and young adults. The disease manifests mainly with the involvement of
lymph nodes and is rapidly fatal. During 1970s, KS was also detected in the USA
and in Europe before the AIDS epidemic era in patients receiving immunosuppressive therapy for renal transplantation and other medical conditions (Tornesello
et al. 2010; Buonaguro et al. 2003). In transplant recipients, KS is reported to be
the second most common tumor after lymphoma to arise and occurs more often
(10 vs. 3% of all transplant-associated neoplasms) in patients who receive
cyclosporin as part of their immunosuppressive regimen. Several studies support
the idea that KS is a disorder of growth factor production (Boshoff et al. 1995;
Boshoff and Weiss 1997, 1998, 2002). Even though HIV protein-tat involvement
in KS is hypothesized to induce cytokines, it was recognized that this may not be
the sole inciting event in KS etiology, since KS also occurs in the absence of HIV
infection.
Although KS is also manifested in a small number of HIV-positive transfusion
patients and intravenous drug users, it is very rare in hemophiliacs, which indicated
that the KS agent is cell-associated or that is inactivated during factor VIII purification.
AIDS-KS is almost exclusively confined to homosexual men with AIDS, bisexual
men and their contacts. Gay and bisexual men are about 20 times more likely than
other HIV-positive patients to develop KS. This has suggested that a sexually transmitted cofactor, inefficiently transmitted by blood or blood products may be required
for KS development (Chang and Moore 1996; Chang et al. 1996; Gaidano et al. 1996;
Moore et al. 1996a, b). The occurrence of KS lesions in homosexual men without
HIV infection supports this hypothesis.
Using representational difference analysis (RDA), Chang et al. (1994) reported
the identification of a novel herpesvirus DNA sequences in AIDS associated KS.
This unique DNA is closely related to HVS as well as to EBV. Currently, this new
herpesvirus is called KSHV or HHV-8. KSHV DNA was present in more than 95%
of AIDS-KS tissues, >15% of the non-KS tissue samples from AIDS patients
(Chang et al. 1994). In KS patients, KSHV DNA was detected only in the skin near
the KS tissue and in some adjoining lymph nodes, but not in other tissues. KSHV
DNA was also detected in all the non-AIDS classical KS tissues from southern
Europe and Africa (Chang and Moore 1996; Chang et al. 1996; Gaidano et al. 1996;
Moore et al. 1996a, b). In addition, PBMC of AIDS patients obtained prior to the
development of KS lesions was positive for KSHV DNA and was negative in
matched HIV positive individuals without KS (Chang and Moore 1996; Chang et al.
1996; Gaidano et al. 1996; Moore et al. 1996a, b). These studies strongly suggested
an association of KSHV with KS.
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Cells of KS Lesions
KS lesions are characterized by proliferating spindle shaped endothelial cells,
neovascular structures, inflammatory cells, and an abundance of inflammatory
cytokines (ICs), growth factors (GFs), angiogenic factors (AFs), and invasive factors.
Whether KS is a true malignancy or a well-characterized consequence of infection
with unique hyperplasia is still a debatable subject. The discrepancy between KS
and other tumors is more evident in the histopathology of the tumor. Most human
tumors are derived from the clonal outgrowth of a single cell type. By contrast, KS
lesions show a variety of cell types. Advanced lesions include a predominance of
spindle-shaped cells, thought to be derived from cells in the endothelial or mesenchymal lineage (Wang et al. 2004). All KS lesions display a proliferation of
aberrant, slit-like neovascular spaces as well as variable quantities of infiltrating
inflammatory cells, including plasma cells, T cells, and monocyte/macrophages
(Boshoff and Weiss 1998; Blauvelt 2001; Wang et al. 2004). KS tumors lack many
of the features of classical tumor cells as they are often oligo- or polyclonal, lack a
fully transformed phenotype in vitro, and do not form tumors upon implantation in
nude mice (Boshoff and Weiss 1998).
In vitro cultures of KS cells generally produce IL-6, granulocyte-macrophage
colony-stimulating factor (GMCF), vascular endothelial growth factor (VEGF),
platelet-derived growth factor (PGF), and basic fibroblast growth factor (BFGF)
(Samaniego et al. 1998). The cells cultured from KS tumors almost always have normal
diploid karyotype, grow for a limited number of passages in culture, and contain a
mixture of different cell types. The dominant cell types are fibroblasts and endothelial
cells, both of which have a spindle shape (Samaniego et al. 1998). These spindle cells
are dependent upon exogenous growth factors and are not fully transformed by most
laboratory criteria (Samaniego et al. 1998). They generally fail to grow in soft agar
and do not induce tumors upon transplantation in immunodeficient mice.
KSHV was found in peripheral blood CD19 positive B cells (Sin et al. 2010;
Dittmer et al. 1999). Using in situ PCR hybridization, KSHV DNA was demonstrated
in the flat endothelial cells lining vascular spaces of KS lesions as well as in typical
KS spindle cells of all forms of KS (Chang et al. 1994). KSHV DNA does not
persist in the cultures of KS tissues and the KS cell lines are not positive for KSHV
DNA and the reason for this is not clear.
KSHV encodes ~86 ORFs of which at least 22 are potentially immunomodulatory and antiapoptotic (Boshoff et al. 1995) and the ORFs are named based on their
homology to HVS. In KS lesion endothelial cells, KSHV is in a latent form with
about 1020 copies of the viral episome per cell and lytic replication is observed in
a low percentage of infiltrating inflammatory monocytes. Only a small proportion
(<10%) of spindle-shaped endothelial cells are positive for KSHV in early KS
lesions. In advanced KS lesions, KSHV is latently expressed in >90% of the tumor
(spindle) cells, indicating that paracrine mechanisms are probably important in the
initiation and progression of KS. In nodular (advanced) lesions, all the spindle cells
contain latent KSHV (Boshoff et al. 1995), suggesting that KSHV latent proteins
145
provide a growth advantage to the infected cells. KS spindle cells are referred to as
the driving force of KS histogenesis (Ensoli et al. 1992).
KSHV and Primary Effusion Lymphoma

PEL, a rare type of B-cell lymphoma detected in HIV+ and HIV patients, is also
called as body-cavity-based lymphoma (BCBL). PEL are presented as malignant
lymphomatous effusions in the pleural, pericardial, or peritoneal cavities, usually
without significant tumor mass or lymphadenopathy/lymph nodal involvement and
these lymphomas occur predominantly in HIV-positive individuals with immunosuppression (Boshoff et al. 1998). KSHV viral DNA was first detected in patients
during a survey of 193 lymphomas with and with out AIDS. KSHV sequences were
detected in eight non-Hodgkins lymphomas (NHLs) in HIV infected individuals,
all of which were EBV positive (AIDS-BCBLs) (Cesarman et al. 1995). These lymphomas appear to represent a distinct group of B cell NHLs, without c-myc rearrangement and with striking characteristics such as absence of most lineage-associated
antigens that are distinct from the vast majority of AIDS-related lymphomas
(Karcher and Alkan 1997). Only two of the eight patients with BCBLs had KS
indicating that KSHV-associated BCBLs can occur independently of KS in patients
with AIDS. BCBL cell lines containing only KSHV were isolated ,which suggests
that KSHV may be involved in this NHL independently of EBV. Unlike KS, PEL
has characteristics of a typical malignancy, with the B cell being of clonal origin.
Circular KSHV episome is seen in every cell in the tumor. These cells grow well in
culture and have been an ideal system to study KSHV replication in vitro.
PEL has poor prognosis and median survival of approximately six months (Carbone
and Gloghini 2007), and there are no specific treatments targeting PEL. PEL is characterized by the expression of KSHV latency proteins LANA-1 (ORF73), vCyclin
(ORF72), v-FLIP (ORF71), Kaposins (ORFK12), LANA-2, K1 and vIL-6 as well as
>18 miRNAs with 15% of cells spontaneously entering lytic cycle (Chandriani and
Ganem 2010 ; Jenner et al. 2003; Klein et al. 2003; Carbone et al. 2009).
KSHV and Multicentric Castlemans Disease

Multicentric Castlemans Disease (MCD) is an atypical lymphoproliferative disorder
(ALPD) defined using clinical and pathologic criteria. MCDs association with KS
was observed during the clinical course of HIV infection. Soulier et al. (1995)
reported the presence of KSHV-like DNA sequence in MCD. This is polyclonal
involving multiple lymphoid organs. KSHV-associated MCDs can occur independently of HIV infection and KS. KSHV-associated MCD is the highly indolent
systemic variant of Castlemans disease (CD) and is also designated as plasmablastic
variant of MCD (Dupin et al. 1999). It is a lymphoproliferation accompanied by
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D. Everly et al.
weight loss and fever and is more commonly diagnosed in HIV-infected patients
and has very poor prognosis (Chadburn et al. 1997; Dupin et al. 1999). Unlike
KSHV-positive PEL cells, the plasmablasts in MCD are KSHV-positive and EBV
negative.
KSHV Biology
In vivo KSHV infects variety of target cells including human B cells (Chandran
2010; Mesri et al. 1996; Akula et al. 2001), macrophages, endothelial cells, keratinocytes and epithelial cells. Several B-cell lines have been established from PEL
tumors bearing either KSHV alone (BCBL-1 and BC-3) or KSHV and EBV (BC-1,
JSC-1, HBL-6). KSHV has also been shown to infect a variety of cell types in vitro
including human B, endothelial, epithelial, and fibroblast cells as well as variety of
animal cells such as owl money kidney cells, baby hamster kidney fibroblast cells,
Chinese hamster ovary cells (CHO) cells and mouse fibroblasts. Infection of human
primary B cells by EBV results in the establishment of latent infection, transformation, and B-lymphoblastoid cell lines. By contrast, primary nonstimulated B cells are
poorly infected with KSHV and infection does not lead to immortalization. Instead,
a lytic replication is reported in activated B cells. In vitro KSHV infection of adherent
target cells does not result in a productive lytic cycle (Naranatt et al. 2004). Instead,
KSHV infection of human endothelial cells (human microvascular dermal
(HMVEC-d), human umbilical vein endothelial cells (HUVEC), human foreskin
fibroblasts (HFF), human endothelial cells immortalized by telomerase (TIME), and
human embryonic kidney epithelial cells (HEK 293) results in the expression of
latency-associated genes and thus providing a reasonable model for studying latency
in vitro (Naranatt et al. 2004). Lytic replication could be induced from these cells by
chemicals or by the lytic switch KSHV-ORF 50 (RTA- replication and transcription
activator). In vitro, the switch from latency to lytic can be achieved by treating
latently infected cells with phorbolesters or sodium butyrate or by constitutively
expressing RTA. The stimuli for in vivo lytic activation are not well understood and
there are evidences for cytokine mediated lytic cycle activation.
KSHV Latent Proteins and Oncogenesis

Like EBV, KSHVs latency program is directly linked to oncogenesis due to its
antiapoptotic role and by increasing cell survival (Table 7.2). The lytic cycle was
presumed not to contribute to oncogenesis as the cells that enter lytic cycle die.
One of the major lytic gene vGPCR coded by ORF74 was demonstrated to have
oncogenic properties (Table 7.2). However, in situ hybridization studies have shown
only 13% of latently infected spindle cells express lytic genes, thus supporting the
importance of latent gene expression in tumor development.
Latency
Latency
Latency
and Lytic
replication
ORF71
(vFLIP/K13)
ORF72
(v-Cyclin)
ORFK12
(Kaposin)
Reported to be present in most spindle cells of all

stages of KS and in PEL KSHV-transforming gene
Multiple transcripts of various lengths being produced from
K12 locus resulting in Kaposin A, B, and C forms
Structurally similar to cellular D-type cyclins and forms

an active kinase complex with cellular CDK6 kinase
Stimulate the expansion of KSHV-infected cells
Blocks KSHV lytic replication
Table 7.2 KSHV proteins with potential involvement in oncogenesis

Gene
Expression
Proposed functions in KSHV life cycle
ORF73
Latency
Tethering viral episome to the host chromosome
(LANA-1)
KSHV episome replication/segregation
Associates with a host cell DNA polymerase and RNA
helicase
(continued)

Inactivates pRb and p53 proteins
Interacts with GSK3-, promotes nuclear -catenin accumulation
Activates aberrant c-Myc stabilization
Cooperates with H-Ras to transform fibroblasts
Complexes with P53 in PEL cells
Stabilizes intracellular activated Notch protein
Inhibits TGF- signaling
Associates with a number of cell cycle proteins and DNA
methyltransferases (Dnmts)
Transcriptional coactivator of c-Jun
Drives cell transformation in vitro
Can inhibit the extrinsic apoptosis pathway by preventing the activation of
caspases
Involved in constitutive NF-kB activation through the IkB kinase (IKK)
complex
Contributes to PEL cell survival
Extends lifespan of spindle cells
Provides the inflammatory phenotype to KS lesions.
Cooperates with Myc to promote lymphoma
Suppresses autophagy
Suppresses CXCR4 by upregulating miR-146a
Regulates the transcription of S-phase genes
Stimulates quiescent fibroblast cells to enter S-phase
Can phosphorylate CDK2 substrates such as ORC1, CDC6, p27Kip1,
histone H1, Bcl-2 and p53
Activates p38/MK2 pathway and to stabilize various
cytokine mRNA containing AREs
Kaposin A can lead to cell transformation in vitro
Latency
v-BCL2
v-GPCR
(ORF74)
Lytic
ORF-K2 (v-IL6) Lytic/latent
Lytic
Latency
K1
miRNAs
Table 7.2 (continued)

Gene
Expression
Expressed in KS lesions and in cell lines derived

from PEL
Plays an antiapoptotic role in virus infected cells
Expressed in only a few cells of KS lesions, but in a
greater number of cells in PEL and MCD lesions
Early lytic gene vIL-6 play important roles in KSHV
replication and pathogenesis
Characterized as viral oncogene
Likely to play a critical role in both the initiation and
promotion of KS tumor development
Can activate the promoter of the lytic cycle switch
ORF50
18 mature miRNAs have been detected so far in

latently KSHV-infected cells
miR-K3 can suppress both viral lytic replication and
gene expression
miR-132 regulates antiviral innate immunity
Contribute to viral-induced reprogramming by
silencing the cellular transcription factor MAF
Deletion of a 14 miRNA cluster from the KSHV
genome significantly enhances viral lytic
replication as a result of reduced NF-B activity
K1 is expressed at very low levels in KSHV-infected
cell lines
Induced during lytic replication
Proposed functions in KSHV life cycle
Powerful signaling capacity

Possesses a cytosolic ITAM motif and mimics signaling through the B-cell
receptor (BCR) Activates tyrosine kinases Lyn and Syk, as well as
PLC-g2 and vav
Akt activation
Angiogenesis and inflammation
Important regulator of cell death
Interacts with cell survival proteins
Enhances cell transformation
Mediates signaling through the gp130 signal transducer
Can stimulate the growth of KSHV-infected lymphoma cells
Promotes hematopoiesis
Acts as an angiogenic factor through the induction of VEGF
Creates tumor friendly milieu through paracrine mechanisms
Secretion of chemokines and growth factors
Activates mitogen- and stress-activated kinases, and induces transcription via
multiple transcription factors including AP-1, CREB, NFAT and NFB
Causes cellular transformation in vitro and leads to KS-like tumors in
transgenic mouse models
Mediates angiogenesis, and inflammation
Inhibit p21 and attenuates cell cycle arrest

miR-K12-11 upregulates xCT expression and increases cell permissiveness
for KSHV infection and protects infected cells from death induced by
reactive nitrogen species (RNS)
Induce IL-6 and IL-10 secretion by macrophages and monocytes
149
LANA-1 (ORF73). KSHV LANA-1 expressed during latency in KS, MCD, and PEL
cells is a large nuclear phosphoprotein with no known mammalian homolog (Rainbow
et al. 1997). It binds the KSHV terminal-repeat (TR) regions and tethers the viral
episome to the host mitotic chromosome through interactions with cellular chromosome-associated proteins (Cotter and Robertson 1999). LANA-1 has similarities to
EBNA-1, which include the presence of central repeat region of variable length
composed of acidic amino acids, essential roles in viral episome maintenance and
segregation. LANA-1 has been shown to perform multiple functions which contribute
to KSHV-associated neoplasia-like inactivation of pRb and p53 proteins (Fujimuro
et al. 2003), upregulation of -catenin, and stabilization of its expression by sequestering
its inhibitor, glycogen synthase kinase 3-. LANA has been shown to physically
interact with cellular proteins, including p53, pRB, RING3, histone H1, ATF4/
CREB2, and members of the mSin3 corepressor complex. LANA interacts with
GSK3-, leading to the accumulation of cytoplasmatic and nuclear -catenin
(Fujimuro et al. 2003), activation of the Tcf/Lef transcription factors and aberrant
c-Myc stabilization (Bubman et al. 2007). LANA-1 can cooperate with H-Ras to
transform fibroblasts (Radkov et al. 2000). LANA-1 is also referred to as the guardian
of KSHV latency, as it modulates several cell-cycle pathways of infected cells.
vFLIP (K13/ORF71). v-FLIP is a homologue of the cellular protein FLIP and the
vFLIP gene is expressed in late stage KS lesions and PEL cells from a polycistronic
mRNA encompassing the latency locus containing an internal ribosome entry site
(IRES) located within the v-cyclin ORF (Thome et al. 1997; Boshoff et al. 1998;
Talbot et al. 1999). KSHV FLIP protein is an important component of KSHV pathogenesis. vFLIP protein has the ability to drive cell transformation in vitro (Sun et al.
2003) and can inhibit the extrinsic apoptosis pathway by preventing the activation of
caspases (Djerbi et al. 1999). Its ectopic expression can lead to constitutive NF-kB
activation through the IkB kinase (IKK) complex (Chaudhary et al. 1999; Keller et al.
2000) and can contribute to survival of PEL cells (Godfrey et al. 2005; Guasparri et al.
2006). v-FLIP contains death-effector domains which interact with the adapter protein
FADD, inhibiting the recruitment and subsequent activation of the protease FLICE by
the CD95 (Fas) death receptor and protects cells from Fas, TNFR-1, and TNF-related
apoptosis-inducing ligand receptor (TRIAL-R)-mediated apoptosis. v-FLIP expression in B-cells leads to development of tumors in mice by preventing death receptorinduced apoptosis triggered by CTL immunosurveillance. Expression of v-FLIP in
spindle cells has been shown to extend their lifespan (Chugh et al. 2005) and provides
the inflammatory phenotype to KS lesions. Finally, activation of NF-kB by v-FLIP
has been shown to oppose lytic reactivation that thereby stabilizes KSHV latency.
vCyclin (ORF72). KSHV-encoded v-cyclin is a human cyclin D homolog (closely
related to cyclin D2). v-cyclin forms a ternary complex with CDK6 and INK4.
Distinct from cellular cyclins, v-cyclin protein has longer half-life. It activates the
cyclin-dependent kinases (CDKs) CDK4 and CDK6, thus phosphorylates pRb,
releases the repression on E2F and that subsequently upregulates the transcription of
S-phase genes, whose products modulate the progression of cells from G1- to S-phase
(Kang and Lieberman 2009). Furthermore, unlike cellular D cyclin/CDK6 complexes, KSHV-cyclin/CDK6 activity is resistant to inhibition by the CDK inhibitors
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D. Everly et al.
(CKI) p16, p21, and p27 (Swanton et al. 1997). v-Cyclin/CDK6 expression can also
phosphorylate histone H1, the CDK2 substrates (p27KIP1, Id-2, cdc25a, E2F-4),
replication proteins (orc1 and cdc6), and thus allows cell to overcome cell cycle
arrest. Ectopic expression of v-cyclin in quiescent fibroblasts prevents CDK inhibitors
(CKIs; p16INK4a, p21CIP1 and p27KIP1)-imposed G1 arrest and stimulates entry
into S-phase (Sharp and Boshoff 2000). Interestingly, v-Cyclin has also been reported
to trigger apoptosis in cells with elevated levels of CDK6 probably by phosphorylating
and inactivating the cellular antiapoptotic factor Bcl-2.
Kaposin. Kaposin or K12-containing mRNAs have been reported in most spindle
cells of all stages of KS and in PEL cells during latency and lytic replication (Staskus
et al. 1997; Sturzl et al. 1997). Translational regulation of the K12 transcript is very
complex and yields a minimum of three protein species (Kaposin A, Kaposin B, and
Kaposin C), of which Kaposin B is the most abundant in BCBL-1 cell lines and is
encoded by the sequences upstream of K12, but not by K12 itself. Kaposin B is a
scaffolding protein and has been shown to perform immunomodulatory functions
such as increasing the expression of certain cytokines by stabilizing their mRNAs
(McCormick and Ganem 2005) via p38 MAPK pathway and inhibiting the decay of
AU-rich elements (AREs) in 39-untranslated regions of mRNAs. Recently, kaposin
locus was shown to code for KSHV micro-RNAs (Cai et al. 2005; Pfeffer et al. 2005;
Samols et al. 2005, 2007), which may be involved in the regulation of viral and
cellular immune responses. Kaposin C is a chimera of direct-repeat sequences DR1,
DR2, and K12. Kaposin A is the predicted product of K12, and has been reported to
be involved in cell transformation (Muralidhar et al. 1998, 2000; Tomkowicz et al.
2005). Kaposin A is a 60-aa transmembrane protein whose overexpression in fibroblasts can lead to their transformation in vitro, suggesting that the molecule can
stimulate signaling pathways linked to growth deregulation (Muralidhar et al. 1998).
The mechanism of Kaposin-mediated transformation is not clear but has been linked
to its ability to bind cytohesin-1 (Moss and Vaughan 2002), an exchange factor for
ADP ribosylation factor (ARF) family GTPases, key regulators of vesicular trafficking
and of the dynamics of the actin cytoskeleton (Moss and Vaughan 2002).
KSHV miRNA. miRNAs are small noncoding RNAs of ~22 nucleotides expressed
by virtually all multicellular organisms and are known to regulate gene expression
either at posttranscriptional level by binding to 3UTRs of target and/or repression
of their translation. miRNAs have been shown to regulate cellular differentiation,
proliferation, apoptosis, metabolism, and immune responses. The KSHV miRNAs
are transcribed during latency and their expression has been confirmed in KS but
their functions are not yet fully characterized. The kaposin transcription unit of KSHV
encodes 12 pre-miRNAs (Cai et al. 2005; Pfeffer et al. 2005; Samols et al. 2005;
Ziegelbauer et al. 2006, 2009; Ganem and Ziegelbauer 2008) and these pre-miRNAs
can engender 18 mature miRNAs (Umbach and Cullen 2010). Some of these
miRNAs appear to function as modulators of the latent-lytic switch (Bellare and
Ganem 2009; Ziegelbauer et al. 2009).
KSHV miRNA (miRK12-5) has been identified to be capable of suppressing
ORF50 mRNA probably by decreasing histone H3 K9 methylation or increasing
151
histone H3 acetylation, and a striking loss of DNA methylation throughout the viral
and cellular genome (Ablashi et al. 2002). KSHV miRNA cluster as a genetic element
activates CpG methylation through an indirect mechanism involving modulation of
DNA methyltransferase (DNMT) gene transcription regulation that helps maintain
the latent state of the viral chromosome (Ablashi et al. 2002). Recent study by Lei
et al. (2010) showed that the deletion of a 14 miRNA cluster from the KSHV genome
significantly enhanced KSHV lytic replication as a result of reduced NF-kB activity.
Expression of miR-K1 has been indicated to efficiently rescue NF-kB activity and
inhibit viral lytic replication, whereas inhibition of miR-K1 in KSHV-infected PEL
cells displayed the opposite effect.
Recent reports identified few potential KSHV miRNA targets as thrombospondin
(THBS; a major regulator of cell adhesion, migration, and angiogenesis), BRCA1associated C-terminal helicase (BACH1; DNA repair protein supporting BRCA1
damage response), leucine zipper transcription factor MAF (musculoaponeurotic
fibrosarcoma oncogene homolog), and BCLAF1 (Gottwein and Cullen 2010;
Samols et al. 2007; Skalsky et al. 2007; Ziegelbauer et al. 2009), which primarily
control viral reactivation, apoptosis, cell survival, immune responses (Umbach and
Cullen 2010), cellular differentiation, and transcriptional reprogramming of host
cells. The KSHV miR-K12-11 seed region shares complete homology to that of
hsa-miR-155, a multitasking immune-specific miRNA controlling B cell maturation,
lymphomagenesis, transformation and cancer (Qin et al. 2010; Gottwein et al. 2007;
Samols et al. 2007; Zhao et al. 2009). High levels of the precursor miRNA mir-24
recently emerged as a biomarker in patient derived KS samples (OHara et al. 2009).
Cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key
inducer of cell cycle arrest has been shown to be direct target for KSHV miR-K1.
Ectopically, expression of KSHV miR-K1 specifically inhibits the expression of
endogenous p21 in KSHV-negative cells and attenuates the cell cycle arrest that
normally occurs upon p53 activation suggesting that KSHV miR-K1 is likely to
contribute to the oncogenic potential of KSHV.
Cytokines and Angiogenic Factors in KS

A marked characteristic of KS is the infiltration of inflammatory cells accompanied
by inflammatory cytokines, angiogenic factors, growth factors, and chemokines,
making it an angioproliferative lesion. The importance of some of these cytokines
and angiogenic factor in establishment of latency and ultimately in tumor progression
is discussed here (Fig. 7.2).
KSHV induced proinflammatory cytokines. A variety of ICs are produced in lesions
from all forms of KS. These include, IL-1b, IL-6, IL-8, Cox-2, TNF, IFNg. IL-1
produced and released by endothelial cells in a KS lesion induces autocrine growth
of the endothelial cells. Studies have also shown that their growth effects are mediated by induction of bFGF production which appears to be the final mediator of KS
cell growth (Ensoli et al. 1994; Samaniego et al. 1995, 1997; Ensoli and Sturzl 1998;
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Host and viral gene regulation during in vivo endothelial cell infection by KSHV
Late phase of signal

pathway activation
Secretion of cytokines
and growth factors
PI3K
Early phase of signal

pathway activation
Activation of
NF-kB, ERK1/2
p38
MAPK
AKT
NF-kB, ERK1/2
Stabilization of
Cytokine mRNA
Cell
Proliferation /anti
apoptosis
Induction and regulation
of host genes
Concurrent
persistent latent and
transient lytic gene
expression
Tube
formation
K12
Anti-inflammatory
Chemokines
cytokines
Pro-inflammatory
Growth and
cytokines
angiogenicfactors
Maintenance of latency
LANA, vFLIP, vCyclin, K12, mRNA
Autocrine/Paracrine action
Cell
migration
Dissolution of
basement membrane
Plasmin generation
Fig. 7.2 Model depicting the early and late phases of KSHV infection of HMVEC-d cells, transcription factor regulation, establishment and maintenance of infection, and cytokine secretion.
Virus binding and entry lead to signal pathway induction, such as FAK, Src, PI 3-K, AKT, PKC-,
MAPK-ERK1/2, and NF-B signal molecules which coincides with viral-DNA entry into the
infected-cell nuclei, concurrent transient expression of limited viral lytic genes, and persistent
latent gene expression. Expression of several host genes including cytokines, growth factors, transcription factors etc., is initiated by AP-1, ERK1/2 and NF-B. Released host factors act in
autocrine and paracrine fashions on the infected, as well as neighboring, cells. The autocrine action
of these factors, along with viral gene expression, probably contributes to the late phase of signal
pathway activation including sustained NF-B activation and phosphorylation of p38 MAPK,
ERK1/2, and AKT required for the maintenance of latency
Faris et al. 1998). IL-1b increases the expression of adhesion factors on endothelial
cells to enable transmigration of leukocytes. IL-1b dependent NF-kB activation
mediates PGE2 release via the expression of COX2 and PGE2 synthase (Catley
et al. 2003).
KSHV mediated COX2 expression is critical for PGE2 release, which in turn is
responsible for the maintenance of latency (George Paul et al. 2010; Sharma-Walia
et al. 2010), IL-1b could be a feed back loop factor responsible for COX-2 induction
via NF-kB. Latency protein vFLIP is known to induce COX2 expression (Matta
et al. 2007) and recent reports have shown KSHV induced COX2 to be a key factor
in latency inflammation, angiogenesis and cell survival (Sharma-Walia et al. 2010).
IL-6 is an autocrine growth factor known to be over expressed in KS tumors, PEL,
MCD (Miles et al. 1990; Yang et al. 1994) and is implicated in the growth and
153
survival of PEL cells (Asou et al. 1998). IL-6 is known to be regulated both by AP-1
and NF-kB (An et al. 2003; Xie et al. 2005). LANA-1 and vFLIP are known to
induce IL-6 secretion via the activation of AP-1 or NF-kB (An et al. 2003, 2004).
IL-8 is another chemokine expressed in KS lesions, by cultures endothelial cells
and by IC activated endothelial cells. IL-8 is a chemoattractant for neutrophils and
stimulates angiogenesis and tumor growth (Koch et al. 1992; Sparmann and BarSagi 2004). In addition, IL-8 has prominent role in endothelial cell migration, it
might also contribute to the angiogenesis found in the KS lesion. Recent studies
have shown that IL-8 plays a pivotal role in pathogenesis of KS and vFLIP mediates
this IL-8 production (Sun et al. 2006; Wilson et al. 2008).
IFNg mediates several immunomodulatory functions including recruitment of
leukocytes to the site of infection, increased inflammation and the regulation of
Th-2 response. IFNg mediates an antiviral activity by decreasing the NF-kB activation
in CMV infected cells (Sedger et al. 1999), whereas KSHV appears to induce IFNg
for its benefit. The administration of IFN and IL-2 has resulted in KS progression or
onset (Monini et al. 1999). IFNg induces the endothelial cells to acquire the same
feature of KS cells, which includes the KS spindle morphology and the specific cell
marker expression (Fiorelli et al. 1998).
KSHV induced growth factors. Leptin is circulating pleotropic hormone involved in
the regulatory process of immunity, inflammation hematopoiesis and angiogenesis.
Administration of leptin in vivo and in vitro leads to the stimulation of proliferation
and angiogenesis of endothelial cells. Leptin is known to stimulate VEGF secretion
in endothelial cells (Misztal-Dethloff et al. 2004; Naranatt et al. 2004). Leptininduced IkBa degradation in astrocytes potentiates the release of IFNg, TNFa and
IL-1b (Raso et al. 2002). It is also known to stimulate the release of IL-6, TNFa,
and prostaglandins via NF-kB and ERK1/2 pathway. Hence, it is evident that one
cytokine induced by KSHV could initiate the secretion of various other cytokine and
growth factors, leading to the activation of signaling pathway responsible for the
establishment of infection and tumor progression.
PDGF is one of the numerous growth factors that regulate cell proliferation and
survival. PDGF plays a role in embryonic development, cell proliferation, cell
migration and angiogenesis. PDGF-b is a potent paracrine-acting mitogen for cultured
endothelial spindle cells that is expressed in vivo by subpopulation of cells that are
intermingled with the spindle cells. Endothelial cells express PDGF b-receptor
suggesting that PDGF-b may activate the proliferation of KS spindle cells by paracrine mechanism. PDGF has also been reported as an autocrine growth factor in KS
mRNA for angiogenic factor receptors such as VEGF receptor (VEGFR 1 and 2),
the angiopoietin receptors Tie1 and Tie2, and the PDGFR b were reported to be
upregulated by vGPCR induction (Jensen et al. 2005).
GMCSF is a pleotropic cytokine that induces the differentiation and proliferation
of granulocyte and macrophage precursor cells. GMCSF is produced by spindle
endothelial cells and by inflammatory cells in a KS lesion. GMCSF is known to influence the proliferation and migration of endothelial cells and enhance the production
of IL-8 and MIP-1a. GMCSF can be also stimulated via NF-kB pathway by IGF and
PDGF, which were also upregulated upon KSHV infection. GMCSF was thought to
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contribute to differentiation of monocytes into macrophages and dendritic cells that

are found in KS lesions (Clark and Kamen 1987). Hence, a controlled interplay is seen
between these cytokines and is probably regulated by KSHV for its own benefit.
KSHV induced angiogenic factors. Angiogenesis, which is the development of new
blood vessels from preexisting vasculature, is fundamental to tumor growth. Such
neovascularization may be stimulated by factors released from tumor cells, tumorassociated inflammatory cells, or the extra cellular matrix (Loffek et al. 2006). These
factors include VEGF, angiogenin, basic fibroblast growth factor (bFGF) family
members, TGF-b, and E-cadherin. Among angiogenic factors, VEGF-A is the bestcharacterized positive regulator, with its distinct specificity for vascular endothelial
cells. VEGF-A is a proangiogenic molecule and has been reported to play a crucial
role in the development of KS and PEL, and may potentially be involved in multicentric Castlemans disease. VEGF-A stimulates endothelial cell proliferation,
migration, differentiation, tube formation, increased vascular permeability, and maintenance of vascular integrity. VEGF-A has been identified in KS lesions, and VEGF-C
has gained attention because of the presence of its receptor VEGFR-3 in KS lesions
and its ability to induce lymphangiogenesis. Coexpression of VEGF-A and bFGF has
been shown in AIDS-KS and classic KS lesions, and the production of these factors
is believed to be induced synergistically by inflammatory cytokines.
Angiogenin can affect vascular endothelium directly and can facilitate the role of
other angiogenic growth factors, which are elevated significantly during disease
progression in patients with carcinoma. Angiogenin a multifunctional protein was
observed to be upregulated in KS lesions (Sadagopan et al. 2009) and PEL tissues.
Angiogenin translocates into the nucleus in endothelial cells and helps in rRNA
transcription. The increased secretion of angiogenin could regulate a KS tumor,
which is predominated by endothelial cells. Angiogenin was found to be upregulated
only in KSHV-associated lymphomas and not in EBV-associated lymphomas signifying its role in KSHV mediated pathogenesis.
Angiopoietin-2 (ang-2) mRNA was observed in KS lesions by insitu hybridization, and its levels are upregulated in KS as determined by gene expression
microarray analysis (Wang et al. 2004; Bureau et al. 2006). Ang2 levels are
increased in the plasma of individuals with KS, its expression is known to increase
with increasing number of lesion and was observed to reduced upon antiretroviral
therapy when KS resolves.
SDF-1 (Stromal derived factor-1) is a chemokine produced by stromal cells as
well as a variety of cells. SDF-1 promotes hematopoietic cell movement and cancer
cell metastasis. It acts as a critical regulator of cell recruitment from the blood
stream to specific tissues by promoting transendothelial migration through
chemokine gradients across the endothelium. VEGF is known to promote constitutive
SDF-1 expression. Once present on the surface of endothelial cell, SDF-1 supports
the lymphocyte arrest and promotes the recruitment of KSHV infected cells to the
skin (Yao et al. 2003). Leptin is known to induce VEGF and VEGF induces SDF-1
secretion. Hence, there exists a loop between these cytokines that is magnificently
modulated by KSHV probably for its advantage.
155
Cell cultures composed of characteristic spindle-shaped tumor cells have been

established from KS lesion explants by the addition of cytokines such as TNFa,
TNFb, IFNg, IL-1, IL-6, GMCSF, and oncostatin M, highlighting the role of these
paracrine factors in KS lesion cell survival. It is believed that KSHV tumorigenesis
and disease progression are predominantly driven by both paracrine and autocrine
mechanisms, where KSHV infection could induce an angiogenic growth factor and
MMPs-rich microenvironment and a strong cytokine network. These events via
their synergistic actions and communications could support continued proliferation
and migration of KSHV latently infected cells (Sharma-Walia et al. 2010; Dupin
et al. 1999; Qian et al. 2007). There seems to be a tight network existing between
several cytokines during KSHV pathogenesis, which is orchestrated by the latency
proteins including LANA, vFLIP, and Kaposin.
KSHV Animal Models

The experimental barrier to working with KSHV has been the lack of suitable animal
model. KSHV goes into latency upon infection and establishment of latency is a
complex phenomenon, which is yet to be fully understood. There are multiple
cytokines playing important roles both during latent and lytic phase of infection, the
exact combination of cytokines in the milieu is not known. Hence, it is difficult to
develop an animal model where the virus upon infection goes into latency and with
a known trigger switches to lytic cycle. Lytic cycle activation could be induced by
addition of agents such as phorbol esters and is limited only to in vitro cell culture
model and cannot be extrapolated in vivo animal model systems.
Xenograft models of KSHV lymphoma have been able to mimic tumors seen in
humans. Initial studies on the establishment of PEL-like tumors was done using
KSHV+, EBV- cell lines BCP-1 and KSHV+, EBV+ cell lines HBL-6, PEL cells in
nonobese diabetic/severe combined immunodeficiency disease Nod/SCID mice
(Boshoff et al. 1998). In this study, all mice injected intraperitoneally with either
BCP-1 or HBL-6 developed lymphomatous effusion similar to PEL, while all mice
injected subcutaneously with HBL-6 formed solid tumor, but only 1 of 4 injected
with BCP-1 formed solid tumor, thus providing evidence about the importance of
the route of injection in the development of a suitable animal model. These studies
opened up a new area of investigation on xenograft model, and lead to the understanding of the involvement of tumor microenvironment (Staudt et al. 2004) and
host signal molecules (Keller et al. 2006) in the development of lymphomas by
other investigators. Dittmer et al. 1999 (Dittmer et al. 1999) used SCID-hu Thy/Liv
mice reconstituted with the liver and thymus of human fetuses to study viral
transcription as well as the susceptibility of the mice to infection with BCBL-1
derived KSHV. In addition, Parsons et al. (2006) investigated the immune response
to KSHV by implanting NOD/SCID mice with functional human hematopoetic tissue
grafts. Furthermore, they have shown that NOD/SCID mice infected with purified
KSHV provide a system for demonstrating latent and lytic replication.
156
D. Everly et al.
Besides these lymphoma animal models, KS animal models were also developed.
Yang et al. (2000) reported that transgenic mice expressing the KSHV- encoded
chemokine receptor (viral G protein-coupled receptor) vGPCR within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically
resemble KS lesions. Grisotto et al. (2006) used doxycycline inducible vGPCR
expressing system and demonstrated that vGPCR expressing cells accumulated in
areas where angioproliferation was observed. KSHV vGPCR expression was sufficient to induce angioproliferative tumors that resembled human KS (Montaner et al.
2003). Guo et al. (2004) produced ORF74 transgenic mice and showed that these
mice develop tumors resembling KS on the tail or legs. The tumors were reported to
be highly vascularised with characteristic CD31+ve spindle shaped cells expressing
VEGF-C. ORF74 expressing SV-40 T antigen immortalized murine endothelial
cells when injected into nude mice produced KS-like tumors (Montaner et al. 2006).
Mutlu et al. (2007) transfected KSHV bacterial artificial chromosome (KSHVBAC36)
into mouse bone marrow endothelial-lineage cells, generated mECK36 that formed
KS-like tumors. In nude mice, mECK36 cells formed vascular spindle cell sarcomas
that displayed KSHV and host transcriptomes reminiscent of KS. They further
demonstrated that siRNA suppression of KSHV vGPCR inhibited angiogenicity
and tumorigenicity.
In a recent study, common marmoset, a New-World primate, injected intravenously with KSHV seroconverted and maintained high antibody titers for more
than one year. Infection of common marmoset with rKSHV.219 via the oral route
developed a KS-like skin lesion with the characteristic spindle shaped cells along
with small blood vessels (Chang et al. 2009). Although animal model studies have
provided useful insight into the understanding of the tumors and had been instrumental in the identification of possible therapeutic targets, mechanisms of latency
establishment and switch from latency to lytic cycle still remain unanswered. The
plausible reasons for this could be that the immune response in human is not completely identical to mouse system and apart from the physical condition, emotional
status and stress-related response in human might be a contributing factor. The
viruses are more adapted than the human race; they tend to acclimatize themselves
to peacefully coexist in the host, establishment of latency, tumorigenicity and cell
death are mechanisms adopted by the virus for a successful infection. Counteracting
million years of virus evolution need intense research and immense understanding
of the virus.
Mareks Disease Virus

Mareks Disease Virus (MDV) or Gallid herpesvirus 2 (GaHV-2) is a highly contagious
herpesvirus whose infection affects predominantly chickens as well as other avian
species such as turkeys, pheasants, quail, and game fowl worldwide. Mortality rates can
be very high in susceptible birds. MD can develop in chickens as young as 3 week of age
which is characterized by the T cell lymphoma infiltrating the nerves and organs resulting
157
in enlarged nerves and tumors in nerve, organ, muscle and epithelial cells leading to
paralysis of legs, wings, and neck, loss of weight and vision impairment.
Following lytic infection, latency is established mainly in activated CD4+ T cells,
which may be transformed with differing efficiencies, depending on the genotype of
the infected chicken, and result in lymphoma formation. Irrespective of the transformation event, infection of feather follicle epithelial cells in the skin by migrating
lymphocytes leads to the production of infectious particles that are shed into the
environment, providing a continuous source of infectious virus. Though MDV is a
highly cell-associated, it is readily transmitted usually via respiratory tract.
The mechanism by which MDV transforms cells is not clearly understood.
Nevertheless, vaccination developed in 1970 has been used successfully as the central strategy for the prevention and control of MD. Since vaccination prevented the
clinical disease, MDV vaccine perhaps can be considered as the first cancer vaccine.
The earlier vaccine contained the antigenically similar turkey herpesvirus, which is
serotype 3 of MDV. While vaccination prevented clinical disease and reduced shedding of infective virus, it did not prevent infection. This resulted in the evolution of
MDV with increased virulence and resistance to this vaccine. Current vaccines use a
combination of vaccines consisting of HVT and gallid herpesvirus type 3 or an attenuated MDV strain. For additional information, please look up the chapter by Parcells
and Morgan of this book, which deals exclusively on MDV and T cell lymphomas.
The above summary will be a starting point to understand EBV ad KSHV role in
cancers, and the readers will benefit from the various chapters of this book detailing
the mechanism of oncogenesis by herpesviruses.
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Chapter 8
Lymphocryptoviruses: EBV and Its Role

in Human Cancer
Santosh Kumar Upadhyay, Hem Chandra Jha, Abhik Saha,
and Erle S. Robertson
Lymphocryptoviruses: Introduction
Lymphocryptovirus (LCV) or g1-herpesvirus is a genus of g-herpesvirinae that
along with Rhadinovirus or g2-herpesviruses constitutes this subfamily of the herpesviridae family. The herpesviridae family is composed of three subfamilies: a-herpesvirinae, b-herpesvirinae, and g-herpesvirinae. Originally, these subfamilies were
created based on their distinct biological properties such as cell tropism; however, the
present classification depends on the genomic properties of each viral species
(Roizmann et al. 1992; Lacoste et al. 2010). Initially, LCVs were thought to be
restricted to human and Old-World primates, whereas Rhadinoviruses were considered to be inhabitants of new-world primates. In addition, geographical severance
was considered to be the reason behind their evolutionary differences. However,
this paradigm was challenged with the discovery of the human Rhadinovirus,
Kaposis sarcoma-associated herpesvirus (KSHV) and a marmoset LCV Callitrichine
herpesvirus 3 (Rivailler et al. 2002a; Lacoste et al. 2010). Regardless of their classifications, these viruses share a common structure, comprised of a 100200-kb
long linear double-stranded genome surrounded by an icosahedral capsid and subsequently by a lipid bilayer envelop derived from hosts cell membrane (Carville
and Mansfield 2008).
There are approximately 30 known members of LCVs. Examples include the
following: EpsteinBarr virus (EBV) or human herpesvirus 4 (HHV4), rhesus monkey
LCV, herpesvirus papio of baboons, and LCVs of African green monkeys, orangutan,
S.K. Upadhyay H.C. Jha A. Saha E.S. Robertson (*)

Department of Microbiology and Abramson Comprehensive Cancer Center,
University of Pennsylvania School of Medicine, 201E Johnson pavilion, 3610 Hamilton walk,
Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine,
201E Johnson pavilion, 3610 Hamilton walk, Philadelphia, PA 19104, USA
e-mail: erle@mail.med.upenn.edu
169
170
S.K. Upadhyay et al.
Table 8.1 Viral species of genus lymphocryptovirus (00.031.3.01.)

Species name
ICTVdB virus code Isolate(s)/strain(s)
Alternate name(s)
Callitrichine
00.031.3.01.009.
Callitrichine
Marmoset
herpesvirus 3
herpesvirus 3
lymphocryptovirus
(Ca1HV-3)
Cercopithecine
00.031.3.01.002.
Cercopithecine
Baboon herpesvirus,
herpesvirus 12
herpesvirus 12
Herpesvirus papio
(CeHV-12)
Cercopithecine
00.031.3.01.003.
Cercopithecine
African green monkey
herpesvirus 14
herpesvirus 14
EBV-like virus
(CeHV-14)
Cercopithecine
00.031.3.01.004.
Cercopithecine
Rhesus EBV-like
herpesvirus 15
herpesvirus 15
herpesvirus, rhesus
(CeHV-15)
lymphocryptovirus
Human herpesvirus 4
00.031.3.01.005.
Human herpesvirus 4
EpsteinBarr virus
(HHV-4)
Pongine herpesvirus 1 00.031.3.01.006.
Pongine herpesvirus 1 Herpesvirus pan
(PoHV-1)
Pongine herpesvirus 2 Orangutan herpesvirus
(PoHV-2)
Pongine herpesvirus
Gorilla herpesvirus
3(PoHV-3)
Source: ICTV database (http://www.ictvdb.org/Ictv/fs_herpe.htm#Genus31)
and gorilla (Table 8.1). The common members of the Rhadinoviruses are Kaposis
sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), herpesvirus
saimiri (HVS), mouse herpesvirus 68 (MHV68), ovine herpesvirus 2 (OvHV-2),
equine herpesvirus 2 (EHV-2), and rhesus monkey rhadinovirus (RRV) (Damania
and Jung 2001; Ackermann 2006; Ehlers et al. 2010). The members of gammaherpesvirinae are lymphotropic. However, some are capable of undergoing lytic replication
in fibroblasts, endothelial and epithelial cells (Damania and Jung 2001). Importantly
in Old-World nonhuman primates, the natural hosts for LCVs, the infection remains
persistent in the blood cells.
Lymphocryptoviruses and Cancer

Similar to EBV, Old-World LCVs can also immortalize primary B-cells in vitro, and
LCV infection has been found to be tightly associated with tumorigenesis in vivo
(Rivailler et al. 2002a). Simian LCV infection of immunocompromised macaques
at various National Primate Research Centers (NPRCs) has been found to be associated with a condition similar to oral hairy leukoplakia, which is frequently seen in
HIV-infected patients and is associated with lytic EBV infection of epithelial cells
on the tongue (Carville and Mansfield 2008). Non-Hodgkin lymphoma (NHL) is
the second most commonly diagnosed malignancy in HIV-infected populations,
and similarly, the incidence of NHL in cynomolgus (M. fascicularis) and rhesus
Lymphocryptoviruses: EBV and Its Role in Human Cancer
171
macaques infected with SIV is 415% and 40%, respectively. M. nemestrina, the
pigtailed macaque, housed at a NPRC developed a mycosis-fungoides-like lymphoma
in association with LCV infection, with characteristic features of a T-cell lymphoma
of the skin revealed on histological examination (Rivadeneira et al. 1999). Like
other malignancies caused by EBV in the human host, nonhuman primates may also
get posttransplant lymphoproliferative disorder (PTLD) on infection with their
respective LCV, during organ transplantation. Out of 160 cases of cynomolgus
macaque renal transplant 9 (5.6%) had evidence of PTLD at the time of necropsy,
28103 days after transplantation (Schmidtko et al. 2002). Studies on EBV infection
of tamarins and owl monkeys revealed that cross species transmission of LCVs
from the natural to inadvertent host may also be associated with oncogenesis and
the development of malignant lymphoma (Carville and Mansfield 2008). EBV, the
prototypic member of the LCV genus, infects most of adult humans and usually
persists asymptomatically for the life of the host. It is the most well studied and the
only identified LCV known to infect humans (Moghaddam et al. 1997).
EBV: LCV Causing Cancer in Human

LCVs naturally infecting Old-World primates are known to be biologically similar
to EBV (Rivailler et al. 2002a). LCV infection is ubiquitous in adult Old-World
nonhuman primates, and they harbor persistent LCV infection in their blood. OldWorld rhesus macaques have been used as animal model for EBV infection, as
experimental infection of naive rhesus macaques with rhesus LCV reproduced acute
and persistent infection similar to EBV infection in humans (Moghaddam et al.
1997). The genome sequence of rhesus LCV showed a high degree of amino-acid
sequence homology (75%) with EBV providing the genetic validation for the similarities between EBV and rhesus LCV infection (Rivailler et al. 2002a, b). EBV
infects more than 95% of the human population within the first decades of life and
in the majority of cases infection is asymptomatic. However, later in life, a proportion of EBV-infected individuals can develop IM, a disease that is characterized by
lymphadenopathy and fatigue, (Damania and Jung 2001). In contrast to most of the
viruses identified as the cause of acute infections, EBV was discovered because of
its isolation from cells obtained from tumor-bearing children with BL. The discovery
of BL in late 1950s by Denis Burkitt was based on the climatic and geographical
distribution of this lymphoma with an environmental or infectious etiology. However,
a decade or more later in 1964, Anthony Epstein, Yvonne Barr, and Bert Achong
identified virus particles in BL cells sent by Denis Burkitt by electron microscopy
(Epstein et al. 1964; Kutok and Wang 2006). Subsequent studies revealed the
association of EBV with a variety of other human tumors such as posttransplant
B-cell lymphomas, HD, and NPC. Cells in these tumors contain characteristic multiple
extrachromosomal copies of the circular viral genome (Young and Murray 2003).
Association of this virus was also evidenced by its unique ability to efficiently transform
resting B-cells in vitro into continuously growing lymphoblastoid cell lines (LCLs)
(Young and Murray 2003).
172
EBV Genome
Among the herpesviruses, the EBV genome was the first that was cloned and
subsequently sequenced (Baer et al. 1984). The genome of EBV is a linear doublestranded DNA of approximately 184 kb as packaged in the virion particle. It has a
series of 500-bp terminal direct repeats (TRs) and internal repeat sequences (IRs)
that divide its genome into short and long unique sequence domains (US and UL)
which contains almost all the genome coding capacity. A BamHI fragment-cloned
library was used for sequencing the entire EBV genome. Thus, any particular region
of the EBV genome is referred with respect to its location within the BamH1-digested
fragment of the genome, to a specific BamHI fragment, from A to Z, in descending
order of fragment size (Young and Murray 2003). Upon EBV infection the linear
termini of the viral genome are joined intracellularly to form circular episomal DNA
(Kaschka-Dierich et al. 1976; Lindahl et al. 1976; Given et al. 1979). The process
of genome circularization creates a signature number of TRs. In the dividing latently
infected host cells, the number of TRs remains constant during episomal replication,
and all the daughter cells derived from a single infected cell have been shown to
have identical numbers of TRs in their EBV episomes (Kutok and Wang 2006).
The TR number can also be used as a marker to determine the clonality of latently
infected host cells (Kutok and Wang 2006). The EBV genome encodes nearly 100
proteins, and genes expressed during the lytic phase of infection have homologues
in other human herpesviruses. However, the genes expressed during latent infection
appear to be unique to EBV and are responsible for targeting a range of cellular
pathways that lead to induction of EBV-associated cancers (Table 8.2).
Structure of EBV
Similar to other herpesviruses, EBV has an outer envelope with glycoprotein spikes,
a toroid-shaped DNA core in a nucleocapsid containing 162 capsomeres, and a protein
tegument between the nucleocapsid and envelope (Epstein et al. 1965; Dolyniuk
et al. 1976a, b; Kieff and Rickinson 2007). EBV capsids from purified enveloped
virus are composed of EBV homologues of five previously purified herpesvirion
proteins, the 18-kDa small capsid protein, the 30-kDa minor capsid protein, the
40-kDa minor capsid protein binding protein, the 155-kDa major capsid protein,
and the 68-kDa portal protein (Kieff and Rickinson 2007). Among the common
herpesvirus tegument proteins, the 350-kDa large tegument protein (BPLF1), the
140-kDa large tegument protein binding protein (BOLF1), the 15-kDa myristylated
protein (BBLF1), the 32-kDa myristylated protein binding protein (BGLF2), the
58-kDa capsid-associated protein (BVRF1), the 58-kDa packaging protein (BGLF1),
the 27-kDa palmitylated protein (BSRF1), and a 47-kDa protein kinase (BGLF4)
are present in EBV. The gammaherpesvirus-specific proteins of the EBV tegument
are the 140-kDa major tegument protein (BNRF1), the 19-kDa BLRF2, the 72-kDa
BRRF2, the 54-kDa BDLF2, and the 42-kDa BKRF4 (Johannsen et al. 2004; Kieff
173
Table 8.2 EBV lytic and latent genes and their functions
EBV Gene
Function
Lytic genes
BZLF1/BRLF1
Transcriptional activators of lytic gene expression
BHRF1
Prevents apoptotic cell death in lytic EBV infection,
Dispensable for B-cell growth transformation and for virus
replication in culture
BCRF1
Homologous to human IL-10; may downregulate the host
immune response during viral replication
BLLF1(gp350/220)
Mediates adsorption between EBV and CD21
BKRF3,BBLF2/3, BMRF1,
Replication factors
BSLF1, and BBLF4
BALF2
Replication factor; single-strand DNA binding protein
BALF5
Replication factor; core DNA polymerase
Latent genes
EBNA1
EBNA2
EBNA3A
EBNA3B
EBNA3C
EBNALP
LMP1
LMP 2A/2B
EBER-1/2
BARTs
Tethers viral genome to the host chromosome, interacts with p53

(antiapoptotic function)
Transactivator for many viral (LMP1 and LMP2A) and cellular
(CD21, c-Myc,CD23) genes, essential for cellular transformation, interacts with RBP- Jk, and modulates Notch
signaling.
EBNA2 antagonist and transcriptional coactivator, modulates
Notch signaling
Interacts with RBP-Jk, modulates Notch signaling
Essential for B-cell transformation by EBV, Ubiquitination of
pRb and p27, modulates cell cycle through interaction with
chk2, c-myc, Cyclin A/E/D1, p53 and Mdm2, chromatin
remodeling by interaction with HDAC1 and 2, Interacts with
RBP-Jk, modulates Notch signaling.
Necessary for the efficient outgrowth of LCLs, transcriptional
activation of viral promoters in association with EBNA2
Mimics the function of the B lymphocyte CD40 receptor and
contributes to the EBV-induced transformation of primary
B cells; Interacts with TRAFs
LMP2A sequesters tyrosine kinases from the BCR, block EBV
lytic cycle in B cells, LMP2B competes with LMP2A
function
Most abundant RNAs in latently infected cells, inhibit PKR and
thus the antiviral effects of the interferons
Different BARTs: interfere with the normal function(s) of
RACK1 protein, modulate LMP1-induced NF-kB signaling,
interact with RBP-Jk, modulate Notch signaling
and Rickinson 2007). Interestingly, a number of cellular proteins such as actin,

HSP70, Cofilin, b-tubulin, enolase, and HSP90 are also contained within the EBV
tegument (Kieff and Rickinson 2007). The lipid envelop contains a number of
glycoproteins including gp350 (BLLF1), gH (BXLF2), gB-N, gB-C, and full-length
gB (BALF4), gp42 (BZLF2), gM (BBRF3), gp78 (BILF2), gN (BLRF1), gp150
(BDLF3), and gL (BKRF2) (Johannsen et al. 2004; Kieff and Rickinson 2007).
174
EBV-Encoded Proteins Implicated in Driving

the Associated Pathologies
During the interaction with its host, EBV goes through various phases of its infection
cycle: (1) it infects B-lymphocytes and induces proliferation of infected cells; (2) it
enters into a latent phase that drives the proliferative phase; and finally, (3) it can be
reactivated to produce infectious progeny by inducing lytic cycle (Bornkamm and
Hammerschmidt 2001). The transforming potential of the EBV genome is maintained within one third of the viral genome, and only a limited number of viral genes
are expressed in EBV immortalized cells (Kempkes et al. 1995; Robertson and Kieff
1995). These include three latent membrane proteins (LMP1, LMP2A, and LMP2B),
six nuclear antigens (EBNA1, 2, 3A, 3B, 3C, LP), and two short nonpolyadenylated
RNAs (EBER1 and EBER2). BARF0 a reading frame reported to be expressed in
NPCs (Hitt et al. 1989) is not required for the B-cell immortalization (Robertson
et al. 1994; Bornkamm and Hammerschmidt 2001). Similarly, viral interleukin 10
(vIL10) earlier thought to have a B-cell growth factor activity, and BHRF1, a viral
Bcl-2 homologue have been found to be nonessential for B-cell immortalization
(Marchini et al. 1991; Bornkamm and Hammerschmidt 2001). EBV-encoded latency
program is crucial for its stable persistence in host. By evading the host immune
system, a program of EBV latency is established primarily in resting B-lymphocytes
(Kumar et al. 2010). Depending on the spectrum of viral genes expressed, the latent
Fig. 8.1 Differential expression of EBV genes in the different latency programs. The expression
pattern of the latent transcripts is a characteristic of the specific latency programs associated with
EBV infection. The associated EBV malignancies that are typically linked to particular latency
programs are shown in italics. Latency 0III are shown and are represented by the inclusive oval
shapes for the different transcripts. Latency IV has more recently been described with an EBNA2+
and LMP1- phenotype but EBNA1, EBERs, LMP2A, and EBNA3s have not been determined and
are shown by a broken line including EBNA2 but may also have other transcripts. PEL primary
effusion lymphoma, PTLD posttransplant lymphoproliferative disease
175
program can be growth and proliferative type (latency III) or more restricted type
(latency 0, I, II, or IV). Specific latency programs have been found to be associated
with the different malignant pathologies associated with EBV infection (Kuppers 2003;
Kumar et al. 2010) (Fig. 8.1).
LMP1
LMP1 is an integral membrane protein of 63 kDa that has three domains: an
N-terminal cytoplasmic tail (amino acids 123), which tethers LMP1 to the plasma
membrane and orientates the protein; six transmembrane-spanning domains loops,
which are involved in self-aggregation and oligomerization (amino acids 24186);
and a C-terminal cytoplasmic domain (amino acids 187386), which carries out
most of the signaling activity for the molecule. This C-terminal domain is further
divided into two subdomains, C-terminal activation regions 1 and 2 (CTAR1 and
CTAR2) on the basis of their ability to activate the NF-kB pathway (Huen et al.
1995; Kutok and Wang 2006). LMP1 is one of the more carefully characterized
molecules and is known for its transforming potential. It transforms rodent fibroblasts and is also essential for the immortalization of primary B-lymphocytes to
LCLs (Wang et al. 1985, 1988; Baichwal and Sugden 1988). Expression of the
LMP1 is also known to induce B-cell lymphoma in transgenic mice (Kulwichit
et al. 1998). By mimicking the function of the B-lymphocyte CD40 receptor,
LMP1 is known to contribute to EBV-induced transformation of primary B-cells
(Gires et al. 1997; Hatzivassiliou et al. 1998; Kilger et al. 1998; Damania and Jung
2001). Similar to CD40 receptor the C-terminal domain of LMP1 is capable of
interacting with many signal transducers such as TNF receptor-associated factors
(TRAFs), TNF receptor-associated death domain (TRADD), and receptor-interacting
protein (RIP) (Devergne et al. 1996; Eliopoulos et al. 1996; Izumi et al. 1997, 1999;
Izumi and Kieff 1997; Sandberg et al. 1997; Devergne et al. 1998; Eliopoulos and
Young 1998). In case of CD40 activation, the signal transduction usually takes place
when CD40 is multimerized by binding with an extracellular ligand (Werneburg
et al. 2001); however, LMP1 is capable of multimerization through its transmembrane
domains, thereby generating a constitutively active signal that results in multiple
downstream activities including the activation of NF-kB and JNK mediating activity
and the induction of numerous cellular genes including bcl-2, bclx, mcl1, and A20
(Laherty et al. 1992; Hsu et al. 1995; Huen et al. 1995; Floettmann et al. 1996; Izumi
et al. 1997; Izumi and Kieff 1997; Eliopoulos and Young 1998; Hatzivassiliou et al.
1998; Eliopoulos et al. 1999; Fries et al. 1999; Takeshita et al. 1999; Damania and
Jung 2001), all critically important for EBV-induced immortalization of B cells.
Recently, LMP1 has been discovered to signal the Janus kinase 3 (JAK3) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways upon the activation of
STAT3 and STAT transactivation activity. LMP1-induced vascular endothelial
growth factor (VEGF) expression via the JAK/STAT and mitogen-activated protein
kinase (MAPK)/ERK signaling pathways is thought to be involved in the invasion
of EBV-positive nasopharyngeal carcinoma (Wang et al. 2010).
176
LMP 2A and 2B
The LMP2 gene encodes two distinct integral membrane proteins, LMP2A and
LMP2B (Laux et al. 1988). As the exons for LMP2 gene are located at both the ends
of linear EBV genome, they could only be expressed from the circular EBV episome
during latent infection (Longnecker et al. 1991). Both LMP2A and 2B have 12
transmembrane domains and a 27-amino-acid cytoplasmic C-terminus in common.
However, LMP2A contains an additional 119 amino-acid cytoplasmic N-terminal
domain (Longnecker and Kieff 1990). Both LMP2A and 2B oligomerize to form
patches within the plasma membrane of latently infected B-lymphocytes where
LMP2B has been observed to reduce the impact of LMP2 expression probably by
imposing a dominant negative effect on LMP2 oligomers (Raab-Traub 2009).
Although LMP2 has been found to be consistently expressed in most of the malignancies associated with EBV, none of the LMP2 proteins are essential for B-cell
transformation in vitro (Longnecker 2000). Two of the tyrosine residues in LMP2A
aminoterminal domain (Y74 and Y85) form an immunoreceptor tyrosine-based
activation motif (ITAM) (Fruehling and Longnecker 1997), which mimic the ITAM
present in the B-cell receptor (BCR). The ITAM present in BCR is known to play
an important role in proliferation and differentiation of lymphocytes by activation
of the src and syk protein tyrosine kinases (PTKs). Phosphorylated ITAMs of
LMP2A compete with ITAM of BCR for binding with these PTKs and thereby
negatively regulate the PTK activity (Fruehling and Longnecker 1997). Thus,
LMP2A is implicated in blocking BCR-mediated calcium mobilization, tyrosine
phosphorylation, and activation of the EBV lytic cycle in B-cells (Miller et al. 1995).
In transgenic mice, LMP2A has been shown to drive the proliferation and survival
of B-cells in the absence of BCR signaling (Caldwell et al. 1998). Inhibition of BCR
signaling by sequestering of tyrosine kinases by LMP2A prevents unwanted antigentriggered activation of EBV-positive B-cells, thereby preventing their entry into the
lytic cycle. However, LMP2A itself is able to stimulate these tyrosine kinases and
thus provide the survival signal to the B-cells (Kuppers 2003). Therefore, LMP2A
modifies B-cell development to favor the maintenance of EBV latency and also
prevents inappropriate activation of the EBV lytic cycle, whereas LMP2B plays the
modulatory role in LMP2A function (Longnecker 2000; Young and Murray 2003).
LMP2A recruits Nedd4-like ubiquitin ligases through phosphotyrosine motifs,
resulting in degradation of LMP2A through a ubiquitin-dependent mechanism
(Ikeda et al. 2000) (Fig. 8.2). LMP2A also interacts with ERK1-MAPK, leading to
self-phosphorylation at two serine residues (S15 and S102), which ultimately contributes to JUN activation (Chen et al. 2002; Young and Rickinson 2004). A recent
study provides more conclusive evidence that LMP2B negatively regulates LMP2A
function in preventing the switch from latent to lytic EBV replication (Rechsteiner
et al. 2008). In EBV-harboring Akata cells, overexpression of LMP2B increased the
magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking,
as indicated by more-enhanced upregulation and expression of EBV lytic genes and
significantly increasing production of transforming EBV (Rechsteiner et al. 2008).
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Fig. 8.2 LMP1 and LMP2-mediated cell signaling in EBV infected B cells. LMP1 deregulates cell
signaling through NF-kB, MAPKKK and PI3K by interaction with TRAF2 in mammalian cells.
LMP2A cell signaling is primarily mediated through PI3K and AKT pathways by activation of the
SRC family kinases, which leads to cell survival and motility
LMP2B also reduces the degree of BCR cross-linking required to induce lytic EBV
infection and restored calcium mobilization upon BCR cross-linking, a signaling
process inhibited by LMP2A (Rechsteiner et al. 2008).
EBNA1
EBNA1 is a DNA-binding nuclear phosphoprotein that is crucial for the maintenance
of EBV episome and its replication. Short dyad symmetry (DS) Cis-acting elements
at the origin of latent replication oriP have been identified as EBNA1 binding sites on
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the viral episome (Rickinson and Kieff 2001). EBNA1 increases the transcriptional
activity from both Cp and the LMP1 promoters (Rickinson and Kieff 2001), while
at the same time negatively regulating its own expression through the Qp promoter
(Nonkwelo et al. 1996). Interestingly, EBNA1 is not presented by MHC class I
molecules so that EBV-positive B-cells expressing this antigen are not recognized
by cytotoxic T-cells and thus increases EBNA1 protein stability, which is possibly
achieved by the internal Gly-Ala repeat domain (Levitskaya et al. 1995, 1997).
A direct role for EBNA1 in oncogenesis has been suggested by studies performed
on transgenic mice, where directing the EBNA1 expression in B-cells resulted in
B-cell lymphomas (Wilson et al. 1996; Young and Murray 2003). EBNA1 along
with another latent antigen EBNA3C has been recently identified to interact with a
tumor metastasis suppressor, Nm23-H1. Using nude mice model, these EBV antigens
have been evidently shown to promote the growth of transformed cells by rescuing
Nm23-H1-mediated suppression (Kaul et al. 2007).
EBNA2
EBNA2 is one of the first EBV-encoded antigen to be expressed following primary
B-cell infection in vitro and also indispensable for cellular transformation. Although
EBNA2 does not bind to DNA directly, it can control many viral and cellular gene
expression, such as that of LMP1, LMP2A, CD21, CD23, CD25, and C-FGR, via
engaging various transcription regulators including RBP-Jk, SPI1/PU.1, TAF40,
TFIIB, TFIIE, TFIIH, and RPA70 (Tong et al. 1995a, b, c; Damania and Jung 2001;
Klein et al. 2010). Most of the EBNA2-responsive promoters possess a common
DNA sequence (GTGGGAA) for binding to RBP-Jk, the master regulator of the
Notch signaling pathway (Grossman et al. 1994). Notch genes encode cell surface
receptors that regulate development of several cell types. In Drosophila melanogaster,
mutations in Notch loci led to abnormalities in the notching of the wing, whereas in
human these can lead to development of T-cell malignancy (Artavanis-Tsakonas
et al. 1995). As the ligand (Jagged 1, 2 in humans) binds to the extracellular part of
the Notch receptor, its intracellular domain is cleaved off and targeted to the nucleus
to complex with RBP-Jk. This complex then transcriptionally activates genes
including Hairy/Enhancer of split (HES) family, cyclin D1, and p21 (Aster et al.
2008; Borggrefe and Oswald 2009). Both EBNA2 and the cleaved intracellular
region of Notch (RAMIC) transactivate the same set of viral and host gene promoters
as they shown to compete for binding to RBP-Jk. This indicates that their interaction
sites on RBP-Jk overlap at least partially (Sakai et al. 1998). Furthermore, interaction
between EBNA2 and RBP-Jk (or CBF1) has been found to be essential for EBVmediated transformation, as a specific deletion of the RBP-Jk binding domain
within the EBNA2 gene in the context of the whole virus renders the virus incapable
of immortalizing B-lymphocytes in vitro (Cohen et al. 1991; Harada et al. 2001).
As little or no RBP-Jk association with Notch1 has been observed in EBV-positive
B-lymphocytes compared to the RBP-Jk associated with Notch1 in T-cell lines,
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EBNA2 is thought to compete for the available pool of RBP-Jk more effectively in
B-cells, offering a potential explanation for the ability of EBV to efficiently infect
and immortalize primary human B-cells in vitro (Callahan et al. 2000).
EBNA2 also plays a role in chromatin remodeling in regulating both viral and
host gene expressions by interacting with numerous modulatory proteins actively
involved in this process. This includes the SNF5 subunit of the SWI/SNF chromatin
remodeling complex (Wu et al. 1996), histone acetyltransferases (HATs) P300/
CBP, PCAF (Wang et al. 2000), and the helicase DP103 (Grundhoff et al. 1999;
Klein et al. 2010). Deletion mutation analysis of the EBNA2 protein showed that the
Pro-rich aminoterminal and a domain within the divergent region mediate the interaction between EBNA2 and hSNF5/INI1 (Wu et al. 1996). EBNA2 engages the
hSNF-SWI complex to generate an open chromatin conformation at the EBNA2responsive target genes, thereby facilitating the activity of the RBP-Jk/EBNA2/
RNA-polymerase II transcription complex (Wu et al. 1996). This hypothesis was
strengthened by the additional observation where antibodies directed against components of the hSNF-SWI complex precipitated chromatin-associated DNA that
contained a targeted EBNA2-responsive element in the context of both episomal
and cellular chromatin. This enrichment could not be observed in the EBV-negative
cells or when the EBNA2-responsive element was mutated (Wu et al. 2000).
EBNA3 Family of Proteins: 3A, 3B, and 3C

The three members of the EBNA3 family, EBNA3A, -3B, and -3C, lie in a tandem
array on the EBV genome. These proteins share an amino-terminal domain with a
2025% homology, but different carboxy-terminal domains. They are hydrophilic
nuclear proteins that contain heptad repeats of Leu, Ile/Val that can act as dimerization
domains (Young and Murray 2003). Genetic studies have revealed that both
EBNA3A and EBNA3C are essential for in vitro B-cell transformation, whereas
EBNA3B is dispensable (Robertson 1997; Young and Murray 2003). Members of
the EBNA3 family participate in transcriptional regulation of both cellular and viral
genes. EBNA3C in particular transcriptionally activates cellular genes, such as
CD21, CD40, Vimentin, and the viral encoded LMP1 (Allday and Farrell 1994;
Silins and Sculley 1994). Moreover, EBNA3C also acts as a transcriptional repressor
from the viral Cp promoter (Radkov et al. 1997). All EBNA3 proteins repress the
EBNA2-mediated transactivation by associating with RBP-Jk and disrupting its
binding to the cognate DNA sequence and also to EBNA2 (Robertson 1997; Young
and Murray 2003). Thus, both cellular and viral promoters containing RBP-Jk
cognate sequences are cooperatively regulated by EBNA2 and EBNA3 family proteins
(Young and Murray 2003). EBNA3C plays a key role in deregulating the cell cycle
and transformation of B-cells in vitro by interfering with the function(s) of a
number of important cell-cycle regulators (Kumar et al. 2010). EBNA3C interacts
with and enhances Cyclin A/CDK2 dependent kinase activity and efficiently
rescues p27-mediated repression of Cyclin A/CDK2 kinase activity by decreasing
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the molecular association between the Cyclin A/CDK2 complex and p27 in
EBV-transformed LCLs (Knight and Robertson 2004). EBNA3C also interacts
specifically with metastatic suppressor protein Nm23-H1 and reverses its ability to
suppress the migration of BL cells and breast carcinoma cells (Subramanian et al.
2001) and was also shown to regulate transcription of a number of genes involved
in regulation of cell migration and, so, demonstrates a transcriptional component of
this interaction (Kuppers et al. 2005; Choudhuri et al. 2006; Kaul et al. 2007, 2009).
EBNA3C is also shown to rescue the pRb-induced flat cell phenotype and target
pRb for proteasome-ubiquitin mediated degradation (Knight et al. 2005a). Further
mechanistic studies revealed that EBNA3C specifically recruits the ubiquitin ligase,
Skp1/Cul1/F-box complex (SCFSkp2), which adds ubiquitin moieties to many cellcycle regulators such as pRb and p27, marking them for subsequent degradation
(Knight et al. 2005a, b). Additional studies also demonstrated that EBNA3C forms
a ternary complex with the p53 tumor suppressor protein and its negative modulator
Mdm2 (Saha et al. 2009; Yi et al. 2009). Mdm2 is a specific ubiquitin ligase that is
known to negatively regulate the tumor suppressor activity of p53 by facilitating its
ubiquitin mediated degradation. Interestingly, EBNA3C has been shown to suppress
the p53s function by stabilizing Mdm2 through deubiquitination (Saha et al. 2009;
Yi et al. 2009). Furthermore, Knight et al. showed that EBNA3C manipulates the
cellular deacetylase activity and thereby the chromatin structure through interactions
with ProTa in association with deacetylases (HDAC1 and HDAC2) and corepressors
(mSin3A and NCoR) (Knight et al. 2003). In a recent study, the vitamin D receptor
(VDR) has been identified as a binding partner of EBNA3A, which subsequently
activates VDR-dependent genes to protect LCLs against vitamin-D3-induced
growth arrest and apoptosis (Yenamandra et al. 2010).
EBNALP
EBNALP (EBV nuclear antigen leader protein) is encoded by a small ORF in the
leader exons of the EBNA transcripts and is composed of repetitive units derived
from repetitive nucleotide sequences in the EBV internal repeat (IR1) sequence
(Bodescot et al. 1986). Therefore, EBNALP protein may vary in size depending on
the number of BamHI W repeats contained by a particular EBV isolate (Young and
Murray 2003). Although EBNALP does not seem to be absolutely required for
B-cell transformation in vitro, mutant viruses for EBNALP have reduced efficiency
for immortalization of B-cells and it is shown to be necessary for the efficient outgrowth
of LCLs (Hammerschmidt and Sugden 1989; Mannick et al. 1991; Allan et al.
1992). EBNALP colocalizes with the promyelocytic leukemia (PML) protein,
which is known to be involved in the sequesteration of transcription factors. It has
been suggested that EBNALP disrupts PML function, which leads to the transcriptional
activation of viral promoters in association with EBNA2 (Allan et al. 1992; Nitsche
et al. 1997; Young and Murray 2003). Both EBNALP and EBNA2 have been found
to form complexes with HDAC4 in an LCL background, where EBNALP coactivated
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transcription by relocalizing HDAC4 and HDAC5 from EBNA2 activated promoters

to the cytoplasm (Portal et al. 2006). EBNALP was suggested to interact with both
pRb and p53 (Jiang et al. 1991; Szekely et al. 1993); however, these interactions
have not been shown to affect either pRb or p53 mediated signaling pathways
(Young and Murray 2003). Recently, studies have shown that EBNALP may form a
ternary complex with p53 and Mdm2, where Mdm2 serves as a bridge (Kashuba
et al. 2010). Although EBNALP has been found to be neutralized by Mdm2 destabilizing the effect on p53, it can efficiently block p53-dependent gene activation in
LCLs, thus providing a potential mechanism for the accumulation of p53 levels in
LCLs without the induction of p53-mediated apoptosis (Kashuba et al. 2010).
EBERs
EBV expresses two small nonpolyadenylated (noncoding) RNAs - EBER1 and
EBER2 in almost all the forms of latency (Fig. 8.1). Although EBERs have not been
found to be essential for the EBV-induced primary B-lymphocytes transformation
(Young and Rickinson 2004), a potential role for them has been suggested, which
relates to the maintenance of viral persistence (Nanbo et al. 2002). Through assembling with autoantigen La and ribosomal protein L22, EBERs are shown to bind and
interfere with the function of the interferon inducible, double-stranded-RNAactivated protein kinase PKR (Takada and Nanbo 2001). As PKR has a role in
mediating the antiviral effects of the interferons, it has been suggested that EBERmediated inhibition of PKR function could be important for viral persistence (Nanbo
et al. 2002). Interestingly, expression of the EBERs in BL cell lines has been found
to increase tumorigenicity, promote cell survival, and induce interleukin-10 (IL-10)
expression (Ruf et al. 2000; Takada and Nanbo 2001; Young and Rickinson 2004).
These studies suggest that EBV genes previously shown to be dispensable for the
B-cell transformation may be involved by contributing in a critical way to the pathogenesis of a number of EBV-associated malignancies (Young and Murray 2003).
BARTs
A group of highly spliced transcripts encoded by the BamHI A region of the EBV
genome, which are abundantly expressed in EBV-associated malignancies, are
commonly referred to as either BamHI A rightward transcripts (BARTs), or
complementary-strand transcripts (CSTs) or BARF0 (Karran et al. 1992; Smith
et al. 2000). The function of most of the BARTs is not conclusively known but their
detection in B-cells from normal donors and in many EBV-associated tumors
suggests that they are likely to have critical roles in viral persistence and in development of the associated pathologies. For example, the RPMS1 CST encodes a nuclear
protein that binds to RBP-Jk and modulates EBNA2-mediated Notch signal transduction (Smith et al. 2000). Another cytoplasmic protein encoded by A73 CST
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interacts with RACK1 protein (Smith et al. 2000). Since RACK1 modulates signaling
from protein kinase C and Src tyrosine kinases, this suggests a possible role for A73
in growth control (Smith et al. 2000). EBV-encoded BART miRNAs also target the
3 UTR of the LMP1 gene and negatively regulate LMP1 protein expression (Lo et al.
2007). These miRNAs have also been found to modulate LMP1-induced NF-kB
signaling and alleviate the cisplatin sensitivity of NPC cells (Lo et al. 2007). Another
important transcript generated from the BamHI A region is BARF1, which encodes
a 31-kDa secreted protein expressed as a latent protein in EBV-associated NPCs and
gastric carcinomas (GCs) (Decaussin et al. 2000; zur Hausen et al. 2000). BARF1
shares limited homology with the human colony-stimulating factor (CSF) 1 receptor
and displays oncogenic activity when it is expressed in rodent fibroblasts and simian
primary epithelial cells (Sheng et al. 2001).
Host Machinery Targeted by EBV to Induce Tumorigenesis

Cell Signaling
EBV is a highly immunogenic virus, as confirmed by the strong response induced
at the time of primary contact, which successfully constrains the virus in a rigorously
latent, immunologically silent status. After spreading through activation of the lytic
cycle, EBV establishes a latent infection in the memory B-cells by negatively regulating the expression of the major immunogenic latent antigens (Merlo et al. 2010).
A large body of evidence has clearly shown that EBV-encoded proteins can critically
modulate the function of major players in different cell-signaling cascades in the
development of associated cancers.
The activation of a signaling cascade leading to the posttranscriptional modification
and nuclear translocation of NF-kB and Rel-family transcription factors modifies
the expression of numerous proteins involved in various phases of cancer development,
including cell proliferation (e.g., cyclin D1 and E), survival (e.g., cFLIP and IAPs),
inflammation and angiogenesis (e.g., IL-1, IL-6, VEGF, ICAM-1, and E-selectin),
epithelial-mesenchymal transition (e.g., vimentin and cathepsin-B and -Z), invasion
and metastasis (e.g., uPA, MMPs 2 and 9, and ICAM-1), and many others (Basseres
and Baldwin 2006). EBV has developed sophisticated mechanisms to hijack the
NF-kB signaling pathway to achieve a successful infection and promote survival
signals. There is a critical interdependence between the host cell and virus to finetune NF-kB-mediated functions, both positively and negatively, which has allowed
survival and efficient proliferation of these tumor viruses during evolution. During
latent EBV infection, NF-kB constitutively activates its downstream targeted genes,
which subsequently allows for viral persistence. The current understanding of the
molecular controls suggests a plausible model whereby the inflammatory microenvironment is sensed by EBV through NF-kB activation in infected cells. Inflammation
may have an adverse effect on the biology of the virus due to the immunogenic
response against EBV-infected cells; however, it has also been reported that EBV
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subtly exploits NF-kB-mediated signaling pathways to elicit the viral latency

program (de Oliveira et al. 2010).
To date, a number of EBV-encoded antigens are well defined and are also shown
to be associated with the deregulation of various cellular signaling pathways. Further
studies are required to identify detailed mechanisms for EBV specific manipulation
of host-signaling pathways in human. The EBV-encoded LMP1 is involved in
several signaling pathways including NF-kB, AP-1, JAK/STAT, PI3K/AKT, and
ERK-MAPK and has been shown to regulate their downstream effects (Dawson
et al. 2003; Mainou et al. 2005; Zheng et al. 2007). LMP1 also activates the PI3K/
AKT/mTOR signaling pathway in B-lymphocytes (Lambert and Martinez 2007).
The mTOR signaling pathway has been identified as a downstream component of
the PI3K/AKT pathway in the LMP2A-transfected NPC cell lines (Moody et al. 2005).
Furthermore, LMP1 can mimic CD40 signaling to induce B-cell activation and
differentiation in vivo (Rastelli et al. 2008) by usurping its activities through interaction with important signaling components such as TRAF1, 2, 3, and 5 involved in
various pathways including the NF-kB, JNK, p38/MAPK, PI3K/AKT, and JAK/
STAT signaling pathways (Uchida et al. 1999). NF-kB activity is repressed by
expression of EBNA1 in numerous carcinoma cell lines suggesting that its activation
is not due to clonal variation or cell line specificity. It has also been reported that a
number of malignancies are associated with chronic activation of NF-kB (Valentine
et al. 2010). In spite of the constitutive expression of several viral antigens during
latent infection, many EBV-associated tumors are poor targets for the cellular
immune response, suggesting that escape from immune surveillance is an early
event in the development of oncogenesis (Dantuma and Masucci 2003).
Chromatin Remodeling
A number of recent studies show that similar to many other tumor viruses, EBV has
also evolved sophisticated strategies to manipulate cellular chromatin-modifying
enzymes to precisely control viral as well as cellular genes expression. The EBV
proteins known to critically regulate the chromatin remodeling mechanism include
latent antigens EBNA2, EBNA3C, BGLF4 (Cotter and Robertson 2000; Knight
et al. 2003; Lee et al. 2007) and the lytic cycle inducer Zta (Deng et al. 2003; Wei
and Zhou 2010) (Fig. 8.3).
EBNA2 activates the viral LMP1 promoter via interaction with many histone
acetyltransferases (HATs), including p300, CBP, and PCAF, whereas the transcriptionally inactive point mutants of EBNA2 lack binding affinity to these HATs and
are unable to activate LMP1 (Wang et al. 2000). EBNA2 has been suggested to
utilize the intrinsic HAT activity for positively regulating viral genes transcription
(Wang et al. 2000). In another study, a phosphorylated fraction of lymphocyte
EBNA2 has been found to be associated with a component of the hSWI/SNF HAT
complex, hSNF5/Ini1 (Wu et al. 1996) (Fig. 8.3). The SWI/SNF complex can activate
or repress transcription of a subset of genes through alteration of the chromatin
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Fig. 8.3 EBV proteins influence the remodeling of the chromatin structure during infection and
development of associated malignancies. This schematic shows the functions of various molecules
of the chromatin remodeling machinery, which have altered functions due to their interaction with
a number of EBV-encoded antigens. EBNA3C is the encoded transcription regulator that binds to
HDACs, ProTa and p300; BGLF4, the kinase seen in the tegument, can be associated with
condensin phosphorylation and TopoII activation; EBNA2 is the major EBV transcription factor
also interacting with p300, CBP, PCAF, and the SWI/SNF complex; and Zta is the immediate early
viral transcription activator in its interaction with HATs including CBP
structure by altering the effects on transcription imposed by nucleosomal packaging

of DNA (Kwiatkowski et al. 2004). Chromatin immunoprecipitation (ChIP) assays
showed that EBNA2 recruits this complex to the LMP2A regulatory segment (Wu
et al. 2000). Moreover, the relocalization of hSWI/SNF complex found to be dependent
on binding of RBP-Jk to its recognition sequence, as well as on EBNA2 expression
(Wu et al. 2000; Hayward 2004). Additionally, EBNA2 also modulates the expression of the CD23 gene expression through recruitment of the hSWI/SNF complex
to its regulatory region (Wu et al. 2000).
EBNA3C also delicately controls chromatin structure by modulating cellular
chromatin remodeling machineries (Radkov et al. 1999; Cotter and Robertson 2000;
Knight et al. 2003). EBNA3C has been found to recruit histone deacetylase (HDAC1)
enzyme activity (Radkov et al. 1999) (Fig. 8.3). Interestingly, as both EBNA3C and
RBP-Jk share similar binding region to HDAC1, it has been suggested that through
recruitment of HDAC activity, EBNA3C serves as a bridge between RBP-Jk and
HDAC1 interaction, to repress the transcription activation from the viral Cp promoter
(Radkov et al. 1999). EBNA3C has also been found to interact with prothymosin a
(ProTa), a cellular protein known to interact with histones and likely to be involved
in the chromatin remodeling machinery (Gomez-Marquez and Rodriguez 1998;
Cotter and Robertson 2000). Moreover, EBNA3C also interacts with the cellular
HAT p300 (Cotter and Robertson 2000).Typically, addition of acetyl groups to the
core histone molecules results in disassociation of the compact chromatin structure,
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which renders the genetic regulatory sites accessible to the transcriptional machinery
(Shiama 1997; Giles et al. 1998). As a result, the interactions of EBNA3C with
ProTa and p300 suggest another important role for EBNA3C in modulating the
chromatin structure for transcriptional activation. On the contrary, EBNA3C expression downmodulates EBNA2-mediated increased HAT activity in a doseresponsive
manner (Cotter and Robertson 2000). In another study, EBNA3C was also shown to
form a complex with ProTa, HDAC1, HDAC2 and the transcriptional corepressors
mSin3A and NCoR (Knight et al. 2003) (Fig. 8.3). Thus, EBNA3C plays an
extremely important role during EBV-mediated immortalization of infected cells by
precisely balancing the overall transcriptional events.
EBV lytic cycle activator Zta is a bZIP protein shown to also have a role in stimulating nucleosomal HAT activity of the CREB binding protein (CBP) (Fig. 8.3). Zta
and CBP colocalize to viral immediate-early promoters and overexpression of Zta has
been found to lead to a robust increase in H3 and H4 acetylation at various regions of
the EBV genome (Deng et al. 2003). In a recent study, in EBV infected B-cells,
the latency-associated viral antigens have been suggested to inhibit expression of the
proapoptotic Bcl-2-family member Bim and enhance cell survival through epigenetic
regulations (Paschos et al. 2009). However, the actual viral latent protein involved in
this phenomenon is yet to be identified (Paschos et al. 2009). Along with controlling
chromatin transcription levels, EBV antigens can also modulate the higher order
chromatin structure formation. The EBV-encoded kinase BGLF4 has been shown to
induce cellular DNA condensation through condensin phosphorylation and topoisomerase II (Topo II) activation (Lee et al. 2007). EBV reactivation leads to cellular
chromatin condensation and interchromosomal space enlargement, and BGLF4 is
considered to be responsible for this activity as its expression in various cell types
caused a prophase-like individualized condensation pattern for chromatin (Lee et al.
2007). Overall, the reprogramming events that occur in infected B-cells facilitate EBV
persistence and the development of EBV-mediated carcinomas.
Cell-Cycle Progression
Cell-cycle progression is exclusively regulated by four major families of proteinsthe cyclins, the cyclin-dependent kinases (CDKs), retinoblastoma family of proteins
(pRb and the related pocket proteins), and the cyclin-dependent kinase inhibitors
(CDKIs) (Johnson and Walker 1999; Obaya and Sedivy 2002). In mammals, there
are at least nine CDKs and 16 cyclins, and each CDK combines with a specific
cyclin to generate an active holoenzyme (Ekholm and Reed 2000; Obaya and Sedivy
2002). The CDKs (CDK1, 2, 4, 6, and 7) are required for cell-cycle progression, and
they are expressed throughout the cell cycle, but their catalytic activity requires
binding to a cyclin, which acts as a positive regulatory subunit for determination of
the target specificity of the kinase. Cyclins can be categorized by the cell-cycle
phase in which they are expressed. The G1 cyclins (Cyclins D1, D2, D3, and E) are
required for G1 to S phase transition, followed by the activation of an S-phase cyclin
(Cyclin A) for the completion of S phase. Further, a mitotic cyclin (Cyclin B) facilitates
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Fig. 8.4 Deregulation of cell cycle phases by the EBV-encoded antigens. EBV-encoded latent
antigens EBNA2, EBNA3C, LMP1, and LMP2 deregulate different phases of cell cycle by
interacting with many important cell cycle regulators. These include the cyclins, cyclin inhibitors,
and the tumor suppressors, which include p53, SKp2, and p16
the entry into mitosis (M) phase from G2 (Morgan 1997; Saha et al. 2010c). The levels
of different cyclins oscillate throughout the cell cycle as a result of coordinated
synthesis and ubiquitin-proteasome-mediated degradation, ensuring the correct
temporal activation of each CDK and imposing directional irreversibility to the
progression of the cell cycle (Fig. 8.4) (Morgan 1997). In the G1 phase, an active
cyclin/CDK complex phosphorylates the pRb and related pocket proteins releasing
the E2F family of transcriptional factors (E2F1-8) and subsequently transactivates
an array of genes accountable for initiating DNA replication facilitating G1 to S
phase transition (Saha et al. 2010c). In the absence of mitogenic signals, CDKs
remain inactive, and are negatively controlled by a number of CDKIs including p15,
p14ARF/p16, p21WAF1/CIP1, and p27KIP1, which ultimately prevent aberrant cell-cycle
proliferation (Saha et al. 2010c).
In normal cells, the integrity of DNA replication is protected. This relies on the
p53 tumor suppressor protein along with other DNA damage response proteins to
repair the damage. In response to various genotoxic stresses, p53 either blocks the
cell cycle to allow repair or induces apoptosis if the cellular injury cannot be repaired
187
(Saha et al. 2009, 2010c). For example, p53 enhances transcription of the specific
CDKI, p21WAF1/CIP1, which in turn interacts with and inhibits CDK2 to arrest the cellcycle progression. Thus, p53 is one of the prime targets for oncogenic viruses and
is the most frequently mutated gene in human cancers (Saha et al. 2010c). Earlier, it
was shown that EBV fails to block p53-mediated apoptosis in LCLs induced by
variety of genotoxic agents, suggesting that, unlike other small DNA tumor viruses,
such as SV40 or HPVs, EBV does not compromise p53 function (Allday 2009). On the
contrary, it has also been shown that EBV can disrupt G1 cell-cycle checkpoint
by negatively acting downstream of p53-mediated pathway (Leao et al. 2007).
However, more recently, it has been clearly shown that EBNA3C can directly bind
to the p53 protein and repress its functions in part by blocking its transcriptional
activity as well as facilitating its degradation through stabilization of its negative
regulator, Mdm2 (Saha et al. 2009; Yi et al. 2009). In addition, EBNA3C has further
been shown to negatively regulate p53-mediated functions by interacting with its
regulatory proteins, the inhibitor of growth family proteins, ING4 and ING5, known
to be frequently deregulated in different cancers (Saha et al. 2010a).
To facilitate cell-cycle progression, EBNA3C also targets the CDKI p27KIP1 and
pRb for ubiquitin-proteasome dependent degradation through the recruitment of the
SCFSkp2 E3 ligase activity (Knight et al. 2005a, b). By disrupting p27KIP1 from Cyclin
A/CDK2 complexes, EBNA3C enhances CDK activity (Knight and Robertson
2004). EBNA3C can also override p16INK4A-mediated suppression during EBVmediated in vitro transformation, consistent with EBNA3C targeting the checkpoint
at the G1/S transition regulated by pRb (Saha et al. 2010c). As a result, similar to
the adenovirus-encoded E1A and HPV-encoded E7 oncoproteins, EBNA3C can
also cooperate with oncogenic mutant H-ras for immortalization and transformation
of rat embryonic fibroblasts (REFs) (Saha et al. 2010c). EBV encodes another oncoprotein LMP1, which when expressed can repress transcription from the p16INK4A
promoter. However, it did not have any significant effect on the p21WAF1/CIP1 promoter
(ONions and Allday 2004). Furthermore, it is known that constitutive expression of
the c-Myc oncoprotein in B-lymphocytes induces overall protein synthesis and cellcycle division. Additionally, c-Myc can also stimulate the expression of D-type
cyclins and cyclin E and downregulate p21WAF1/CIP1 and p27KIP1 (ONions and Allday
2004; Saha et al. 2010c). The EBV-encoded latent antigen, EBNA2 directly activates c-Myc further increasing transcription of the cyclin D2 gene, whose enhanced
expression is generally found in EBV-associated lymphomas (Saha et al. 2010c).
Earlier reports have shown that in vitro EBV infection or its transforming antigen
LMP1 upregulates only cyclin D2 but not cyclin D1 gene expression in primary
B-lymphocytes as well as BL cells (Saha et al. 2010b). However, by contrast, Cyclin
D1 protein level has been shown to be significantly expressed in a number of EBVpositive LCLs (Kim et al. 2002; Park et al. 2004) or SCID mice lymphomas (Murai
et al. 2001). Surprisingly, these abovementioned studies did not directly set out to
explore the contribution of Cyclin D1 in EBV-mediated B-cell oncogenesis.
Nonetheless, more recent studies have shown that EBNA3C stabilizes as well as
enhances the kinase activity of the Cyclin D1/CDK6 complex, as well as the nuclear
localization of Cyclin D1 to bypass the G1 restriction point (Saha et al. 2010b).
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Importantly, this particular study provides evidence that shows that EBNA3C
specifically targets Cyclin D1 activity, which subsequently nullifies pRb-mediated
growth suppression (Saha et al. 2010b). This describes a fundamental mechanism
by which EBV can deregulate the mammalian cell cycle in EBV-associated human
cancers (Saha et al. 2010b).
Diagnosis of EBV Infections and Malignancies

EBV is the causative agent of infectious mononucleosis and also contributes significantly to the pathologies of associated benign and malignant lesions. These include
oral hairy leukoplakia, inflammatory pseudotumor, HD, NHL, NPC, and GC (Kumar
et al. 2010). Molecular diagnosis of these diseases is very important for monitoring
and treatment of patients affected by these diseases. In biopsy tissues, in situ hybridization for EBV-encoded RNA transcripts is considered the gold standard for identification of EBV-related histopathological lesions and is routinely used with LMP1
immunostaining to detect latent EBV infections. Except for the case of immunocompromised patients, high serological titers may serve as tumor marker for EBV-related
malignancies (Gulley 2001). Because EBV elicits a rapid antibody response, testing
acute-phase serum samples for the presence of EBV specific antibodies provides a
means for detection of the early phase of EBV infection. Antibody tests for EBV can
measure the presence and/or the concentration of at least six specific EBV antibodies.
By evaluating the results of these different tests, the stage of EBV infection can be
determined (http://www.cdc.gov/ncidod/diseases/ebv.htm). EBV viral load testing
from blood samples by quantitative PCR is now a promising test for early diagnosis
and monitoring patients with PTLD (Tsai et al. 2008). Another new and powerful
approach of diagnosis of EBV-related pathologies is using gene expression profiling,
which provides the added advantage of subclassifying EBV-related diseases and
more comprehensive monitoring in response to therapy (Gulley 2001).
Treatment Options for EBV-Associated Malignancies

Treatment options for EBV-mediated tumors may include manipulating the balance
between outgrowing EBV-infected B cells and the EBV-cytotoxic T lymphocytes
(CTL) response, or targeting the B cells with monoclonal antibodies, chemotherapy
or radiation therapy.
Immunotherapy
EBV-associated tumors express viral encoded antigens and are excellent antigenpresenting cells, expressing high levels of immune system costimulatory molecules
(Heslop 2009). Therefore, one therapeutic option is to manipulate the immune
189
system to target and eradicate these malignancies. Immunotherapy with EBV-specific

CTLs has proved effective in PTLDs, which are highly immunogenic tumors
expressing type III latency (Fig. 8.1). The malignant cells in HD and NPC express
type II latency and hence a more restricted pattern of EBV antigens that suggests
less immunogenicity (Straathof et al. 2003).
Use of Cytotoxic T Cells

EBV-Specific T Cells
EBV-specific CTLs could be generated from EBV transformed LCLs. In a study
where donor-derived EBV-specific CTLs have been administered to more than 100
patients after hematopoietic stem cell transplantation (HSCT), the results suggest
that this approach is highly effective as prophylaxis in high-risk patients with a
history of PTLD or patients receiving selective T-cell depletion (Heslop et al. 1996;
Rooney et al. 1998; Heslop 2009).
Unmanipulated Donor T Cells

EBV-specific T-cell response can also be provided by infusing unmanipulated donor
lymphocytes from EBV-seropositive HSCT cell donors. This approach has been
shown to have a more than 70% response rate in HSCT patients with established
PTLD (Heslop 2009). However, it has an associated risk of inducing severe or fatal
Graft-versus-host disease (GVHD), as the frequency of alloreactive T cells in the
cell product has been found to be more than a log higher than the frequency of
virus-reactive T cells (Heslop et al. 1994; OReilly et al. 1997). In an approach to
circumvent this problem, T cells have been transduced with the thymidine kinase
(TK) suicide gene, which can be activated by infusion of ganciclovir if the recipient
develops GVHD. This approach has shown promising results in early-phase trials
(Heslop 2009).
Targeting B-Cells
Antibody Therapy
One strategy of prevention and treatment of EBV-associated tumors could be to
eliminate EBV-infected B cells. In this approach, B cell-specific surface antigens
present on the EBV transformed malignant cells are targeted with antibodies.
The most commonly used antibody for this purpose is a chimeric murine or human
190
monoclonal anti-CD20 antibody, Rituximab (Straathof et al. 2003). This antibody

has been used as prophylaxis and treatment for PTLD after HSCT, with promising
results showing initial response rates between 55% and 100% (Kuehnle et al. 2000;
van Esser et al. 2002; Powell et al. 2004; Brunstein et al. 2006). However, as CD20
expression is not confined to the malignant cells, normal B-cells are also targeted
resulting in marked immunosuppression. Fatal viral infections have also been
reported after rituximab therapy (Suzan et al. 2001; Heslop 2009).
Antiviral Agents
For the treatment of EBV infected patients, nucleoside analogs, which target the
virus-specific enzyme, thymidine kinase (TK) expressed in lytically infected cells,
are currently in use (Heslop 2009). However, as EBV tumors usually show a latent
pattern of gene expression and thus lack the TK expression, it makes antiviral therapy
alone ineffective as an antineoplastic therapy (Heslop 2009).
Radiation Therapy and Surgery

When the virus associated tumor is confined to a single site, radiation and/or surgery
can be effective. Surgery and radiation may also have a role in managing local
complications because of malignancies, such as compression of vital organ structures
(Heslop 2009).
Chemotherapy
Chemotherapy with regimens used in lymphoma therapy, such as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), could be a therapeutic
option for patients who do not respond to immune manipulation or rituximab
(Heslop 2009). However, chemotherapy has the risk of putting the patient at
increased risk of infection particularly when they have preexisting immune suppression (Heslop 2009).
Vaccination Against EBV

The development of an EBV vaccine may be protective against primary infection
and hence presumably reduce the burden of EBV-associated cancers. EBV neutralizing
antibodies mainly targeting the major virus surface glycoprotein gp350/220 and
191
several vaccine candidates based on gp350/220 have been successfully developed

(http://www.who.int/vaccine_research/diseases/viral_cancers/en/index1.html). Live
recombinant vaccinia virus vectors have also been used to express the gp350/220
antigen and have been found to confer protection in primates and elicit antibodies in
EBV-negative Chinese infants (World health organization Web site: http://www.
who.int/vaccine_research/diseases/viral_cancers/en/index1.html). Soluble recombinant
gp350/220 produced in CHO cells was found to be safe in humans; however, they
need strong adjuvants to elicit acceptable immunogenicity (codevelopment by
MedImmune, GSK and Henogen) (World health organization Web site: http://www.
who.int/vaccine_research/diseases/viral_cancers/en/index1.html). Phase II clinical
trials of this candidate vaccine are under way. Clinical trials of an EBNA-3A
peptide are also being conducted in Australia (World health organization Web site:
http://www.who.int/vaccine_research/diseases/viral_cancers/en/index1.html)
Conclusion
Capability of lifelong asymptomatic persistence in their host keeps LCVs among
the most successful category of viruses. Deregulated immune system of host,
however, makes the hibernating virus exert its pathogenic role, mainly contributing
to induction of a range of cancers in the host. Its infectivity in the human host makes
EBV one of the most well-studied LCV. Extensive molecular studies revealed that
EBV-virulence is distributed among a number of proteins and RNA, encoded by its
genome, with specific antigens contributing more to the critical functions than
others. EBV proteins identified as essential for host-cell transformation have been
thought to provide the scope or molecular blueprint for target based drug design to
treat the EBV-induced pathologies. However, to date the therapies implicating the
manipulation of the immune system have appeared to be more progressive and
promising. Combinatorial therapy designed on the basis of the clinical condition of
patient may be more useful in the majority of the cases, as it may have a higher
potential for success.
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Chapter 9
Nonhuman Primate Gamma-herpesviruses

and Their Role in Cancer
Ryan D. Estep and Scott W. Wong
Introduction
The Gamma-herpesvirinae are a subfamily of lymphotropic herpesviruses that infect
and replicate mainly in lymphoid cells and are capable of causing cellular transformation. Importantly, viruses belonging to this subfamily have been associated with oncogenesis in both humans and nonhuman primates, and have been linked to such human
diseases as nasopharyngeal carcinoma, Burkitts lymphoma, Kaposis sarcoma, multicentric Castlemans disease, and non-Hodgkins lymphoma. The contributions of
gamma-herpesvirus infections to the development of other cancers are also a possibility,
and thus, an intense focus has been placed on these viruses to better understand the
mechanisms by which they induce oncogenesis. Overall, the gamma-herpesviriniae
represent examples of fine tuned virushost relationships, in which typical infection of
natural host species is relatively harmless, and only under specific and much less
frequent circumstances, may be capable of promoting the development of cancer.
The gamma-herpesvirus subfamily can be further divided into the lymphocryptovirus (or g-1) genus and the rhadinovirus (or g-2) genus, based on genomic
organization and sequence homology. In humans, the lymphocryptovirus genus is
R.D. Estep
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th
Avenue, Beaverton, OR 97006, USA
S.W. Wong (*)
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th
Avenue, Beaverton, OR 97006, USA
Division of Pathobiology and Immunology, Oregon National Primate Research Center,
Beaverton, OR, USA
Department of Molecular Microbiology and Immunology, Oregon Health & Science University,
Portland, OR, USA
e-mail: wongs@ohsu.edu
201
202
0
R.D. Estep and S.W. Wong

10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180 190
Kilobases
EBV/RhLCV
HVS
KSHV
RRV
Fig. 9.1 Alignment of specific primate gamma-herpesviruses that have been sequenced. Conserved
blocks of genes are indicated (black) along with genes that are unique (white) to the each gammaherpesvirus. The genomes of EpsteinBarr virus and rhesus lymphocryptovirus are represented as
one, as they are nearly identical (Rivailler et al. 2002)
represented by EpsteinBarr virus (EBV), while the rhadinovirus genus is represented by Kaposis sarcoma-associated herpesvirus (KSHV). Lymphocryptoviruses
with similarity to human EBV have been identified in many species of Old- and
New-World monkeys (Dunkel et al. 1972; Kalter et al. 1972; Landon and Malan
1971; Levy et al. 1971; Naito et al. 1971), and similarly, rhadinoviruses have been
identified in many primates (Damania and Desrosiers 2001; Greensill et al. 2000;
Lacoste et al. 2000a, b; Melendez et al. 1968; Simas and Efstathiou 1998). Species
specificity appears to preclude infection of nonhuman primates with human gammaherpesviruses (Renne et al. 2004; Wang et al. 2001), thus preventing the study of the
mechanisms of pathogensis of these human viruses in primate model systems.
However, due to the discovery of highly related primate counterparts of these human
viruses, and their ability to promote the development of similar disease sequelae as
their human counterparts, these primate viruses provide relevant model systems for
understanding and deciphering the oncogenic mechanisms of these herpesviruses
in vivo. Rhesus macaque lymphocryptovirus (LCV), Herpesvirus saimiri (HVS),
and Rhesus macaque rhadinovirus (RRV) are discussed in the following sections as
models of primate gamma-herpesviruses oncogenesis that are important for understanding mechanisms of primate herpesvirus oncogenesis. To illustrate the relatedness of the simian gamma-herpesviruses to the human, the genomes of the viruses
are depicted in Fig. 9.1.
Herpesvirus Saimiri
Herpesvirus saimiri (HVS, saimirine herpesvirus 2) is the prototypical rhadinovirus,
the natural host of which is the squirrel monkey (Saimiri sciureus). The virus was
originally isolated from a kidney cell culture of a healthy squirrel monkey (Melendez
et al. 1968) and was also identified in peripheral blood cells of persistently infected
Nonhuman Primate Gamma-herpesviruses and Their Role in Cancer
203
squirrel monkeys by coculture with permissive cell lines such as owl monkey kidney
(OMK) cell line (Desrosiers and Falk 1982). In the wild, most squirrel monkeys are
naturally infected with HVS within the first two years of life and remain asymptomatically and persistently infected for the remainder of their lives, without the development of any overt disease (Melendez et al. 1968). Although infections of squirrel
monkeys with HVS are generally asymptomatic, the virus can cause fatal T cell
lymphomas in other New-World primates, including, marmosets, tamarins, and owl
monkeys, as well as Old-World monkeys, including rhesus macaques and cynomolgus
monkeys (Fickenscher and Fleckenstein 2001), suggesting that in its natural host
HVS is not oncogenic, but when introduced into other nonhost species, the virus has
the potential to produce cancer.
HVS strains are classified into three subgroups (A, B, and C), based on their
pathogenic qualities and genomic similarity. While most genes are well conserved
amongst different HVS strains, there is extensive sequence divergence at the left
end of the HVS L-DNA of the different subgroups. Subgroup C viruses possess the
highest oncogenic properties, and are the only subgroup that is capable of immortalizing
human, rabbit, and rhesus monkey lymphocytes, and causing fulminant lymphomas
in both Old-World and New-World primates. The genomes of HVS strains A11 and
C488 have been fully sequenced (Albrecht et al. 1992; Ensser et al. 2003), with both
HVS genomes containing 7677 open reading frames (ORFs). The genomes of all
HVS strains possess homologues of several cellular genes, which are thought to
have been pirated from host cell genes during evolution of the virus. These genes
include those involved in various processes, such as regulation of cellular growth
and proliferation (e.g., viral cyclin), nucleotide metabolism (e.g., viral dihydrofolate
reductase [DHFR]), complement cascade activation (e.g., viral complement control
protein homologue [CCPH]), apoptosis (e.g., viral Bcl-2), and cytokine signaling
(e.g., viral IL-17 and vGPCR) (Fickenscher and Fleckenstein 2001). All of these
genes could potentially contribute to the oncogenic potential of the virus, although
evidence appears to suggest that they are not required for T cell transformation and
pathogenicity, and are likely more important for persistence of the virus within its
natural host species. However, a gene encoding a protein with limited homology to
any cellular protein is located near the left end of the genome of all three HVS
subtypes. This protein, termed the Saimiri transformation-associated protein (Stp),
has been shown to play a critical role in direct transformation of T cells by HVS
in vitro and in vivo (Duboise et al. 1998; Jung and Desrosiers 1991). Interestingly,
although Stp of HVS subgroup A and C (StpA and StpC) are both transforming
in vitro, with StpC displaying a higher transforming potential than StpA, Stp of
HVS subtype B (StpB) does not appear capable of inducing transformation (Choi
et al. 2000; Jung et al. 1991). These findings correlate closely with the oncogenic
potential of the different HVS subtypes in vivo and indicate the importance of Stp
to viral oncogenesis (Tsygankov 2005). StpC has been extensively studied and has
been found to be a perinuclear membrane protein that is capable of interacting with
and activating host cell signaling pathways, including the Ras-ERK pathway and
NF-kB pathways (Jung and Desrosiers 1995; Lee et al. 1999; Tsygankov 2005).
Thus, as a result of alteration of normal cellular signaling, expression of StpC may
204
result in cellular transformation. In addition to Stp, Subgroup C strains also encode

another transforming protein termed the tyrosine kinase-interacting protein (Tip),
which has also been found to be important for viral transformation. The tip gene is
located directly downstream from the stp gene and is transcribed as part of a single
bicistronic mRNA that encodes both proteins. Tip was initially identified as a
protein that interacted with a T cell-specific tyrosine kinase Lck (Biesinger et al.
1995; Fickenscher and Fleckenstein 2001). Indeed, Tip has been shown to activate
Lck (Wiese et al. 1996) and, in addition, has been found to be capable of activating
STATs, NF-AT, and the nuclear mRNA export protein Tap (Hartley and Cooper
2000; Isakov and Biesinger 2000; Lund et al. 1997; Yoon et al. 1997). Although Tip
is also capable of inducing T cell lymphomas in transgenic mice (Wehner et al.
2001), the exact role that Tip may play in cellular transformation remains unclear
(Tsygankov 2005). However, based on in vivo studies using Stp and Tip deleted
forms of HVS, it is apparent that both proteins are essential for T cell transformation
of subgroup C viruses in vivo and in vitro (Duboise et al. 1998). The fact that subgroup
C viruses encode two highly oncogenic viral proteins may also partly explain why
these viruses are more oncogenic in vivo than subgroup A and B viruses.
Although there is no true counterpart to HVS that has been identified in humans,
the value of this animal model is still extremely important. The mechanisms by
which HVS is capable of existing within its host population with no apparent disease
manifestation yet remains capable of inducing lymphoma in other nonhost species
suggest a close coevolution of virus and host that allows viral persistence and spread
with no overt symptoms in the host. Understanding the mechanisms by which both
the virus and natural host maintain this close relationship, and what exactly prevents
induction of viral-related disease, provides an important model to help understand
species restriction in terms of oncogenic herpesviruses, and could provide insight
into potential targets for treatment and prevention of herpesvirus-induced cancers.
Rhesus Macaque Lymphocryptovirus

Several lymphocryptoviruses have been identified in both Old- and New-World
monkeys based upon cross-reactivity with antibodies directed against human EBV
proteins, due to the conservation of sequences between these viruses (Dunkel et al.
1972; Kalter et al. 1972; Landon and Malan 1971; Levy et al. 1971; Naito et al. 1971).
Rhesus macaque LCV (RhLCV), or Cercopithecine herpesvirus 15, is emerging as a
useful animal model for studying EBV infection and disease development in vivo using
the rhesus macaque as a model (Carville et al, 2008). RhLCV, like EBV in humans, is
spread through oral secretions, and is generally ubiquitous within captive rhesus
macaque populations, with essentially all animals seroconverting by the time they
reach 2 years of age (Carville and Mansfield 2008). Two distinct lineages of RhLCV
(RhLCV1 and RhLCV2) have been identified (Cho et al. 1999), and the complete
sequence of the RhLCV1 genome has recently been determined (Rivailler et al. 2002).
The RhLVC genome is 171,096 nucleotides in length, with a GC content of 62%,
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Fig. 9.2 Lymphoma and retroperitoneal fibromatosis (RF) in RRV-infected rhesus macaques.
(a) Hematoxylin and eosin (H&E) stain of lymphoma isolated at biopsy from the breast of animal
19185, original magnification 1,000. (b) Combined in situ hybridization of lymphoma with RRV
cosmid probe (purple) and immunohistochemistry with mouse anti-CD20 (brown) and anti-CD3+
(gray) demonstrates that RRV is present in CD20+ cells comprising the lymphoma, original
magnification 1,000. (c) Same as (b), except cosmid vector control replaces the RRV cosmid
probe. (d) H&E stain of lymphoma isolated from liver of animal 19286 during necropsy, original
magnification 200. (e) Same as (b) except RRV cosmid probe detected by immunofluorescence
(purple) with no evidence of CD20 staining. (f) Similar to (c), this is the control for panel (e).
(g) H&E stain of RF attached to the stomach of animal 18483, original magnification 400.
(h) Combined in situ hybridization with RRV cosmid probe (purple) and immunohistochemistry
with anti-CD20 (brown) and anti-CD3 (gray) shows that RRV is not present in the CD20- or CD3positive cells, original magnification 1,000. Arrows point to RRV-positive cells that have spindlelike morphology. (i) Identical to (h), except vector control replaces the RRV cosmid, original
magnification 1,000. (This research was originally published in Blood. Orzechowska et al. (2008)
Rhesus macaque rhadinovirus-associated non-Hodgkins lymphoma: animal model for KSHV
associated malignancies. 112:42274234. the American Society of Hematology)
which closely compares to the genome of EBV. Further, the overall nucleotide homology between the RhLCV and EBV genomes is 65%, and the genetic organization of
these viruses is colinear (Fig. 9.2). RhLCV encodes eighty open reading frames (ORFs),
all of which share some level of homology to genes in human EBV, with the average
homology between EBV and RhLCV ORFs being 75.6% (Rivailler et al. 2002).
206
Human EBV is generally spread via oral secretions, and initially infects and
replicates in the oropharynx, with the subsequent development of a generally
asymptomatic and persistent latent infection of peripheral blood B lymphocytes.
In addition to the typical disease associated with primary infection, EBV is capable
of inducing transformation of infected cells, and has been linked to the development
of several human malignancies, including Burkitts lymphoma, Hodgkins lymphoma,
and nasopharyngeal carcinoma (Fields et al. 2007). Previous attempts to infect
nonhuman primates with human EBV have been generally unsuccessful and may
largely be due to species-specific restrictions that prevent LCVs from immortalizing
B cells other than those derived from their natural host species (Moghaddam et al.
1998; Wang et al. 2001). Thus, infection of nonhuman primates with LCVs specific
to the host species in question has been examined as a more relevant experimental
model system to better understand EBV infection in vivo. Recently, RhLCV has
emerged as a feasible model, allowing for the study of LCV infection and pathogenesis
in rhesus macaques.
RhLCV infection of rhesus macaques has been shown to result in disease manifestations very similar to those associated with EBV infection in humans. For example,
experimental oral inoculation of nave rhesus macaques with RhLCV results in the
development of lympadenopathy, lymphocytosis, and an increase in CD23+ B cells,
all of which are symptoms of primary EBV infection and mononucleoisis in humans
(Moghaddam et al. 1997). Further, RhLCV is also capable of infecting peripheral
blood B cells, in which it establishes a persistent latent infection, with occasional
reactivation of virus from these cells, followed by shedding of virus in oral secretions.
Thus, primary infection of RhLCV mirrors very closely the symptoms of primary
EBV infection in humans. In addition to disease associated with acute RhLCV
infection, several RhLCV-associated malignancies have also been shown to occur in
rhesus macaques coinfected with SIV, and are similar to EBV-associated cancers
that frequently occur in AIDS patients. For example, B cell tumors can develop in
SIV-infected rhesus macaques that display many similarities to some Non-Hodgkins
lymphomas (NHLs) seen in AIDS patients that are associated with EBV infection
(Carville and Mansfield 2008; Shibata et al. 1993). SIV-associated NHLs show a
similar pattern of development to those seen in HIV-infected patients, and direct
examination of these lymphomas for the presence of RhLCV sequences demonstrates a strong association of the virus with their development (Carville and
Mansfield 2008; Habis et al. 1999, 2000). In addition to lymphoma, other manifestations such as squamous epithelial proliferative lesions have also been found to
develop in SIV-infected rhesus monkeys and appear similar to oral hairy leukoplakia
that is commonly associated with EBV infection in humans. Indeed, these lesions
have been found to contain EBV-like sequences by immunohistochemistry and in
situ hybridization, suggesting a causative role for RhLCV in this disease in rhesus
macaques (Baskin et al. 1995). Also of importance, experimental studies involving
inoculation of LCV-seronegative macaques with RhLCV-immortalized B cells indicate that RhLCV infection of immunnosuppressed animals results in the induction
of lymphomagenesis (Rivailler et al. 2004). Taken together, these studies provide
strong evidence that RhLCV infection and disease development in rhesus macaques
207
closely parallels EBV infection in humans, and thus provides a novel system to
allow for the analysis of the mechanisms of LCV pathogenesis in vivo.
Several EBV-encoded genes have been implicated as major regulators of viral
transformation and oncogenesis, namely, the latently expressed EpsteinBarr nuclear
antigens (EBNAs) and latent membrane proteins (LMPs) (Farrell 1995). Although
these genes are normally important for establishment of latency and persistence of
virus within infected cells, under certain conditions expression of these genes can also
result in cellular transformation, leading to aberrant cell growth and ultimately the
development of viral-related cancers (Young and Murray 2003). Importantly, RhLCV
possesses homologues of these EBV genes, suggesting that these two highly related
viruses may have similar mechanisms of transformation in vivo (Wang et al. 2001).
Indeed, in vitro studies of several RhLCV oncogenes indicate that they may function
similarly, if not identically, to their EBV counterparts. For example, RhLCV LMP2A
protein, which is 62% identical to EBV LMP2A, has been shown to be capable of
activating cellular signaling pathways and inhibiting differentiation of epithelial cells,
suggesting similar properties to its EBV counterpart and a possible role in pathogenesis (Siler and Raab-Traub 2008). RhLCV EBNA genes have also been examined and
have been shown to share similar structural and functional similarities to the homologous EBV genes (Jiang et al. 2000; Peng et al. 2000). Further, analysis of tumors from
RhLCV-infected rhesus macaques indicates that expression of RhLCV homologs of
EBV transforming genes are associated with development of malignant lymphomas
(Blaschke et al. 2001). Therefore, as is the case for EBV, expression of transforming
viral genes during infection may result in the development of viral cancers in vivo.
In addition to the known transforming genes in EBV, other viral factors conserved
between EBV and RhLCV with potential roles in oncogenesis are likely yet to be
identified. This is evidenced by the recent discovery of several noncoding viral
microRNAs (miRNAs) in RhLCV that share significant homology to those found in
EBV (Cai et al. 2006). miRNAs have been identified in numerous organisms and
viruses and are capable of posttranscriptionally regulating gene expression via interactions with target mRNAs (Gottwein and Cullen 2008). Importantly, miRNAs have
also been proposed to be involved in the development of cancer (McManus 2003), and
given that viral miRNAs play roles in regulating the expression of both viral and cellular genes, it is plausible that in some cases, viral miRNAs such as those encoded by
RhLCV may be involved in viral oncogenesis (Dykxhoorn 2007; Gottwein and Cullen
2008). Further analysis will be necessary to determine the exact role of RhLCV and
EBV miRNAs in viral oncogenesis.
Since RhLCV is capable of inducing disease manifestations in infected rhesus
macaques similar to those seen in EBV-infected humans, and given the high genetic
similarity of these viruses, RhLCV provides a highly relevant and functional animal
model system that allows for in vivo analysis of the mechanisms of EBV-associated
pathogenesis and oncogenesis. Further experimental analysis of RhLCV using this
system may lead to a better understanding of the exact mechanisms of viral transformation, and allow for the determination of roles of individual viral genes in oncogenesis. Most importantly, the use of the RhLCV/rhesus macaque model will allow for
208
the development and testing of potential vaccine strategies and therapeutics to prevent
or treat LCV-induced malignancies.
Rhesus Macaque Rhadinovirus

Rhesus macaque rhadinovirus (RRV) is a g-herpesvirus of rhesus macaques, which
has been shown to be closely related to human herpesvirus 8 (HHV-8)/Kaposis
sarcoma-associated herpesvirus (KSHV). KSHV is the causative agent of Kaposis
sarcoma (KS), as well as B cell lymphoproliferative disorders (LPD) primary effusion
lymphoma (PEL), multicentric Castlemans disease (MCD), and some non-Hodgkins
lymphomas (Cesarman et al. 1995; Chang et al. 1994; Oksenhendler et al. 2002;
Soulier et al. 1995), diseases that are frequently associated with HIV infection.
An RRV strain (RRV17577), was obtained from a simian immunodeficiency virus
(SIV)-infected macaque that had developed B cell hyperplasia, and upon isolation
and characterization of this herpesvirus isolate, the RRV17577 genome was found to
be essentially colinear with KSHV, retaining many of the ORFs believed to be
involved in the pathogenesis of KSHV (Searles et al. 1999) (Fig. 9.2). Specifically,
67 of 79 ORFs are similar to those in KSHV. The general structure of the RRV17577
genome is similar to other herpesviruses and consists of a long unique region (LUR)
of ~131 kb in length, flanked on both ends by regions of terminal repeats.
RRV is a natural pathogen of macaques, with greater than 90% of captive rhesus
macaques having been found to be seropositive for RRV (Damania and Desrosiers
2001). RRV transmission between susceptible hosts is believed to occur via shedding
in saliva, and similar to KSHV, RRV is capable of establishing a latent infection in
B cells (Bergquam et al. 1999). KSHV infection of primates has thus far proven
unsuccessful, and due to the high genetic similarity and pathogenic properties to
KSHV, RRV infection of rhesus macaques has become a widely utilized animal
model to study KSHV disease development in vivo, particularly in the setting of
SIV induced immunodeficiency. Experimental inoculation of rhesus macaques with
RRV17577 that have previously been infected with SIVmac239 promotes the development
of B cell hyperplasia, persistent lymphadenopathy, splenomegaly, and hypergammaglobulinemia (Bergquam et al. 1999; Wong et al. 1999). The hyperplastic
lymphoproliferative disorder (LPD) in RRV-infected macaques closely resembles
the plasma cell variant of multicentric Castlemans disease B cell hyperplasia seen
in humans infected with KSHV. More recently, RRV infection has also been associated with lymphomagenesis and a mesenchymal cell proliferative lesion in SIVinfected rhesus macaques (Orzechowska et al. 2008). Thus, RRV infection of
immune compromised rhesus macaques is an invaluable model for KSHV-associated
disease development that is utilized to dissect the mechanisms of viral pathogenesis
and to develop potential targets for vaccine development against KSHV.
Numerous genes in KSHV have been implicated in oncogenesis, and a majority
of these genes are conserved in the RRV genome (Searles et al. 1999; Wen and
Damania 2010). Many of these viral oncogenes are homologues of cellular genes,
209
from which they are believed to have been pirated during evolution of the virus, and
are capable of affecting numerous processes, such as cellular proliferation (e.g.,
viral Cyclin) (Li et al. 1997), apoptosis (e.g., viral Bcl-2) (Sarid et al. 1997), and
immunoregulation (e.g., viral interferon regulatory factors/vIRFs) (Gao et al. 1997).
In general, the main function of these viral genes is likely not to induce transformation
per se, but rather to help establish an environment that favors viral infection, replication,
and persistence within an infected host. However, due to the fact that these genes
dysregulate normal cellular function and immune control, their expression may also
result in cellular transformation and the development of cancer. Examination of
several RRV homologues of KSHV genes has indicated that these genes retain similar
function to their KSHV counterparts and, thus, are likely to play similar roles in
regard to oncogenesis in vivo. For example, the KSHV encoded vIL-6 molecule has
been shown to be a functional homologue of cellular interleukin 6 (IL-6), and
expression of this protein has been associated with KS, PEL, and MCD (Staskus
et al. 1999). RRV also encodes a vIL-6 homologue, and studies indicate that RRV
vIL-6 is capable of promoting B cell proliferation in vitro (Kaleeba et al. 1999) and
that expression of this viral protein is associated with the development of lymphoproliferative and mesenchymal cell proliferative disorders in RRV-infected rhesus
macaques (Orzechowska et al. 2008, 2009) (Fig. 9.3). Another RRV gene, ORF74,
which encodes the viral G protein-coupled receptor (GPCR), shares sequence similarity to the KSHV vGPCR, a protein that has been suggested to play a major role
in viral oncogenesis (Arvanitakis et al. 1997; Bais et al. 1998; Cannon 2007;
Cesarman et al. 1996). The vGPCRs of KSHV and RRV are homologues of CXCR2,
the cellular receptor for IL-8, and are thus capable of regulating cellular signaling
pathways in cells in which they are expressed. Like the KSHV vGPCR, the RRV
vGPCR has been shown to possess constitutive and ligand-dependent signaling
abilities, promote cytokine secretion, and induce transformation of cells in which it
is expressed (Estep et al. 2003). Thus, the RRV vGPCR is likely to contribute to the
development of RRV-associated malignancies in a fashion similar to the vGPCR of
KSHV. Other RRV genes with no apparent cellular counterparts are also likely to
play a role in viral transformation. For example, the R1 protein of RRV, which
shares similarity to KSHV K1, has been shown to be transmembrane signaling
proteins with oncogenic potential in vitro and in vivo (Damania et al. 1999, 2000).
In addition, RRV encodes several microRNAs miRNAs that may be involved in
pathogenesis, with the majority sharing no apparent homology to those that were
similarly identified in KSHV (Schafer et al. 2007; Umbach et al. 2010). Importantly,
all of the RRV miRNAs that have been identified have been found to be expressed
within B cell lymphoma and retroperitoneal fibromatosis tissues isolated from RRVinfected macaques, suggesting that the expression of these miRNAs may be involved
in the development of RRV related cancers (Umbach et al. 2010). Further in vivo
analysis of the roles of the numerous RRV-encoded oncogenes will be the focus of
future research, particularly through the creation of recombinant forms of RRV with
mutated or deleted forms of these genes, and the use of the rhesus macaque model
of infection for the assessment of effects on disease development (Estep et al. 2007).
By studying the function of these viral oncogenes in the context of an in vivo infection
210
Fig. 9.3 Detection of vIL-6 in RRV-associated lymphomas, RF and MCD. Lymphoma from RM
19185 was stained with murine monoclonal antibodies specific for RRV vIL-6 and CD20 (a),
lymphoma from animal 19286 was stained with murine monoclonal antibodies specific for RRV
vIL-6 and goat anti-human IgM, and nuclei (c). (b) and (d) are isotype controls, original magnification 630. Panel (a), vIL-6 (green) and CD20 (red); panel (b), isotype controls for vIL-6 and
CD20; panel (c), vIL-6 (green) and IgM (red), and nuclei (blue); and panel (d), isotype controls for
vIL-6 and IgM. RF from animal 18483 was stained for vIL-6 (green), vimentin (red), and nuclei
(blue) (panel e), original magnification 630. MCD lesion from animal 19455 was stained for
vIL-6 (green), CD20 (red) and nuclei (blue) (panel f), original magnification 630. (This research
was originally published in Blood. Orzechowska et al. (2008) Rhesus macaque rhadinovirusassociated non-Hodgkins lymphoma: animal model for KSHV-associated malignancies.
112:42274234. the American Society of Hematology)
211
in a primate model that closely recapitulates KSHV infection of humans, the contributions of individual genes to viral pathogenesis can be examined, and this in turn
will provide unique insight into the roles they and their KSHV counterparts may
play in oncogenesis.
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Chapter 10
Rhadinoviruses: KSHV and Associated

Malignancies
Susann Santag and Thomas F. Schulz
Kaposis Sarcoma Herpesvirus

Kaposis Sarcoma Herpesvirus (KSHV), also termed human Herpesvirus 8 (HHV8),
was discovered by Y. Chang and P. Moore in a Kaposis Sarcoma tissue of an AIDS
patient (Chang et al. 1994). KSHV is the only known Rhadinovirus capable of
infecting humans. It is known to cause three human neoplastic diseases and may be
involved in other clinical manifestations.
Origin and Evolution

KSHV is today of low prevalence (05%) in Northern Europe (Preiser et al. 2001),
most of North America (Baillargeon et al. 2001; Kouri et al. 2004; Engels et al. 2007)
and most of Asia (de Sanjose et al. 2009), of intermediate prevalence (520%) in
countries around the Mediterranean Sea (Whitby et al. 1998), in parts of South
America (Mohanna et al. 2005) and in some ethnic populations [e.g. some South
American Indian tribes (Biggar et al. 2000; Whitby et al. 2004; Mohanna et al. 2007),
South Siberia (Cassar et al. 2010) or Xinjiang in China (Dilnur et al. 2001)] but of
high prevalence (2080%) in most countries of sub-Saharan Africa (Dedicoat and
Newton 2003; Klaskala et al. 2005; Adjei et al. 2008).
There is good evidence that KSHV has co-evolved with human populations.
Thus, sequence variation, mainly in the variable K1 gene of KSHV, has been used
to define five lineages (AE) and additional subgroups (Hayward 1999). Of these,
lineage B and subtype A5 are commonly found in sub-Saharan Africa, lineage D in
S. Santag T.F. Schulz (*)

Institute of Virology, Hannover Medical School, Hannover, Germany
e-mail: Schulz.Thomas@mh-hannover.de
215
216
S. Santag and T.F. Schulz
old Australasian populations (Cassar et al. 2007) and lineage E in native South
American populations (Biggar et al. 2000). This pattern suggests that KSHV spread
across the globe with migrating human populations. Why KSHV should have
become infrequent in many populations and geographic areas, while remaining
highly prevalent in some, remains unexplained.
Transmission and Risk Factors

Horizontal transmission via saliva seems to be a common route of infection (Vieira
et al. 1997; Cattani et al. 1999; Dedicoat et al. 2004; Casper et al. 2006). In endemic
areas, infection occurs frequently during childhood and increases with age (Mayama
et al. 1998; Whitby et al. 2000; Cunha et al. 2005; Plancoulaine et al. 2000; MalopeKgokong et al. 2010).
In addition, sexual transmission of KSHV contributes to its spread in endemic
countries (Butler et al. 2009) and appears to be the main transmission route among
individuals at increased risk of sexually transmitted disease both in endemic and
non-endemic countries. Less important routes of infection include injection drug
use and blood borne transmission (including transmission via transfusion) as well as
organ transplantation (Atkinson et al. 2004; Hladik et al. 2006; Cannon et al. 2009;
Vamvakas 2010).
KSHV Associated Diseases

Table 10.1 summarises the disease manifestations that have been attributed to KSHV
infection. For three neoplastic diseases, Kaposis Sarcoma (KS), primary effusion
lymphoma (PEL) and the plasma cell variant of multicentric Castlemans disease
(MCD), a causative role of KSHV is accepted. A recent evaluation of the available
epidemiological and molecular evidence by an IARC (International Agency for
Research on Cancer)/WHO working group led to the classification of KSHV as a
group I carcinogen (Bouvard et al. 2009). Reported manifestations of primary
KSHV infections are based on relatively few case reports and small studies (see
below). In addition, a long list of clinical conditions has been attributed to KSHV
infection, but hardly any have been corroborated by a sufficient number of laboratories
for such associations to have become generally accepted.
KSHV Primary Infection

Several case reports suggest that KSHV primary infection in HIV-infected patients or
organ transplant recipients can occasionally lead to fever, arthralgia, cervical lymphadenopathy, splenomegaly and pancytopenia, which spontaneously resolves within
10
Rhadinoviruses: KSHV and Associated Malignancies
217
Table 10.1 Spectrum of KSHV-associated diseasesa

Stage of infection/KSHV association Disease
Primary infection
Mostly asymptomatic; on rare occasions infectious
mononucleosis-like illness, KS, MCD or rare cases of
bone marrow failure in immunosuppressed patients
(section KSHV Primary Infection)
Accepted causative role/strong
KS (Newton et al. 2006) (all four types) (section
association
Kaposi Sarcoma)
Primary effusion lymphoma (PEL) (Cesarman et al.
1995) (section Primary Effusion Lymphoma)
Plasma cell variant of multicentric Castlemans disease
(MCD) (Soulier et al. 1995) (section Multicentric
Castlemans Disease)
Plasmablastic lymphoma arising from MCD (Dupin
et al. 2000; Oksenhendler et al. 2002; Dargent et al.
2007; Ustun et al. 2009)
Unconfirmed associationb
Multipe Myeloma (Rettig et al. 1997; Said et al. 1997);
could not be confirmed by others (MacKenzie et al.
1997; Brander et al. 2002)
Mesenchymal tumours (1 case KSHV-positive out of
76) (Kazakov et al. 2002)
Hemophagocytic syndrome (Luppi et al. 2002); could
not be confirmed by others (Mikala et al. 1999)
Other lymphomas (Lazzi et al. 1998; de Sanjose et al.
2004; Lazzi et al. 2006)
Large-cell centroblastic lymphomas (Hansen et al.
2000)
Autoimmune bullous dermatoses:
Pemphigus vulgaris and pemphigus folicaeus (Tye
1970; Memar et al. 1997)
Bullus phemigoid (Gaspari et al. 1997)
Idiopathic pulmonary arterial hypertension (IPAH)
(Cool et al. 2003); could not be confirmed by others
(Henke-Gendo et al. 2005; Katano et al. 2005)
Sarcoidosis (Di Alberti et al. 1997); could not be confirmed by others (Lebbe et al. 1999)
Kikuchis disease (Huh et al. 1998); could not be confirmed by others (George et al. 2003; Cho et al. 2007)
Prostate cancer (Hoffman et al. 2004); could not be
confirmed (Jenkins et al. 2007)
a
KSHV association was based on PCR, IgG antibody responses, in situ hybridisation on tumour
cells and immunohistochemistry on tissue sections
b
On the basis of KSHV detection by PCR or immunohistochemistry in some, but not most, studies
the course of several weeks (Oksenhendler et al. 1998; Luppi et al. 2000). In a study
of HIV1-negative men, primary infection was associated with mild, non-specific
signs and symptoms of diarrhoea, fatigue, localised rash and lymphadenopathy
(Wang et al. 2001). Goudsmit and colleagues showed that within a Dutch MSM (men
who have sex with men) cohort, KSHV seroconversion in HIV-infected patients
218
follows a brief, low-grade viremia which was mostly asymptomatic (Goudsmit et al.
2000). In an endemic region (Egypt), Andreoni and colleagues observed that primary
KSHV infection in immunocompetent children can be symptomatic and associated
with a transient febrile skin rash (Andreoni et al. 2002). These results were confirmed
by a study analysing primary g-herpesviral infection among Zambian children,
showing a weak association between the history of rashes in children with primary
KSHV infection (Minhas et al. 2010). These findings indicate that generally mild
symptoms can occur during primary infection of immunocompetent individuals and
that these can be more severe in immunocompromised patients.
Kaposi Sarcoma
In 1872, the Hungarian dermatologist Moritz Kaposi published a description of an
entity termed idiopathic multiple pigmented sarcoma of the skin (Kaposi 1872),
which was afterwards renamed Kaposis sarcoma (KS). He described several cases
of elderly patients with skin lesions, typically on the lower extremities. In his histological examination, Moritz Kaposi noted a picture of small cell carcinoma,
with cells appearing in masses and clumps. All patients described by Kaposi died
of Kaposis sarcoma. For more than a century, this disease was known as a rare and
low-grade malignancy in Europe and North America. By 1950, the total number of
cases reported was approximately 600 (McCarthy and Pack 1950). Until 1980, KS
had incidence rates of 0.020.06/100,000 in the USA, seen mostly in persons of
European descent (Safai and Good 1981) and up to 1/100,000 (men) or 0.27/100,000
(women) in Italy, with higher incidence rates in Sicily (Geddes et al. 1994).
In 1981, several groups observed an epidemic of KS in young, homosexually
active men (Friedman-Kien 1981; Gottlieb et al. 1981; CDC 1981). The course of
disease in these patients was often fulminant and malignant. Friedman-Kien noted
several opportunistic infections and suspected this to be the tip of the iceberg
(Friedman-Kien 1981). He was proved correct in the following years and AIDS-KS,
also called epidemic KS, is until today one of the first manifestations of AIDS.
It took again more than 10 years until KSHV, the infectious agent necessary to
develop KS, was discovered in 1994. The epidemiological evidence for a causative
role of KSHV in the pathogenicity of KS is extensive and convincing and supported
by numerous cohort and casecontrol studies. KSHV DNA could be detected in most
KS lesions, independently of HIV status (Dupin et al. 1995; Dictor et al. 1996) and
within these lesions, KSHV could be localised to the spindle cells, the hallmark of
the tumour (section Clinical Features and Treatment) (Boshoff et al. 1995). There
is a strong correlation between KSHV seroprevalence and KS incidence. Worldwide,
seroprevalence for KSHV is high in AIDS risk groups that also show an increased
incidence of KS, and is low in groups in whom KS is rare (Whitby et al. 1995; Gao
et al. 1996; Moore et al. 1996; Rezza et al. 1999). Furthermore, KSHV seroprevalence is high in organ transplant recipients who developed KS after transplantation
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(Parravicini et al. 1997; Regamey et al. 1998). Globally, KS is more common in

regions with an intermediate or high prevalence of KSHV (e.g. Mediterranean region,
Africa), whereas the incidence of KS is low in regions with lower prevalence of
KSHV (e.g. the USA, Northern Europe) (Gao et al. 1996). Both casecontrol and
prospective cohort studies strongly link infection with KSHV to KS (Chang et al.
1994; Melbye et al. 1998; Grulich et al. 1999; Sitas et al. 1999; Newton et al. 2003).
The detection of KSHV DNA in peripheral blood often precedes the development of
KS lesions and is associated with an increased risk of the subsequent appearance of
KS lesions (Moore et al. 1996; Laney et al. 2007). In addition, several cohort studies
on AIDS- or iatrogenic KS demonstrated that high antibody titres against KSHV
prior to diagnosis are associated with an increased risk of KS (Regamey et al. 1998;
Renwick et al. 1998; Frances et al. 1999; OBrien et al. 1999; Newton et al. 2006).
In general, 90100% of patients with KS have high antibody titres to KSHV, independent of ethnicity or geographic localisation (Chatlynne and Ablashi 1999).
Different Forms of KS
Clinically and epidemiologically, four variants of KS have been defined. They are
histologically indistinguishable and represent variants of the same disease.
Classical KS
Classical KS represents the original form of KS described by Moritz Kaposi, which
occurs mainly in elderly men with Mediterranean or eastern European Jewish ancestry.
Even though KS was first described as an aggressive tumour (Kaposi 1872), this
form is today known to be a rare, mainly indolent and usually involves the skin of
the lower and higher extremities as well as the trunk. Internal involvement does
occur in approximately 10% of all cases. Classic KS is more common in men than
in women with a malefemale ratio between 15:1 and 3:1 (Antman and Chang 2000;
Geddes et al. 1994) and occurs predominantly in elderly people with a mean age at
diagnosis between 65 and 70 years (DiGiovanna and Safai 1981).
Endemic KS
Endemic KS is more aggressive, may involve lymph nodes in addition to the skin
and affects HIV-negative people and children (Slavin et al. 1970; Ziegler and
Katongole-Mbidde 1996). It occurs mainly in certain parts of central and eastern
Africa. The endemic form appeared before the HIV epidemic and was well documented in Uganda in the 1960s (Hutt and Burkitt 1965).
220
Iatrogenic KS
The incidence of KS is about 100- to 500-fold increased in patients receiving immunosuppressive treatment after solid organ transplantation in comparison to the general
population (Zavos et al. 2004; Tessari et al. 2006; Grulich et al. 2007). As a result
of the different KSHV prevalence rates, KS occurs in less than 1% of transplant
recipients in the USA and Western Europe (Farge 1993) but increases up to 4% of
transplant patients in Saudi Arabia (Qunibi et al. 1998).
Epidemic (AIDS-) KS
Epidemic or AIDS-KS is the most common and most aggressive form of KS. It not
only involves the skin and lymph nodes but often also disseminates to the inner
organs, including lung, gastrointestinal tract, liver and spleen and is frequently fatal.
AIDS-KS became the most common HIV-associated malignancy in the 1990s
particularly affecting young homosexual men who had a 20-fold higher risk of KS
development compared to other HIV transmission risk groups (Beral et al. 1990).
Cohort studies revealed a high incidence of KS among homosexual men who were
infected with both HIV and KSHV at baseline: the 10-year probability to develop KS
was nearly 50% (Martin et al. 1998; Renwick et al. 1998). Even though adjusted
incidence rates for AIDS-associated KS declined from 15.2 in years 19921996 to 4.9
during 19971999 after the introduction of effective anti-HIV therapy (Carrieri et al.
2003), this form of KS is still one of most common HIV-associated malignancies
(Dal Maso et al. 2009). Owing to the widespread HIV epidemic in Africa, KS is currently the most common cancer in men in Africa (Dedicoat and Newton 2003).
KS and Its Co-factors

Globally, HIV-infection is associated with an approximately 3,000-fold increase in
risk for KS and is its most important co-factor (Grulich et al. 2007). The risk for an
HIV- and KSHV-coinfected person to develop KS increases with immunosuppression
(Mbulaiteye et al. 2003). HIV-infected homosexual men who subsequently acquire
KSHV have an approximately 50% chance to develop AIDS-KS within 510 years
(Renwick et al. 1998; OBrien et al. 1999; Stein et al. 2008).
HIV aggravates KSHV pathogenicity probably on several levels: Immune suppression is a potent risk factor, as indicated by the 200-fold increase of KS in transplant
recipients relative to the general population (Vajdic et al. 2006; Grulich et al. 2007).
However, given that in industrialised countries, KS is approximately 3,000-fold
more common in HIV-infected patients than in the general population, other aspects
of HIV infection may promote KS development. These range from inflammatory
cytokines to the HIV transactivating protein Tat (Aoki and Tosato 2007), which was
reported to activate lytic replication of KSHV (Zeng et al. 2007) and to accelerate
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the tumorigenic effects of the KSHV G-protein coupled receptor (vGPCR) (Pati
et al. 2001; Guo et al. 2004).
The importance of the immune system is also illustrated by the fact that KS can
regress after reduction of immunosuppressive treatment in solid organ transplant
recipients (Moray et al. 2004) and the improvement of KS in AIDS patients after the
introduction of highly active antiretroviral therapy (HAART) (Dupont et al. 2000;
Bower et al. 2009). In addition, mild immune suppression was suggested as a risk
factor for classic KS (Brown et al. 2006b).
Although the incidence of KS correlates broadly with the worldwide variation of
KSHV prevalence (section Kaposi Sarcoma), several exceptions exist: Seroprevalence for KSHV is comparable in the African countries Uganda, Zambia, Gambia
and Ivory Coast, but juvenile and endemic KS is found mainly in East Africa, around
the great lakes (Gompels and Kasolo 1996; Dedicoat and Newton 2003). Furthermore,
even though KSHV prevalence is high in Papua New Guinea, KS prevalence is low
in this country (Rezza et al. 2001; Dedicoat et al. 2004). These observations point to
a role of other still unknown factors involved in the aetiology of KS. These co-factors
may involve host genetic polymorphisms, e.g. in VEGF or IL8-receptor genes
(Brown et al. 2006a; Zanetti et al. 2010) or certain HLA types (Alkharsah et al. 2007).
In addition, environmental factors, such as volcanic soil, durum wheat, blood-sucking
insects and plant-derived chemicals, that can reactivate KSHV from latency in
tissue culture, have been suggested as possible co-factors (Whitby et al. 2007;
Ascoli et al. 2009; Pelser et al. 2009; Goedert et al. 2010).
Clinical Features and Treatment

KS ranges from indolent to aggressive forms associated with significant mortality.
In classical KS, typical cutaneous lesions consist of purple-blue or reddish-brown
plaques and nodules, appearing initially on the hands and feet and progressing up
the extremities over a period of years to decades. Extracutaneous spread can occur.
More aggressive forms disseminate to lymph nodes and to the inner organs including
the pulmonary and gastrointestinal tract, liver and spleen.
KS is a vascular tumour with a complex histology. All KS lesions are composed
of spindled-shaped tumour cells (the hallmark of the tumour), atypical endothelial
cells and an inflammatory cellular infiltrate (Fig. 10.1). The spindle-shaped tumour
cells express endothelial markers including CD31 and CD34 (Nickoloff 1991,
1993), but also markers of the lymphatic endothelium such as VEGFR3 (Dupin
et al. 1999). However, some cells express markers for dendritic cells, macrophages
or smooth muscle cells. These observations suggest that the cells are either derived
from a pluripotent mesenchymal progenitor or KS lesions might represent a heterogeneous population of cells. KSHV can influence the differentiation pattern of vascular
endothelial cells towards that of lymphatic endothelial cells (Wang et al. 2004a).
In addition, there are some suggestions that KSHV can induce endothelial
mesenchymal transition (P. Ojala et al., pers. comm.).
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Fig. 10.1 Kaposis sarcoma. Histological section of a lymph node metastasis from an 45-year-old
HIV-positive patient. (a) Staining with haematoxylineosin (250 magnification) shows vascular
slits and elongated spindle cells. (b) Staining with an antibody to KSHV LANA shows the presence of LANA in the nuclei of numerous spindle cells (brown staining, 125 magnification). Image
kindly provided by Dr. Buesche, Dept. Pathology, Hannover Medical School
Most cells are latently infected and express only a limited number of viral genes
including the latency-associated nuclear antigen-1 (LANA), viral cyclin (vcyclin)
and viral FLICE inhibitory protein (vFLIP) (section Viral Proteins Involved in
Tumorigenesis); only in a small proportion of the cells does KSHV undergo lytic
replication (Staskus et al. 1997).
KS lesions evolve from early (inflammatory) patches to plaques that develop
further to tumour nodules. All three kinds of lesions can be detected in one KS-patient
in parallel.
Treatment of AIDS-KS initially involves an efficient control of HIV replication
by HAART (Dupont et al. 2000; Bower et al. 2009). For transplant KS a reduction
in immune suppression can be attempted, but has to be balanced against the risk of
transplant rejection (Moray et al. 2004). In general, local therapy including radiotherapy is applied for mild to moderate local KS whereas systemic chemotherapy
has to be applied in case of aggressive progression including visceral involvement.
Although the productive replication of KSHV responds to some anti-herpesviral
drugs such as gancyclovir or cidofovir (Casper 2006), the efficacy of these drugs
against established KS lesions is limited. HIV-infected patients treated with gancyclovir for other indication (usually Cytomegalovirus CMV disease) have a lower
risk of developing AIDS-KS (Glesby et al. 1996), suggesting that a reduction of
productive KSHV replication can prevent the progression to KS.
More recent strategies to treat KS target cellular growth and angiogenic pathways,
such as vascular endothelial growth factor (VEGF), platelet-derived growth factor
(PDGF) and matrix metalloproteinases (MMPs) that are dysregulated by the virus.
Rapamycin, the inhibitor of mTOR, showed promising results in the treatment of
iatrogenic KS (Stallone et al. 2005). Further experimental compounds include COL3,
an inhibitor of MMPs (Dezube et al. 2006) and imatinib, a tyrosine kinase inhibitor
of c-kit and PDGF (Koon et al. 2005). However, continued efforts will be essential to
further optimise existing strategies and find new targets for future therapy.
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KS: A True Malignancy or a (Chronic) Inflammatory,

Proliferative Disorder?
KS lesions consist of spindle-shaped cells of endothelial origin, but infiltrated plasma
cells, lymphocytes and other inflammatory cells are also present. Early KS lesions
contain only a small number of spindle cells (less than 10%) and a large number of
infiltrated immune cells. The number of spindle cells increases within the development of the lesion towards the later nodular stage, during which KSHV is present in
more than 90% of spindle cells, but not in normal vascular endothelium (Dupin et al.
1999). Spindle cells explanted from KS tumours release several growth factors and
cytokines such as IL-1, bFGF, VEGF, interleukin 8 (IL-8) and monocyte chemotactic
protein-1 (MCP-1) (Salahuddin et al. 1988; Ensoli et al. 1989), which may play a
role in the aberrant angiogenesis seen in KS lesions. Injection of cultured KS cells
into mice causes a strong angiogenic reaction of mouse cell origin, but no tumour
growth (Salahuddin et al. 1988), suggesting that they do not represent classical
transformed cells that are able to grow in immunodeficient mice. However, the
interpretation of this observation is complicated by the fact that cultured KS cells
lose KSHV after a few passages and the reported lack of tumorigenicity of cultured
KS spindle cells could, therefore, conceivably be linked to the absence of KSHV.
Clonality studies suggest that KS is an oligoclonal and multifocal disease (Judde
et al. 2000): in one study nearly 80% of the KS lesions were oligoclonal, with evidence
of clonal and oligoclonal KS lesions within the same patient (Duprez et al. 2007).
Taken together, these observations lead to the widely held view that especially the
early stages of KS are defined by a benign and inflammatory-driven, proliferative
process rather than by true tumorigenesis. From this early, mainly polyclonal stage, the
lesion might depending on immunosuppression or other still unknown circumstances
proceed towards a true malignancy. Therefore, KS could be termed an inflammatorydriven tumorigenic process or a paracrine neoplasia (Mesri et al.). These issues are
dealt with in more detail in other chapters of this book and also in recent reviews
(Douglas et al. 2007; Mesri et al. 2010; Riva et al. 2010). A more detailed discussion of
viral proteins thought to be involved in KS pathogenesis is provided in section
Mechanisms of Tumorigenesis in KSHV-Associated Diseases of this chapter.
KSHV-Associated Lymphomas
In addition to KS, KSHV is also associated with two B-cell-lymphoproliferate
diseases, primary effusion lymphoma and multicentric Castlemans disease.
Primary Effusion Lymphoma

Shortly after the discovery of KSHV as causative agent of KS, E. Cesarman and
colleagues identified KSHV DNA in a subgroup of AIDS-related non-Hodgkin
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lymphomas (NHL) (Cesarman et al. 1995). These malignancies were subsequently

called primary effusion lymphoma (PEL) (Nador et al. 1996). PEL is a very rare
subgroup of B-cell NHL, comprising 3% of AIDS-related lymphomas (Little
et al. 2001). Owing to the rarity of the disease, only case reports and no systematic
casecontrol or cohort studies are available. Epidemiological evidence for the role
of KSHV in PEL is, therefore, limited to the detection of KSHV in all reported
cases of PEL.
In addition to KSHV, more than 90% of all AIDS-PEL cases show co-infection
with EBV. However, only a restricted set of latent EBV genes is expressed
(Horenstein et al. 1997) and the role of EBV in PEL is not fully understood. Gene
expressing profiles of EBV-positive and EBV-negative PEL as well as other NHL
have revealed the important role of KSHV in the oncogensis of PEL, as its presence
selects for a very distinct cellular gene expression and a clearly different lymphoma
type (Fan et al. 2005).
Even though the majority of PEL cases is found in HIV-positive patients, several
HIV-negative PEL patients have been described. These patients are likely to have an
underlying immunosuppression (e.g. after solid organ transplantation iatrogenic
PEL). PEL has also been diagnosed in two elderly HIV-negative men in Italy, a
country with intermediate prevalence for KSHV (Ascoli et al. 1999). Weakened
immune functions in old age may, therefore, contribute to PEL development in geriatric patients. HIV-negative PEL share several features with AIDS-associated PEL
at the morphologic and molecular level as well as the immunophenotype, but are
characterised by onset at an older age and a less aggressive clinical course are
reported (Ascoli et al. 1999).
Solid PEL
Even though the majority of PEL cases have been reported as lymphomatous effusions in the absence of a solid tumour mass (section Primary Effusion Lymphoma),
a few patients with KSHV-positive extracavitary solid tissue lymphomas have been
observed (Dotti et al. 1999). These solid variants which can occur before, after or
independently of effusion lymphoma often appear at extranodular sites and are similar
to the effusion type with regard to morphology, immunophenotype and molecular
characteristics. Therefore, these two variants have been grouped as a single clinicopathologic entity and the extracavitary form is classified as solid, whereas the effusion
type is classified as classic PEL. The hallmark of both variants and their distinctive
feature in comparison to other NHL is the presence of KSHV.
Clinical Features and Therapy

PEL is clinically characterised by the lymphomatous growth in a liquid phase in
body cavities such as pleural, pericardial or peritoneal spaces, mainly without identifiable associated extracavitary tumour mass. Morphologically, PEL cells are large
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in size, have a basophilic cytoplasm and round to irregular nuclei. The cells exhibit
a range of appearances from immunoblastic to plasmablastic to anaplastic. Gene
expression profiles revealed that PEL cells express a distinct pattern of genes,
distinguishable from other NHL or normal B-cells and indicating a plasmablastic
derivation with immunoblastic features (Jenner et al. 2003; Klein et al. 2003).
PEL cells have a characteristic phenotype with expression of the lymphocyte
marker CD45, the activation markers CD30, CD38, CD71 and epithelial membrane
antigen (EMA), as well as markers associated with plasma cell differentiation such
as CD138/Syndecan-1 and MUM1/IRF4 (Carbone et al. 2001). However, classical
B-cell markers (CD19, CD20) or T-cell markers (CD2, CD3, CD5 and CD7) are not
detectable in general (Nador et al. 1996), but may be expressed occasionally on
tumour cells (Brimo et al. 2007).
Immunoglobulin gene rearrangements define the B-cell origin of PEL cells
(Nador et al. 1996). Rearranged immunoglobulin genes show high levels of somatic
mutation in their heavy chain variable (VH) regions, suggesting a postgerminal
B-cell origin (Matolcsy et al. 1998). Furthermore, gene expression profiles showed
an expression pattern of PEL distinct but related to both immunoblasts and plasma
cells (Jenner et al. 2003; Klein et al. 2003). In conclusion, it is likely that the lymphoma
is derived from a transition state between antigen-selected germinal centre B-cells
and terminally differentiated plasma cells.
The prognosis of PEL is extremely poor with a median survival of less than 6
month. No widely used standards for therapy have been established and the disease
is not curable. The use of the antiviral therapy HAART is associated with a better
prognosis (Boulanger et al. 2005) and, therefore, recommended for HIV-positive
PEL patients. Some case reports showed prolonged remission of PEL with antiretroviral therapy alone (Hocqueloux et al. 2001). These findings imply an important
role of immune reconstitution in the control of this aggressive lymphoma (Chen et al.
2007). As for other NHL, a systemic combination therapy based on cyclosphamide,
doxorubicin, vincristine and prednisone (CHOP) is frequently used. Novel
approaches include antiviral components such as cidofovir (De Clercq 2003). Many
antiviral drugs are most effective during viral lytic phase; therefore, their effect
might be improved with the additional administration of drugs such as valproate, an
inducer KSHV of lytic replication (Klass et al. 2005). Other options for treatment,
such as reactivation of the p53 pathway using the murine double minute 2 (MDM2)
inhibitor Nutlin 3a to induce apoptosis in PEL tumour cells (Sarek et al. 2007), are
actively discussed at the moment.
The Role of KSHV in PEL Pathogenesis

How KSHV promotes the development of PEL is still under intense investigation.
As in KS, most PEL tumour cells are latently infected, but a small percentage of
infected cells enters the productive replication cycle at any one time. During latency,
the same viral proteins found in KS are also expressed in PEL. In addition, one of
the KSHV interferon regulatory factor (IRF) homologues, vIRF-3 (also called
226
LANA-2), is expressed during latency in B-cells and there is also substantial expression
of the lytic protein vIL6, a homologue of human interleukin 6 (IL-6) (section Viral
Proteins Involved in Tumorigenesis). Using RNAi, several viral proteins have been
shown to be essential for the continuous proliferation and survival of PEL cell lines.
While silencing LANA by RNAi did not induce apoptosis but reduced the copy
number of the viral episome, presumably due to its role in episomal persistence
(Godfrey et al. 2005), targeting vFLIP and vcyclin by RNAi caused efficient
apoptosis in all PEL cell lines tested (Guasparri et al. 2004; Godfrey et al. 2005).
Furthermore, knockdown of vIRF3 (LANA-2) resulted in repressed proliferation of
PEL cells and activation of caspase-3 and -7 (Wies et al. 2008).
PEL are devoid of gene rearrangements such as chromosomal translocations
including bcl-6, bcl-2 and c-myc, which are associated with other aggressive B-cell
NHLs (Cesarman et al. 1995; Nador et al. 1996). However, mutation in the 5
noncoding regions of BCL-6 are found in a fraction of PEL (Carbone et al. 2000).
Multicentric Castlemans Disease

Castlemans disease (CD), also called angiofollicular lymph node hyperplasia, is a
polyclonal lymphoproliferative disease. It was originally described by Benjamin
Castleman in 1956 (Castleman et al. 1956). CD represents several clinical and pathological entities, it can affect a single lymph node (localised or unicentric CD) or can
be generalised (multicentric form). Multicentric Castlemans disease (MCD) presents
with lymphadenopathy and frequently multi-organ involvement; it is associated with
systemic manifestations such as inflammation and B-cell hyperreactivity and is more
aggressive. MCD is less common than localised CD and occurs in the HIV-negative
population in the sixth decade, but in HIV-positive patients also at younger age.
Following the histopathology of CD, the disease can similarly be divided into two
groups: the hyaline vascular type is more common and tends to be localised, whereas
the plasma-cell variant is only rarely localised but often multicentric. Furthermore,
mixed types exist, complicating an accurate classification of individual cases.
MCD and KSHV
The incidence of MCD increased with the emergence of the HIV epidemic. Several
groups described the co-existence of KS and MCD in patients (Rywlin et al. 1983;
Lachant et al. 1985) and shortly after the detection of KSHV in KS, J. Soulier and
colleagues could demonstrate the presence of KSHV DNA in HIV-positive and
HIV-negative MCD samples (Soulier et al. 1995). KSHV could be detected in 13%
of patients with localised CD and 75% of patients with MCD (Bowne et al. 1999).
Immunohistochemical studies revealed that KSHV is found in the plasmablasts
within MCD lesions; these plasmablasts occur as isolated cells in the mantle zone
of B-cell follicles and seem to be absent in KSHV-negative MCD (Dupin et al. 1999;
Parravicini et al. 2000). KSHV-infected plasmablasts have germ line immunoglobulin
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genes but are lambda light chain restricted and appear to represent pre-germinal
centre nave B-cells that are polyclonal but monotypic (Du et al. 2001). These plasmablasts can aggregate and form microlymphomas or even plasmablastic lymphoma.
HIV-positive patients with KSHV-dependent MCD have a 15-fold higher incidence
of NHL, e.g. plasmablastic lymphoma, compared to the general HIV-positive population (Oksenhendler et al. 2002).
The viral expression pattern in KSHV-associated MCD differs from KS and PEL,
which show mainly expression of latent proteins. In the affected B-cells in KSHVassociated MCD, viral cytokines such as vIL-6 are expressed in addition to the viral
latent proteins LANA, v-cyclin, vFLIP and vIRF3 (Parravicini et al. 2000).
The Role of IL-6 in MCD

The characteristics of MCD are partly due to cytokine dysregulation, which includes
increased levels of the pleiotropic cytokine IL-6 (Yoshizaki et al. 1989; van Kooten
et al. 1993; Oksenhendler et al. 2000); IL-6 is known to be involved in B-cell stimulation, mediates B-cell differentiation and promotes the growth of B-cell associated
diseases. Anti-IL-6 or anti-IL-6-receptor (IL-6R) antibodies have been shown to
abrogate CD-related symptoms and led to involution of the affected lymph node(s)
(Nishimoto et al. 2000). Furthermore, hIL-6 transgenic mice develop CD-like
syndromes, which can be reversed with the application of antibodies against hIL-6R
(Katsume et al. 2002).
KSHV vIL-6 mimics many biological functions of its human homologue IL-6
(Viral proteins involved in Tumorigenesis) and might play an important role in the
apparent B-cell proliferation in MCD (Aoki et al. 1999; Uldrick et al. 2010). It has
been hypothesised that activation of hIL-6 pathways by vIL-6 might transform nave
B-cells into plasmablasts, leading to the lymphoproliferative diseases associated
with KSHV, including MCD (Du et al. 2001).
Clinical Features and Treatment

MCD has multiple manifestations including fever, weakness, severe fatigue and lymphadenopathy, followed by splenomegaly and hepatomegaly. MCD in HIV-positive
patients is more aggressive with clinical features including severe constitutional symptoms, generalised lymphadenopathy and frequent bone marrow involvement.
The diagnosis is mostly established by lymph-node biopsy. While surgical excision
is often the first line therapy for local CD, treatment for MCD is not standardised
and due to its comparative rarity, no randomised clinical trials have yet been performed. Corticosteroids are given to improve symptoms in case of acute aggravation.
Several cases were reported to have had a partial or complete response following
treatment with rituximab (Corbellino et al. 2001; Nicoli et al. 2009), an antibody
against the B-cell surface antigen CD20, which is used as therapy for B-cell lymphomas and autoimmune diseases (Lim et al. 2010). Targeting hIL-6 or its receptor
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hIL-6R with specific antibodies (tocilizumab, siltuximab) also showed partial or

complete response in several cases. Other strategies include the application of
immunomodulatory agents (such as INF-a) or systemic chemotherapy, e.g. the
combination therapy CHOP. In line with the role of KSHV in MCD development,
treatment with the antiviral agent gancyclovir led to remission of KSHV- and HIVassociated MCD (Casper et al. 2004); however, the antiviral agent cidofovir was
shown not to be effective against HIV-associated MCD (Berezne et al. 2004).
HAART is used as first line therapy for HIV and it could recently be shown in a
systematic review of the literature that life expectancy in HIV- associated MCD
cases has improved in the HAART era; however, a direct role could not be established
and the effect of HAART might be explained by the immune reconstitution in
treated patients (Mylona et al. 2008).
Mechanisms of Tumorigenesis in KSHV-Associated Diseases

General Mechanisms of KSHV-Mediated Tumorigenesis
In the absence of HIV or iatrogenic immune suppression, KSHV-associated disorders (Table 10.2) are rare, even in populations with a high KSHV seroprevalence.
Thus, it is probable that KSHV infection represents only one of several events
involved in pathogenesis. In KS, PEL and also MCD, the majority of infected cells
is latently infected; however, lytic replication takes place in a fraction of infected
cells. In KS and PEL, this fraction generally makes up less than 510% but may
reach 525% of the infected cells in MCD (Parravicini et al. 2000). Several KSHV
proteins have been shown to have transforming and oncogenic properties in in vitro
and in vivo assays (section Viral Proteins Involved in Tumorigenesis, Table 10.3).
Latent proteins may directly promote the growth of infected cells, whereas viral
proteins only expressed during the productive replication cycle are suspected to act
mainly via autocrine or paracrine mechanisms.
Several virus-encoded proteins share high homology with cellular partners
(Table 10.3) and are thought to have been acquired from the host in the distant past
during co-evolution of a KSHV progenitor within mammalian hosts. These viral
homologues often mimic, or may go beyond, the effects of their cellular counterparts.
Their expression often enables infected cells to manipulate their environment
(Choi et al. 2001).
Distinct expression patterns of KSHV proteins are detected in different KSHV
associated diseases (Parravicini et al. 2000). It is unclear if these differences are due
to cell type or tissue specific control of KSHV gene expression. The exact contribution of individual proteins to the development of KSHV-associated diseases is not
well understood in spite of experimental evidence that some of them, in particular
the proteins encoded by open reading frame (orf) K1, K9 (an interferon regulatory
factor homologue), 74 (a chemokine receptor homologue with constitutive signalling activity) as well as Kaposin A and LANA may have transforming properties
(Viral proteins involved in Tumorigenesis).
Endothelial cells
Spindle-shaped cells
of endothelial origin, monocytes
Cellular origin
Infected cells
Table 10.2 Comparison of KSHV-associated diseases

KS (all forms)
Neoplasm
Inflammation-driven neoplastic process/
paracrine neoplasia
Clinical presentation
Predominantly in immunodeficient
patients but also in elderly immunocompetent individuals (classic KS),
sometimes systemic symptoms,
prognosis depends on extent of disease
Sites
Mainly skin, frequent involvement of
oral cavity and viscera
KSHV
Present in all cases
B-cells, transition state between

antigen-selected germinal centre
B-cells and terminally
differentiated plasma cells
B-cells
Related to immuno-blasts
and plama cells
Null-phenotype: often lack
B-cell markers
Express B-cell activation
markers (CD30, CD38)
Express plasma cell markers
(CD138, MUM1)
Body cavities, extranodular sites

for solid form
Present in all cases (diagnostic
criterion)
Predominantly in immunodeficient
patients, systemic symptoms,
poor prognosis
PEL (effusion and solid form)

Malignant tumour
(continued)
B-cells
Plasmablasts
Express B-cell markers (such as CD20)
Lack activation markers
Monotypic IgMl expression
Present in most HIV-associated and ~50%

of HIV-noninfected cases of the plasma
cell variant of MCD
B-cells, nave, pre-plasma state
Lymph nodes, spleen
KSHV-associated MCD
lymphoproliferative disease; can rarely
develop into plasmablastic lymphoma
Predominantly in immunodeficient patients,
systemic symptoms, poor prognosis
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229
Most cells latently infected, mainly

latent proteins expressed, a small
percentage shows evidence
of productive lytic replication
Absent
EBV co-infection
Mono- and oligo-clonal

Not known
KS (all forms)
Expression of viral
proteins
Clonality
Mutations in infected
cells

Monoclonal
No chromosomal abnormalities
but mutation in the 5-noncoding
regions of BCL-6
Most cells latently infected, mainly
latent proteins expressed, a small
percentage shows evidence of
productive lytic replication, vIRF3
(LANA-2) expressed
Frequent
PEL (effusion and solid form)
Rare
Most cells latently infected, mainly latent

proteins expressed, latent and lytic
genes (in 525% of infected cells)
expressed, vIRF3 (LANA-2) expressed
Polyclonal
Not known
KSHV-associated MCD
230
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231
Table 10.3 Selection of KSHV proteins and miRNAs involved in oncogenesis

Protein (gene)
Latent/lytic Cellular homologue Mode of action
LANA (ORF 73)
Latent
None
Inhibits p53 and pRB-E2F
pathway, upregulates hTERT,
interacts with Brd2/4, traps
GSK-3b thereby deregulation
of Wnt-pathway; transforming
activities (in presence of
h-Ras)
vcyclin (ORF 72)
Latent
D-type cyclin
Constitutively activates CDK6,
increases cellular proliferation,
causes genomic instability,
cytokinesis defects and
polyploidy
vFLIP/K13 (ORF 71)
Latent
FLIP
Activates NFkB; antiapoptotic
function, induces endothelial
spindle cell formation
Kaposin A (ORF K12) Latent
None
Interacts directly with cytohesin-1,
transforming activities in
rodent fibroblasts
LANA2/vIRF3 (ORF
Latent
IRFs
Expressed only in B-cells,
K10.5)
inhibits p53, inhibits
apoptosis in PEL cells,
antagonises IFN
K1/VIP (ORF K1)
Lytic
None
Induces NFkB, NFAT and bFGF,
prevents death-receptor
mediated apoptosis, activates
PI3K, Akt, Vav and Syk
kinases, induces MMP-9 and
VEGF in endothelial cells,
causes immortalisation of
primary endothelial cells,
vascular tumours in transgenic
mice
vGPCR (ORF 74)
Lytic
IL-8 receptor
Activates MAPK and PI3K
pathways, increases VEGF
and VEGF receptor 2
(VEGFR-2) secretion,
transforms murine NIH3T3
cells and immortalises human
endothelial cells in tissue
culture (see also text)
vIL-6/K2 (ORF K2)
Lytic
IL-6
Induces human IL-6 secretion,
triggers JAK/STAT-,
MAPK-dependent pathways,
autocrine/paracrine signalling,
supports B-cell proliferation
vBCL2 (ORF16)
Lytic
BCL2
Forms heterodimer with human
bcl-2, inhibits bax-mediated
apoptosis
(continued)
232

Protein (gene)
Latent/lytic
Viral miRNAs (miR-K1 Latent
to miR-K12)
Cellular homologue
miR-K12-11:
miR155
Mode of action
Suppress lytic replication,
influence cell differentiation
and angiogenesis, downregulate several tumour suppressors, thereby involved in
KSHV-mediated oncogenesis:
miR-K1: targets cyclindependent kinase p21
miR-K3: targets NFIB
miR-K9*: targets KSHV
ORF50 (RTA)
miR-K11: targets NFkB
regulator IkBa
miRK12-3 and -7: target C/
EBPb isoform p20/LIP
miR-K12-7: targets the NK
cell ligand MICB
miR-K12-10a: targets
TWEAKR
miR-K12-11: targets e.g.
BACH-1 and IkB kinase e
(IkKe)
miR-K12-6 and -11: target
transcription factor MAF
Abbreviations: LANA latency associated nuclear antigen, pRB retinoblastoma protein, E2F E2
transcription factor, hTERT human telomerase reverse transcriptase, BRD2/4 Bromodomaincontaining protein 2/4, GSK-3b Glycogen synthase kinase-3 b, Ras rat sarcoma protein, CDK6
Cyclin-dependent kinase 6, FLIP FLICE inhibitory protein, NFkB nuclear factor kappa-lightchain-enhancer of activated B-cells, IFN interferon, VIP variable ITAM-containing protein, NFAT
Nuclear factor of activated T-cells, bFGF basic fibroblast growth factor, PI3K phosphoinositide
3-kinase, MMP-9 Matrix metallo protease-9, VEGF vascular endothelial growth factor, GPCR G
protein-coupled receptor, IL interleukin, MAPK Mitogen-activated protein kinase, VEGFR-2
VEGF-receptor 2, JAK Janus kinase, STAT signal transducer and activator of transcription, MAPK
mitogen-activated protein kinase, BCL2 B-cell lymphoma 2, NFIB nuclear factor I/B, C/EBP b
CCAAT-enhancer-binding protein b, MICB MHC class I polypeptide-related sequence B, TWEAKR
tumour necrosis factor-like weak inducer of apoptosis receptor protein, BACH-1 BTB and CNC
homology 1- basic leucine zipper transcription factor 1
It could be shown in different experimental systems that KSHV can induce features
linked to tumorigenesis: KSHV infection of primary endothelial cells induces cell
survival and angiogenesis without resulting in the transformation of endothelial cells
(Wang and Damania 2008). Even though KSHV infection can also not transform
mature B-cells in culture (Kliche et al. 1998), KSHV is required for survival of PEL
cells in culture (Godfrey et al. 2005; Guasparri et al. 2004; Wies et al. 2008) and
individual KSHV proteins possess transforming capacities in different experimental
systems as well as in transgenic mice. KSHV targets several tumour suppressor
proteins, such as p53 and pRB, thereby promoting cell proliferation and survival, which
facilitate tumour development; this includes also inhibition of apoptosis and the interference with cell cycle regulation, caused by several latent and lytic proteins (see below).
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233
Genetic instability is another hallmark of tumorigenesis, which is found in KS

and PEL. KSHV infection can induce chromosomal instability and the proteins
LANA and v-cyclin might be partially responsible (Si and Robertson 2006;
Verschuren et al. 2002; Koopal et al. 2007). Furthermore, DNA damage response
(DDR), a critical mechanism for the maintenance of genomic integrity, is influenced
by several KSHV proteins, which either activate DDR (Koopal et al. 2007) or prevent
response after damage has already occurred (Shin et al. 2006).
Another key aspect in neoplastic development is the stimulation of angiogenesis
and the accompanying secretion of cytokines. Tumour angiogenesis is a complex
process involving different pro-angiogenic factors, which are induced in KSHV
infection, including VEGF, bFGF, IL-8, MMPs and cyclooxygenase-2 (COX-2)
(Sodhi et al. 2000; Prakash et al. 2002; Brinkmann et al. 2007; Qian et al. 2007;
Sharma-Walia et al. 2010).
Viral Proteins Involved in Tumorigenesis

LANA
The latency associated nuclear antigen-1 (LANA) is expressed during latency in
KS, PEL and MCD cells. In addition to its role in episomal persistence and suppression of lytic replication (Verma et al. 2007), LANA contributes to tumorigenesis
though interference with several pathways, e.g. p53-mediated transcriptional activation and apoptosis (Friborg et al. 1999), pRB/E2F- as well as GSK3b/b-cathenin
mediated cell cycle regulation (Radkov et al. 2000; Fujimuro and Hayward 2003).
Furthermore, LANA interacts with RING3/Brd2 (Viejo-Borbolla et al. 2005) and in
cooperation with the cellular oncogene h-Ras is able to transform primary rat fibroblasts (Radkov et al. 2000).
vcyclin
Viral cyclin (vcyclin), expressed during latency in the majority of infected cells
(Davis et al. 1997), is a homologue of cellular D-type cyclins, important regulators
of the cell cycle. vcyclin associates with cellular cyclin dependent kinases (Cdk),
preferentially with Cdk6 (Godden-Kent et al. 1997), and phosphorylates pRB
in vitro and in vivo (Chang et al. 1996). The vcyclin-CDK6 complex has an extended
set of substrates compared to the cellular cyclinD-CDK6 complex, including histone
H1, p21(KIP1), cdc25A and BCL-2 (Godden-Kent et al. 1997; Mann et al. 1999;
Ojala et al. 2000). Furthermore, the vcyclin-CDK6 complex is more resistant to the
inhibitory effects of CDK inhibitors (CDKIs) such as p16(INK4), p21(CIP) and
p27(KIP) (Swanton et al. 1997) and can inactivate p27(KIP1) through phosphorylation (Sarek et al. 2006). In addition to the cell cycle regulatory effects, expression of
vcyclin induces replicative stress, which can lead to senescence and activation of
DNA damage response (Koopal et al. 2007) and it was also shown to affect latency
234
by phosphorylation of nuceleophosmin (Sarek et al. 2011). However, the physiological role of vcyclin during KSHV infection is still not understood.
vFLIP
The viral FLICE (Fas-associated death-domain-like IL-1 b-converting enzyme)
inhibitory protein (vFLIP) inhibits, similarly to cellular FLIP proteins, death receptormediated apoptosis, thereby providing a survival advantage to latently infected
cells. Additionaly, vFLIP effectively suppress autophagy, thereby regulating cell
death (Lee et al. 2009). vFLIP is a potent activator of the NFkB pathway. It binds
the kB kinase-g (IKKg) (Liu et al. 2002), which leads to the induction of many
cytokines (Sakakibara et al. 2009) and induces the spindle shape morphology of
KSHV-infected endothelial cells (Grossmann et al. 2006).
miRNAs
During latency, KSHV expresses 18 mature miRNAs, which originate from 12 viral
pre-miRNAs (Cai et al. 2005; Pfeffer et al. 2005; Grundhoff et al. 2006). The viral
miRNAs target host and viral mRNAs, are involved in the suppression of lytic
replication and can influence cell differentiation, cell proliferation and survival as
well as angiogenesis.
miR-K12-11 is an orthologue of cellular miR155 and downregulates an extensive
set of common targets, including the tumour suppressor BACH-1 (Gottwein et al.
2007; Skalsky et al. 2007) and the IkB kinase e (IKKe) (Liang et al. 2011), thereby
regulating interferon (IFN) signalling and suppressing antiviral immunity. miR-K11
targets IKBa, an inhibitor of the NFkB pathway (Lei et al. 2010). Cytokine secretion
in macrophages and monocytes is affected by miR-K12-3 and -7; these miRNAs have
been shown to downregulate the negative regulator C/EBPb p20 (LIP) leading to
induction of IL-6 and IL-10 (Qin et al. 2009). miR-K1 represses expression of p21, a
cyclin dependent kinase with known tumour suppressor functions (Gottwein and
Cullen 2010). Effects of further KSHV miRNAs are listed in Table 10.3. These varied
effects of KSHV miRNAs point to a multifaceted involvement in KSHV biology.
vIL-6
This homologue of human IL-6 (Neipel et al. 1997) can be detected in KS, PEL and
MCD samples (Parravicini et al. 2000). vIL-6 binds directly to the human gp130
receptor without the need of the hIL-6 receptor a-chain (hIL-6R), thereby activating
the JAK/STAT pathway and further hIL-6 induced pathways (Osborne et al. 1999),
leading also to increased VEGF expression. Owing to the changed requirements for
receptor binding, vIL-6 can theoretically activate every cell in the body expressing the
gp130 signalling chain, which is shared by many cytokine receptors. However, its
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235
binding affinity is lower compared to hIL-6 (Aoki et al. 2001) and vIL-6 expression in
KSHV infected cells is limited. The expression of vIL-6 in a minority of infected cells
suggests a paracrine model for vIL-6 (Liu et al. 2001). Furthermore, vIL-6 can
activate hIL-6 (Mori et al. 2000). vIL-6 potentially contributes to KSHV-related disease progression by continued activation of IL-6-stimulated growth and anti-apoptotic
pathways; its role especially in MCD is mentioned in section Clinical Features and
Treatment. Further details on vIL-6 are reviewed elsewhere (Suthaus et al. 2010).
vGPCR
This early lytic protein is a constitutively active member of the family of CXC
chemokine G protein coupled receptors (GPCR) (Arvanitakis et al. 1997) and is a
homologue of the human angiogenic IL-8 receptor (Cesarman et al. 1996). Several
studies showed the oncogenic potential of this protein, which can activate both
MAPK and PI3K pathways, leading to the activation of several cell-signalling
networks (Sodhi et al. 2000; Montaner et al. 2001). vGPCR has been shown to
transform murine NIH3T3 cells and immortalise human endothelial cells (Montaner
et al. 2001; Bais et al. 2003). A transgenic mouse that enabled endothelial cell-specific
infection revealed that vGPCR was the only viral gene analysed able to cause vascular
tumours in a subset of mice (Montaner et al. 2003). However, while interpreting these
effects obtained with overexpressed vGPCR, it has to be taken into account that in
culture, KSHV cannot immortalise primary endothelial cells. VEGF and VEGF receptor 2 (VEGFR-2) expression is increased in vGPCR-induced tumours in vitro and
in vivo (Sodhi et al. 2000; Guo et al. 2003). vGPCR is expressed only in the minority
of productively infected cells in KS, PEL or MCD, leading to hypothesis that vGPCRmediated effects are driven by spontaneous productive (lytic) reactivation in the background of latency. These data implicate that vGPCR signals via autocrine or paracrine
mechanisms, thereby influencing KSHV driven oncogenesis.
K1/VIP
The lytic transmembrane signalling protein K1/VIP (variable immunoreceptor
tyrosine-based activation motif (ITAM) containing protein) displays a high degree
of variability between different KSHV isolates (Hayward 1999; Zong et al. 1999;
Biggar et al. 2000) (section Origin and Evolution). Expression of the K1 gene in
rodent fibroblasts led to morphologic changes and focus formation indicative of
transformation (Lee et al. 1998). Furthermore, transgenic mice expressing the K1
gene under transcriptional control of the SV40 promoter developed tumours with
sarcomatoid features and malignant plasmablastic lymphoma (Prakash et al. 2002).
K1 expression leads to the activation of different signalling pathways including the
PI3K/Akt pathway, thereby inactivating pro-apoptotic factors (Tomlinson and
Damania 2004; Wang et al. 2006). K1 blocked Fas-mediated apoptosis was shown
to increase cell survival (Wang et al. 2007). Furthermore, K1 expression leads to
236
increased phosphorylation of syk and phospholipase Cg2, as well as increased activity

of NFkB and NFAT (Lagunoff et al. 1999). In addition, K1 was reported to both
augment and repress reactivation in B-cells (Lagunoff et al. 2001; Lee et al. 2002)
and to downregulate the B-cell antigen receptor (BCR) surface expression by inhibition of its intracellular transport (Lee et al. 2000). The K1 protein induces expression
of angiogenic and invasion factors including VEGF, suggesting a role of K1 in
KSHV pathogenesis via paracrine mechanisms (Wang et al. 2004b).
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Chapter 11
Retroperitoneal Fibromatosis Herpesvirus

and Kaposis Sarcoma-Like Tumors in Macaques
Laura K. DeMaster and Timothy M. Rose
Kaposis Sarcoma
Kaposis sarcoma (KS) is a vascular neoplasm that was first identified in elderly men
in the Mediterranean region. Classical KS commonly presents as a relatively benign
multifocal lesion on the skin of the extremities. A highly aggressive, epidemic form
of KS emerged in association with HIV and the acquired immunodeficiency syndrome
(AIDS) epidemic and was largely confined to men who have sex with men (Antman
and Chang 2000). AIDS-associated KS was frequently fatal and involved lymph
nodes, viscera, and mucosa, in addition to the skin. An endemic form of KS, found
in children and young adults in Sub-Saharan Africa prior to the AIDS epidemic, has
now reached epidemic proportions in association with AIDS. Women and children
are frequently affected, with a high tumor burden, rapid disease progression, and
limited life expectancy. Finally, organ-transplant recipients and patients receiving
immunosuppressive therapy are at higher risk of developing classical KS.
Although KS occurs with various morphological forms during the time course of
disease, all forms present with characteristic elongated spindle-shaped tumor cells.
Early KS lesions show scattered spindle cells in a background of neovascular tissue
with obvious inflammatory infiltration. In advanced KS lesions, the spindle cells
proliferate and dominate the lesion. Most spindle cells express endothelial markers
CD31 and CD34 and the lymphatic endothelial markers VEGFR-3 and podoplanin,
L.K. DeMaster
Department of Global Health, University of Washington, Seattle Childrens Research Institute,
Seattle, WA, USA
T.M. Rose (*)
Department of Pediatrics, University of Washington, Seattle, WA, USA
Seattle Childrens Research Institute, 1900 Ninth Ave, Seattle, WA 98101, USA
e-mail: trose@u.washington.edu
251
252
L.K. DeMaster and T.M. Rose
suggesting that spindle cells derive from the lymphatic, rather than vascular, endothelial
lineage. However, KS spindle cells can also express markers for smooth muscle cells
and macrophages suggesting possible pluripotency of the spindle cell precursor.
KSHV Is the Etiological Agent of All Forms of KS

In 1994, KS-associated herpesvirus (KSHV) was first identified in KS lesions of HIVinfected individuals (Chang et al. 1994). Based on sequence analysis, KSHV was
classified as a gammaherpesvirus within the rhadinovirus genus. Significant genomic
colinearity was detected between KSHV and other tumorigenic gammaherpesviruses,
including EpsteinBarr virus (EBV), a member of the lymphocryptovirus genus, and
herpesvirus saimiri (HVS), the rhadinovirus prototype of New World primates. Both
EBV and HVS are able to transform cells in vitro. While EBV is associated with Burkitts
lymphoma, nasopharyngeal carcinoma, and hairy leukoplakia in its natural host, HSV
induces lymphomas and leukemias in nonnatural host species of New World primates.
A causal linkage between KSHV and KS is now widely accepted and was established through a combination of epidemiological and molecular studies. Populationbased studies show strong geographic localization for KS tumor incidence. Serologic
assays developed against viral antigens revealed that KSHV distribution is consistent
with KS disease epidemiology. In most of the world, KS is a rare disease, but classical and endemic KS have long been recognized in the Mediterranean and in regions
of Sub-Saharan Africa. Correspondingly, KSHV seroprevalence is low in North
America (25%), where the disease incidence is low, and high in the Mediterranean
and regions in Sub-Saharan Africa (3080%), where the disease incidence is high
(Ablashi et al. 2002). High seroprevalence was also observed in populations known
to be at increased risk of KS, such as men who have sex with men (Kedes et al.
1996; Moore and Chang 1998).
Molecular evidence linking KSHV to KS disease initially focused on association
of viral genetic material with KS tumor tissue. Putative KSHV DNA sequences
were detected in KS tumor tissue by representational difference analysis (RDA)
(Chang et al. 1994). These viral sequences were found in all KS tissue samples from
HIV-infected patients but not in unaffected controls. Furthermore, KSHV DNA was
found at higher levels in KS lesions than in nontumor tissues of KS patients, demonstrating a strong association of viral DNA with KS tumors. An estimation of the
viral copy indicated that each KS tumor cell contained two viral genomes (Chang
et al. 1994). The presence of KSHV genomic DNA in tumor cells has now been
demonstrated for all epidemiological forms of KS (Boshoff et al. 1995; Moore and
Chang 1995).
Evidence of viral gene expression in tumor cells provided additional support for
a causal role of KSHV. Although viral transcripts are consistently detected in KS
tumor cells, initial findings revealed a highly restricted expression of KSHV latent
genes, demonstrating that tumor cells were predominantly latently infected (Zhong
et al. 1996; Staskus et al. 1997; Dittmer et al. 1998). One of the major gene products
11
Retroperitoneal Fibromatosis Herpesvirus and Kaposis Sarcoma-Like Tumors
253
expressed during latent infection is open reading frame (ORF)73, which encodes
the latency-associated nuclear antigen (LANA) (Kedes et al. 1997; Rainbow et al.
1997). KSHV LANA has a number of functions within infected cells, including
maintenance of the viral genome and deregulation of cell cycle and tumor-suppressor
pathways (Ballestas et al. 1999; Friborg et al. 1999; Radkov et al. 2000). Nearly all
KS tumor cells express nuclear LANA protein, and LANA is now considered a
histological hallmark of latent KSHV infection.
While latency is the predominant program of infection in KS spindle cells, latent
infection of KS tumor cells is unstable in vitro, as cultured cells rapidly lose viral
genomes when passaged (Grundhoff and Ganem 2004). Pleural effusion lymphomas
(PELs) have been identified in AIDS patients that are latently infected with KSHV and
carry the viral episome at a high copy number (Cesarman et al. 1995). PEL cell lines
that maintain KSHV genomes after continuous culture have been derived from the
lymphomas. Treatment with sodium butyrate or phorbol esters induces lytic activation
of the latent KSHV genomes resulting in viral replication and release of infectious
virions. PEL cell-derived virus has proven to be an invaluable tool in KSHV research.
Though many studies have demonstrated the importance of individual viral genes
to KS tumor formation, an in-depth understanding of KSHV pathogenesis has been
hindered by the lack of relevant animal models. Unfortunately, introduction of
KSHV into heterologous animals, such as macaques, has failed to produce a useful
animal model for KS (Renne et al. 2004).
Retroperitoneal Fibromatosis: A KS-like Disease of Macaques

Retroperitoneal fibromatosis (RF) is a multifocal, fibroproliferative syndrome associated with retrovirus-induced simian immunodeficiency in macaques. RF was first
recognized as a disease syndrome in 1976 (Giddens et al. 1979), and epidemic
outbreaks of RF occurred at the Washington National Primate Research Center
(WaNPRC) and other primate centers (King et al. 1983) in the 1980s, which resulted
in a 1% overall mortality and 10% mortality in juvenile macaques (Tsai et al. 1985).
RF disease was originally associated with a form of simian AIDS (SAIDS) caused
by infection with simian D-type retrovirus-2 (SRV-2) (Tsai 1993). RF lesions are
highly proliferative and arise primarily in the peritoneum, in the area of the ileocecal junction and adjacent mesenteric lymph nodes, and secondarily as KS-like lesions
in the skin (Giddens et al. 1985; Tsai 1993). Similarly to KS, RF tumors have a
characteristic spindle-shaped tumor cell (Tsai 1993). Lesions have a significant
vascular component and are infiltrated by inflammatory immune cells. The morphological similarities between RF lesions in macaques and KS lesions in humans were
noted early on, and RF, especially in its skin-associated form, was characterized as
a KS-like lesion (Tsai 1993). Additional cases of RF continue to occur in captive
macaque populations in association with natural infections of SRV-2 or experimental
infections with SIV or SIV/HIV hybrid viruses (Shibata et al. 1997; Bielefeldt-Ohmann
et al. 2005).
254

RFHVMn
RFHVMm
KSHV
100
100
HHV6
95
EHV2
72
HCMV
51
87
HVS
100
100
HSV1
EBV
VZV
Fig. 11.1 Phylogenetic analysis of partial DNA polymerase sequences of RFHVMn and RFHVMm
amplified from RF tumor samples. RFHVMn and RFHVMm cluster with KSHV within the rhadinovirus genus of the subfamily of lymphotrophic gammaherpesviruses. Gammaherpesviruses:
(Rhadinovirus genus) RFHVMn, RFHVMm, KSHV, equine herpesvirus 2 (EHV2), herpesvirus
saimiri (HVS); (lymphocryptovirus genus) EpsteinBarr virus (EBV); alphaherpesviruses: herpes
simplex virus 1 (HSV1), varicella zoster virus (VZV); betaherpesviruses: human herpesvirus 6
(HHV6), human cytomegalovirus (HCMV). Bootstrap analysis using 100 replicates is shown.
Adapted from Rose et al. (1997), Copyright American Society for Microbiology, J Virol 71, 1997,
41384144
The epidemic nature of RF suggested that the disease might have an infectious
etiology, and this hypothesis was supported by experimental transmission studies
using inocula of RF tissue (Giddens et al. 1985). Due to the strong association between
SAIDS RF and SRV-2 infection, it was initially hypothesized that the type D retrovirus
SRV-2 was the causative agent of RF (Tsai et al. 1985). After the discovery of KSHV
in KS tumor tissue, however, the morphological and histological similarities between
RF and KS suggested the involvement of a herpesvirus in RF pathogenesis. Using
consensus degenerate hybrid oligonucleotide primers (CODEHOP) (Rose et al. 1998)
that were highly sensitive and broadly reactive with the different members of the
herpesvirus family (VanDevanter et al. 1996), novel herpesvirus DNA sequences were
identified in RF tissue of two species of SRV-2-infected macaques (Rose et al. 1997).
Initial alignments revealed a 70% nucleotide identity with the DNA polymerase gene
of KSHV. Subsequent studies with CODEHOP PCR primers targeting the conserved
herpesvirus glycoprotein B gene identified nucleotide sequences in the RF tissue that
displayed 75% identity with the glycoprotein B gene of KSHV (Bosch et al. 1998).
Phylogenetic analyses demonstrated that the novel macaque virus sequences were
highly related to each other and to KSHV and clustered with members of the lymphotropic rhadinovirus genus of herpesviruses (Fig. 11.1). This indicated that the amplified
DNA sequences were derived from novel KSHV-like rhadinoviruses of macaques,
11
255
which were designated as retroperitoneal fibromatosis-associated herpesvirus (RFHV)

from pig-tailed macaques (Macaca nemestrina, RFHVMn) and rhesus macaques
(M. mulatta, RFHVMm) (Rose et al. 1997).
Genomic Conservation Between KSHV and RFHV

The complete genome sequence of KSHV was determined from cosmid and phage
libraries prepared from KSHV-infected PEL cell lines, revealing the presence of
more than 90 viral genes (Russo et al. 1996). The majority of these genes encoded
structural and enzymatic proteins that were highly conserved with other herpesviruses, especially the gammaherpesviruses. However, several divergent regions
dispersed within blocks of conserved sequences were identified. These divergent
loci contained a large number of genes with homology to cellular regulatory genes
(Russo et al. 1996). These viral homologs of cellular genes have been shown to
function in the disruption of several cellular processes, including apoptosis, cell
cycle, and antiviral immunity, and are thought to contribute to the unique biology of
KSHV and function in the development of KS tumors (Nicholas et al. 1997).
The complete sequence of the RFHV genome has not yet been determined.
Similar to the situation with KSHV-infected KS tumor cells, culture of tumor cells
derived from RF tumors results in the loss of the viral genome. Furthermore, no
RFHV-infected lymphomas have been identified that would contain high numbers
of the viral episome. Thus, sequence analysis of RFHV has relied upon the use of
CODEHOP-based PCR to amplify regions of interest from DNA isolated from
archived RF biopsies.
The sequence of a 4.3-kb region of the genomes of RFHVMn and RFHVMm,
including the DNA polymerase gene and flanking sequences, has been determined
(Rose et al. 2003). This region corresponds to the divergent locus B of KSHV that
contains a number of viral homologs of cellular genes known to play a role in the
pathogenesis of KSHV. As shown in Fig. 11.2, the RFHV genomes are colinear with
the KSHV genome, containing the glycoprotein B gene (ORF8), the DNA polymerase gene (ORF9), ORF10, ORF RF2 (the viral interleukin-6 (IL-6) homolog),
ORF 02 (the viral dihydrofolate reductase (vDHFR) homolog), ORF RF3 (the K3
MIR1 homolog), and ORF70 (the viral thymidylate synthase (vTS) homolog). Both
RFHVMn and RFHVMm genomes lack an ORF 11 homolog. Though the function
of ORF 11 is not yet clear, sequence conservation suggests that it is evolutionarily
related to ORF 10. Phylogenetic analysis of the sequences in divergent locus B
revealed a consistent clustering with the corresponding gene from KSHV, substantiating the original close phylogenetic relationship between RFHV and KSHV
determined using the DNA polymerase gene (Rose et al. 2003).
Sequence analysis of the divergent locus B region of RFHV revealed the conservation of two genes directly implicated in the pathogenesis of KSHV. First, the
RFHV genome contained a viral homolog of the cellular IL-6 cytokine that has
strong sequence homology to the KSHV vIL-6. The KSHV vIL-6 induces STAT
256
l)
11
K2
(v
10
R
F
(D
R
F
6)
IL
B
(g
Po
R
F
02
O
(
R
F vDH
K3
F
(M R)
O
R
I
R
F
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70
(v
TS
)
KSHV
RF2
RF3
RF2
RF3
RFHVMm
RFHVMn
vDHFR
R2
RRV
vDHFR
ORF12
HVS
Fig. 11.2 Relative organization of ~7.7 kb of the RFHV genome between the glycoprotein B (gB)
and viral thymidylate synthase (vTS) genes in comparison to other primate rhadinoviruses, including
KSHV (human), RRV (rhesus macaque), HVS (South American squirrel monkey). Missing genes
are indicated with a dashed line, and noncontiguous regions of the genome are indicated with a
double-slanted line. Copyright American Society for Microbiology, J. Virol 77, 2003, 50845097
signaling through direct interaction with the gp130 signal transducer. This is
believed to enhance the growth and survival of cells latently infected with KSHV,
thus contributing to virus-induced neoplasia (Chen et al. 2009). The RFHV vIL-6
sequence is 35% conserved with KSHV vIL-6 and the primary hydrophobic sites of
protein interaction with gp130 are maintained, suggesting that RFHV vIL-6 would
display the same binding affinities and function as KSHV vIL-6 (Rose et al. 2003).
The RFHV genome also contains a homolog of the KSHV modulator of immune
recognition (MIR). MIR1 homologs exist in several herpesviruses and are thought to
play a critical role in immune evasion and viral persistence (Stevenson et al. 2000;
Lehner et al. 2005). Two MIR homologs, ORFK3 (MIR1) and ORFK5 (MIR2), have
been identified in KSHV. Both MIR1 and MIR2 function as ubiquitin ligases to
downregulate major histocompatibility class I (MHC-I) and other immune molecules
on the surface of infected cells (Coscoy and Ganem 2000; Ishido et al. 2000). The MIR1
homolog, RF3, shares positional homology with K3, and considerable sequence
similarity exists among the zinc finger domains of RF3, K3, and K5 as well as within
C-terminal conserved regions (Rose et al. 2003). Like the KSHV MIR1, the RFHV
MIR1 homolog is able to downregulate cell surface MHC-1, suggesting that it also
plays an important role in virus-induced neoplasia (Harris et al. 2010).
The RFHV ORF73 LANA homolog was targeted for sequence analysis due to
the critical role of KSHV LANA in viral persistence and tumorigenesis. As shown
in Fig. 11.3, RFHV LANA is similar in size and structurally analogous to KSHV
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257
Fig. 11.3 Comparison of rhadinovirus ORF73 LANA homologs. (a) RV1 lineage rhadinoviruses:
KSHV (human; NP_572129) and RFHVMn (pig-tailed macaque, ABH07414). (b) RV2 lineage
rhadinoviruses: RRV (rhesus macaque, AAD21406) and MneRV2 (pig-tailed macaque,
ABH07415). (c) New World rhadinoviruses: HVS (squirrel monkey; NP_040275, AtHV3) (spider
monkey; NP_048045). Murine rhadinovirus: MHV68 (vole; NP_044913). Adapted from Burnside
et al. (2006). Reprinted from Virology 354, Burnside et al., 103115, 2006, with permission from
Elsevier
LANA (Burnside et al. 2006). It contains an N-terminal serine/proline-rich region,

a large internal glutamic acid-rich repeat region, and a conserved C-terminal domain.
KSHV LANA binds to both host chromatin and to the KSHV viral DNA, thus tethering the viral episome to chromatin. LANA, therefore, plays an important role in
the maintenance of viral infection during latency (Ballestas and Kaye 2001). The
amino acid sequences critical for KSHV LANA binding to chromatin have been
identified (Wong et al. 2004). Analysis of the RFHV LANA sequence revealed the
complete conservation of these residues, suggesting that RFHV LANA would serve
a similar role in the maintenance of the RFHV genome during latency (Burnside
et al. 2006).
The large internal glutamic acid-rich repeat in RFHV LANA contained multiple
repetitions of the consensus sequence EEPEPEPE (Burnside et al. 2006). A monoclonal antibody (mAB247) developed against an EPEPEP repeat in the procyclin
protein of trypanosomes was found to react specifically with RFHV LANA expressed
as a recombinant protein in COS-7 cells, which was targeted directly to the nuclei.
RFHV LANA was also recognized by the anti-KSHV LANA LN53 monoclonal
antibody which binds to EQEQ repeats in the large glutamic acid-rich repeat region
in KSHV LANA, suggesting similarities in the structure of the repeats (Burnside
et al. 2006).
258
Macaques and Other Old World Primates Are Host

to Two Distinct Lineages of KSHV-Like Rhadinoviruses
Subsequent to the discovery of RFHV, it was found that pig-tailed and rhesus macaques
are host to additional closely related rhadinoviruses with similarity to KSHV. A novel
virus called rhesus rhadinovirus (RRV) was isolated by two different groups from
healthy (Desrosiers et al. 1997) and SIV-infected (Wong et al. 1999) rhesus macaques.
Both isolates of RRV have been sequenced, with only minimal nucleotide differences
observed (Searles et al. 1999; Alexander et al. 2000). Limited sequence information
available from a rhadinovirus isolated from a pig-tailed macaque designated M. nemestrina rhadinovirus 2 (MneRV2) revealed a close phylogenetic relationship with
RRV (Auerbach et al. 2000; Schultz et al. 2000; Burnside et al. 2006; Bruce et al.
2009). Sequence analysis revealed that MneRV2 and RRV were distinct from
RFHVMn and RFHVMm, even though they infected the same macaque species.
Two distinct KSHV-like herpesviruses have also been identified in many Old World
primate host species, including African green monkeys, mandrills, gorillas, and chimpanzees (Greensill et al. 2000; Strand et al. 2000; Lacoste et al. 2001). Phylogenetic
analysis of partial DNA polymerase sequences revealed that these viruses cluster into
two distinct lineages (Schultz et al. 2000;Greensill et al. 2000). RFHVMn and
RFHVMm cluster with KSHV and other closely related Old World primate viruses
within the RV1 rhadinovirus lineage while RRV and MneRV2 cluster with a distinct set
of other Old World primate viruses within the RV2 rhadinovirus lineage. No RV2 lineage virus has been identified in humans to date. The complete sequences of the ORF59
DNA polymerase processivity factors from RV1 and RV2 rhadinoviruses from chimpanzees and three species of macaque were determined and compared to the KSHV
ORF59 (Bruce et al. 2009). Sequence and phylogenetic analysis clearly demonstrated
that each primate species was host to a distinct RV1 and RV2 rhadinovirus and that
individual animals were often coinfected with both types of rhadinovirus (Fig. 11.4).
Genetic Similarities Between RV1 and RV2 Rhadinoviruses

Due to their ability to replicate in primary rhesus fibroblast cell lines and produce
significant quantities of infectious virus, the two isolated RRV variants have been
completely sequenced (Searles et al. 1999; Alexander et al. 2000). The RRV genomes
were highly homologous and colinear with the KSHV and RFHV genome sequences.
Gene sequence comparisons revealed a strong conservation of nucleotide and amino
acid sequences between homologous genes of RFHV and RRV. Nucleotide sequence
identity averaged 66% over the more highly conserved herpesvirus genes, including
the DNA polymerase and glycoprotein B genes. Significantly higher nucleotide
sequence identity reaching 88% was detected in numerous regions of these genes.
Amino acid identity between RFHV and RRV sequences ranged from 19% (vIL-6)
to 69% (vTS), demonstrating a close evolutionary relationship.
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259
Fig. 11.4 RFHVMn and RFHVMm are the macaque homologs of KSHV and phylogenetically
cluster with members of the RV1 lineage of Old World primate rhadinoviruses. Protein maximum
likelihood analysis of the complete sequences of homologs of the ORF59 DNA polymerase processivity factor from the known macaque, chimpanzee, and human RV1 and RV2 Old World primate rhadinoviruses. The ORF59 homolog of the New World primate rhadinovirus, herpesvirus
saimiri (NP_040261), was used as an out-group. Bootstrap values for 100 replicate samplings and
the scale for substitutions per site are provided. From Bruce et al., Virol J 2009 6:205, BioMed
Central
Homologs of nearly every KSHV gene were identified in the RRV genomes.
However, as shown in Fig. 11.2, some divergence between the RV1 and RV2 rhadinoviruses was present. In the divergent locus B, the RRV genome contained vIL-6 and
vTS homologs, but lacked a homolog of the MIR1 ORF K3. Since MIR1 plays an
important role in immunoevasion, the lack of this gene suggests that RRV may utilize
a different pathway for maintenance of infection during latency. Analysis of the RRV
and MneRV2 ORF73 LANA homologs also showed significant differences with
the RV1 LANA homologs of KSHV and RFHV. The RV2 LANA homologs lacked the
conspicuous, large, internal, glutamic acid-rich repeat region found in KSHV and RFHV
260
LANA (Fig. 11.3). Furthermore, critical residues in the chromatin-binding motif of

KSHV were not conserved in the RV2 LANA homologs, suggesting a difference in
the tethering function of LANA during latency (Burnside et al. 2006).
Prevalence of RFHV and RRV in Captive Macaque Populations

Serological studies in different National Primate Research Centers have detected
antibodies to rhadinovirus antigens in 9398% of the macaque populations (Desrosiers
et al. 1997; White et al. 2009). However, none of these studies distinguished whether
the antibodies were cross-reactive to both RV1 and RV2 macaque rhadinovirus antigens. Therefore, the actual seroprevalence of RFHV or RRV in these colonies is
unknown. A real-time quantitative PCR (qPCR) assay was developed that targets
common gene sequences within the RV1 variants from pig-tailed (RFHVMn),
rhesus (RFHVMm), and fascicularis macaques (RFHVMf), but does not react with
RV2 variants (Bruce et al. 2006). Additional qPCR assays have been developed
that target either RRV alone (White et al. 2009) or common gene sequences within
the RV2 variants from pigtail (MneRV2), rhesus (RRV), and fascicularis (MfaRV2)
macaques (Bruce et al. 2005). The latter study demonstrated the important lack of
cross-reaction with RV1 variants. Using these assays, studies in the California
National Primate Research Center (rhesus macaques) and Washington National
Primate Research Center (pig-tailed macaques) determined the prevalence of RFHV
DNA in 3542% of the animals, RRV/MneRV2 DNA in 4881% of the animals,
and coinfections in 2042% of the animals (White et al. 2009; Rose et al. unpublished
data). These studies indicate that macaque RV1 and RV2 rhadinovirus infections are
endemic in the National Primate Research Centers.
Evidence for an Etiological Association of RFHV with RF

Although RFHV was originally detected in RF tumors from different macaque
species, the high prevalence of the closely related RV2 rhadinoviruses in the same
species of macaques highlights the need to carefully distinguish between the two
viral lineages during an assessment of etiology. Initially, the lineage-specific qPCR
assays, described above, were used to quantitate the levels of the RV1 and RV2
rhadinoviruses in a set of archived RF tumor samples at the Washington National
Primate Research Center that had a corresponding nonaffected spleen sample for
comparison (Bruce et al. 2006). Six RF cases were identified, all associated with an
SAIDS-like syndrome. Five animals were naturally infected with SRV-2. One
SRV-2 animal had been experimentally infected with an SIV/HIV hybrid virus. The
other animal was SRV-2 negative, but had been experimentally infected with a
pathogenic SIV. All six animals contained multifocal fibrous nodules on the skin or
on visceral organs, diagnosed as RF. qPCR analysis revealed that all six animals
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261
Fig. 11.5 Viral load of macaque rhadinoviruses in RF tumor lesions. The levels of RV1 (RFHVMn/
RFHVMm) and RV2 (MneRV2/RRV) were determined in RF tumor lesions and nonaffected spleen
samples from SAIDSRF pigtail (RF-14, 6) and rhesus macaques (RF-5), respectively, using RV1and RV2-specific TaqMan qPCR assays. The results were normalized to cell number by comparison
to a single-copy cellular gene. Viral load is expressed as genome equivalent copies per million cells.
From Bruce et al. 2006, J. Gen. Virol. 87, 35293538, Society for General Microbiology
were coinfected with both RV1 and RV2 rhadinoviruses. However, the RV1 levels
were on average 1,000-fold higher than the RV2 levels in the RF tumor samples
(Fig. 11.5). Furthermore, the RV1 levels in RF tumor samples were 1,000-fold
higher than the corresponding levels in spleen, showing a strong tumor association.
The RV1 tumor load was determined to be 1.8 106 viral copies per million cells
while the spleen load was 2.9 103 copies per million cells. In contrast, the RV2
tumor load (mean of 1.6 104 copies per million cells) was lower than the load in
the spleen (mean of 2.4 104 copies per million cells) (Bruce et al. 2006), showing
no tumor association.
262
Recently, a case of RF was reported in a rhesus macaque that had been

experimentally infected with SIV and RRV (Orzechowska et al. 2008). No RFHV
was detected in the lesion by PCR; however, the sensitivity or specificity of the PCR
assay was not indicated. This study detected RRV in higher amounts in tissue
derived from the RF lesion than in other tissue; however, no quantitative analysis of
the viral load was determined. A similar situation was seen in the RF2 animal
(Fig. 11.5), where the RV2 viral load was higher in RF tissue than spleen. However,
the viral load for the RF sample was only 2.1 104 copies per million cells, indicating that maximally only 1 in 50 cells in the lesion could be infected (Bruce et al.
2006). In Orzechowska et al., in situ hybridization of RRV cosmid clones representing 50% of the viral genome showed localization to cells within the RF lesion with
a spindle morphology, suggesting that the tumor cells were infected with RRV
(Orzechowska et al. 2008). However, no controls were included that demonstrate
the specificity of this hybridization, an important issue given the close sequence
similarity between the RV1 and RV2 rhadinovirus DNA.
To determine whether the spindle tumor cells in RF lesions are infected by
RFHV, immunohistochemical analysis of RF sections was performed using both the
anti-EP repeat and anti-KSHV LANA LN53 monoclonal antibodies which react
with recombinant RFHV LANA in Western blot and immunofluorescence analysis
and are nonreactive with recombinant RRV or MneRV2 LANA (Burnside et al.
2006). Both monoclonals showed strong specific nuclear staining of spindle cells in
the RF tumor lesions (Fig. 11.6). Essentially, every spindle cell in the tumor was
positive for RFHV LANA demonstrating that the tumor cells were latently infected
by RFHV. Coupled with the qPCR data, this indicated that each tumor cell contained approximately two viral episomes. This is similar to the latent infection of
KS spindle cells by KSHV (Boshoff et al. 1996; Ablashi et al. 2002; Curreli et al.
2003) and is consistent with an etiological association of RFHV with RF.
Immunohistochemical analysis of nontumor lymph nodes from a macaque with
RF, using the LN53 anti-KSHV LANA antibody, revealed distinct staining of occasional small lymphocytes (Bielefeldt-Ohmann et al. 2005). This correlates with the
qPCR data of Bruce et al., from the spleen samples of the RF animals, which suggested that only 1 in 300 spleen cells were infected (Bruce et al. 2006). Although no
IHC staining was determined for the RV2 rhadinoviruses, RRV, or MneRV2, in the
RF tumor lesions or spleen samples in either of the studies by Bielfeldt-Ohmann
et al. or Bruce et al., the level of RV2 virus determined by qPCR was not consistent
with an infection of the tumor cells and more likely was due to an inflammatory
infiltrate containing latently infected lymphocytes. In the single RF case identified
by Orzechowska et al., spindle cells within the RF lesion reacted with a monoclonal
antibody raised against the RRV vIL-6 (Orzechowska et al. 2008). However, the
abnormal nuclear localization of this reactivity and the lack of specificity controls
for RFHV vIL-6 rendered these results inconclusive. Both Orzechowska et al. and
Bielefeldt-Ohmann et al. detected strong vimentin staining in the cytoplasm of the
RF tumor cells with no desmin staining suggesting that the RF lesions, in both
cases, were proliferative mesenchymal lesions (Bielefeldt-Ohmann et al. 2005;
Orzechowska et al. 2008).
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263
Fig. 11.6 Immunohistochemical localization of RFHV LANA in RF tumor cells. Paraffin-embedded

RF tumor (a, b) and normal jejunum (c, d) tissues were deparaffinized, subjected to antigen
retrieval, incubated with the LN53 anti-KSHV LANA monoclonal antibody (a, c) or the anti-EP
monoclonal antibody (b, d), and counterstained with hematoxylin. Inserts show magnification of
positive nuclei in spindle-shaped tumor cells. Reprinted from Virology, 354, Burnside et al. 103115,
2006, with permission from Elsevier
Conclusion
Different species of macaque are infected with RFHV, the macaque homolog of the
human herpesvirus, KSHV. Although its complete sequence is not known, RFHV
exhibits a close similarity to KSHV in gene sequence and content. Its major latency
protein, LANA, is recognized by the KSHV LANA monoclonal antibody and is
expressed in spindleoid tumor cells in RF lesions, a KS-like neoplasm in macaques.
In all cases examined, RFHV LANA was expressed as a major nuclear antigen in
essentially all of the spindle-shaped tumor cells in RF lesions, demonstrating that
the tumor cells were latently infected with RFHV. This was corroborated by qPCR
analysis which detected elevated levels of the viral genome in RF lesions, which
were determined to be approximately two viral genomes per cell. These results
parallel the association of KSHV with KS tumor cells. While much work remains to
show a conclusive etiological association, the current data suggest that the macaque
RV1 rhadinovirus, RFHV, plays a major role in the induction of RF tumors in the
background of a retrovirus-induced immunosuppression. In all cases examined, RF
264
tumors were detected in animals also infected with a closely related rhadinovirus
belonging to the RV2 lineage of Old World primate rhadinoviruses, including RRV
and MneRV2. The role of the RV1 rhadinovirus and its interactions with the coinfecting RV2 rhadinovirus remain to be delineated.
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immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing.
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Chapter 12
Murine Gammaherpesvirus-Associated
Tumorigenesis
Kathleen S. Gray and Samuel H. Speck
Introduction
The gammaherpesviruses subfamily of herpesviruses is distinguished from their
alpha and beta relatives by sequence homology, tropism for lymphocytes, and strong
association with tumorigenesis in several host organisms. For the human gammaherpesvirses, EpsteinBarr virus (EBV) is linked to numerous malignancies, including Burkitts lymphoma and Hodgkins lymphoma, as well as nasopharyngeal and
gastric carcinomas. Likewise, Kaposi Sarcoma-Associated herpesvirus (KSHV)
is the etiologic agent of Kaposis sarcoma, a tumor of mixed lymphocyte and
endothelial cell origin, and is associated with primary effusion lymphoma (PEL)
and multicentric Castlemans disease (MCD), two malignancies of lymphoid origin.
Herpesvirus saimiri (HVS), a natural pathogen of nonhuman primates, is linked to
lymphomagenesis in New World primates and induces fulminant T-cell lymphomas
in rabbits. Although gammaherpesvirus-related tumors are relatively rare, their
incidence is greatly increased in immunocompromised individuals. For example,
prevention and treatment of posttransplant lymphoproliferative disorder (PTLD)
or lymphoma is now an important aspect in the clinical management of transplant
and AIDS patients.
The association of gammaherpesviruses with human disease has made their
study an area of active interest for nearly 6 decades. Until more recently, the breadth
of studies was limited by the narrow host tropism of EBV and KSHV, making it
difficult to examine major aspects of pathogenesis including primary infection and
subsequent events leading to lymphomagenesis. The identification, initial characterization, and sequencing of murine gammaherpesvirus 68 (MHV68), a naturally
K.S. Gray S.H. Speck (*)

Department of Microbiology and Immunology, The Emory Vaccine Center,
Emory University School of Medicine, Atlanta, GA 30322, USA
e-mail: sspeck@emory.edu
267
268
K.S. Gray and S.H. Speck
Fig. 12.1 Schematic depicting unique and conserved g2-herpesvirus genes. As shown, most of the
genes are highly conserved among the rhadinoviruses (g2-herpesviruses) MHV68, KSHV, and
HVS. The majority of these encode replication-associated proteins essential for virus replication
and virion assembly. However, there are a number of genes that are not well conserved among the
rhadinoviruses, and many of these have demonstrated roles in viral latency (note the presence of
several unique genes within each annotated genome). Despite limited sequence homology, several of these genes encode a protein of orthologous function and are interspersed among regions of
high sequence conservation. The genomes are not drawn to scale (genome size for MHV68, KSHV,
and HVS is approximately 120, 138, and 113 kb, respectively) and compensatory adjustments
were made for the additional copies of Orf75 at the right-end of the MHV68 genome. Essential,
attenuated, and nonessential genes were determined by two independent transposon mutagenesis
analyses of the MHV68 genmome (Gong et al. 2009; Song et al. 2005)
occurring rodent gammaherpesvirus, has provided the basis for a model system
using MHV68 infection of laboratory mice to study key aspects of gammaherpesvirus pathogenesis.
MHV68 shares considerable overall sequence homology with EBV, KSHV, and
HVS and encodes homologs of several genes that exhibit transforming properties in
human and nonhuman primate systems (Fig. 12.1). These include genes affecting cell
cycle progression, survival, proliferation, and immune evasion as discussed below.
In addition, MHV68 infection of laboratory mice, as well as wild rodents, shares
several important immunological characteristics with EBV and KSHV infection,
including the kinetics of the adaptive immune response, latent infection of B lymphocytes, and a requirement for an intact immune system to limit viral replication
and associated pathologies.
Although MHV68 is becoming increasingly well accepted as a model to study
several aspects of gammaherpesvirus pathogenesis, some speculation exists in the
field as to its usefulness in the further understanding of gammaherpesvirus-induced
transformation. EBV has clear transforming potential, and is ubiquitously used to
generate immortalized B cell lines in diverse areas of study. Likewise, KSHV also
has transforming potential, and the association of EBV and KSHV with human
malignancy is well-established.
12 Murine Gammaherpesvirus-Associated Tumorigenesis
269
Despite the high level of sequence conservation among MHV68 and the human
gammaherpesviruses, it has been argued that no direct evidence exists to corroborate
the classification of MHV68 as a DNA tumor virus. However, as we discuss below,
MHV68 encodes several proteins that alter the biology of infected cells with consequences highly reminiscent of transformed cells. The inability to efficiently infect
primary mouse B cells with MHV68 in vitro has complicated efforts to demonstrate
commonalities between the mouse and human gammaherpesviruses, and therefore,
true MHV68 transforming potential has not been conclusively assessed. Importantly,
however, MHV68 infection promotes lymphoproliferative disease in both immunocompetent mice as well as mice with specific immune defects (see Table 12.1). Initial
studies aimed at evaluating MHV68-mediated tumorigenesis demonstrated that
slightly more than 10% of infected mice of various genetic backgrounds developed
malignant disease; around 10% of these mice had lymphoproliferative disease (LPD),
and around 4% developed high-grade lymphomas (Sunil-Chandra et al. 1994).
Characteristics of tumors in these studies mimic pathologies observed in human disease and provide further justification for the use of MHV68 to examine the contribution of gammaherpesviruses to lymphomagenesis. Subsequent experiments using
cell lines derived from MHV68-positive lymphomas have also revealed fundamental
similarities to human gammaherpesviruses with regard to tumorigenesis, latency,
and reactivation (Husain et al. 1999; Moser et al. 2005; Robertson et al. 2001;
Usherwood et al. 1996b; Wu et al. 2000). In addition to MHV68, five other viruses
isolated from wild rodents (MHV60, MHV72, MHV76, MHV78, and MHV-Sumava)
are highly genetically similar to MHV68 and have also been associated with the
development of lymphomas in mice (Blaskovic et al. 1980; Hrabovska et al. 2010;
Mistrikova et al. 2004; Mrmusova et al. 2002; Pappova et al. 2004) The following
sections review several key aspects in progression to MHV68-associated lymphoproliferative disease and lymphoma. We discuss putative viral oncogenes, as well as
oncogenic signaling pathways induced by MHV68 infection not yet ascribed to specific viral proteins. We also address the role of the immune system in controlling
MHV68-associated disease, both directly (by way of cell-mediated cytotoxicity) and
indirectly (by controlling inflammation-induced damage resulting from persistent
lytic viral replication). Finally, we discuss the potential contributions of the MHV68
system to furthering the understanding and treatment of human malignancies associated with gammaherpesvirus infection.
MHV68 Encoded Proteins with Possible Direct Roles

in Tumorigenesis
Viral proteins have long acted as harbingers for cellular oncogene discovery. Many
tumor-virus oncogenes function by altering and exploiting key aspects of cellular
biology to favor self-propagation and promote survival of the host cell. As a consequence of viral gene expression and perhaps secondary mutagenic events, infected
cells may come to exhibit several or all hallmarks of transformation: self-sufficiency
in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless
Table 12.1 Characteristics of lymphoma or lymphomagenic disease associated with MHV68 infection
Viral genome
Time to disease
Pathology
Mouse strain
Cell type/Site of pathology positive?
(months)
Incidence
References
Lymphoproliferative disease
C57Bl/6, Balb/c
CB/CCa/lung, K, L, spl,
Yes (ISH)
5.528
5% (n = 220)c
Sunil-Chandra et al. (1994)
MLN, intestine
B lymphocytesa/spl, lung
Yes (ISH-vtRNA) 67
50% infected micee Tarakanova et al. (2005)
Hyperplasia
Balb/c-b2Md
(Lymphoid or
B10Br
B lymphocytes/lung
Yes (ISH)
21
0.5% (n = 220)
follicular)
C57Bl/6-IFNgR/f Lymphocytes/Lung
n.d.g
212
35% (n = 20)
Lee et al. (2009)
Lymphoma
Balb/c-b2M
IB/CBa,b/spl, lung
Yes (ISH-vtRNA) 11.6 (avg)j
29% infected miceh Tarakanova et al. (2005)
& L metastases
B220+ and CD3+
CBA, C57Bl/6,
Yes (ISH)
5.528
4% (n = 220)
lymphocytesa,b/spl, L, K,
Balb/ce
lung, MLN, pancreas,
ovary, K, adrenal gland
Pulmonary lymphoma C57Bl/6-IFNgR-/-f B220+/lungi, LN, heart,
Yes (RT-PCR)
<12k
45% (n = 20)l
Lee et al. (2009)
(pathology similar
brain
to LyG)
Abbreviations: L Liver, MLN mesenteric lymph node, spl spleen, K kidney, LN lymph node, IB immunoblast, CB centroblast, LyG lymphomatous
granulomatosis
a
plasmacytic markers present
b
evidence of clonality in tumors (determined by Ig transcript analysis)
c
Total incidence of LPD: 60% (mice treated with cyclosporine A) versus 20% (untreated mice)
d
disease severity affected by absence of v-cyclin, v-bcl2, and M1 genes
e
50% infected mice (versus 0% mock-infected); 67% infected (versus 22% mock-infected) developed LH or lymphoma
f
Mice infected with both wild-type and v-cyclin-deficient virus
g
Lungs from infected mice at day 90 infected mice were positive for polIII, but not Rta, transcripts by ISH
h
29% infected mice developed lymphoma (versus 6% mock infected)
i
Seven of nine B cell lymphomas presented in lung
j
Compare to 15.8 months (mean) time to disease for mock-infected mice
k
Forty-five percent of infected mice developed B cell lymphoma after 1 year
l
Developed frank tumors
270
271
replicative potential, sustained angiogenesis, tissue invasion and metastasis, and

genome instability. In the following section, we review putative MHV68 oncogenes
encoding proteins that may facilitate the acquisition of transforming characteristics.
v-Cyclin
General characteristics. Sequencing of the MHV68 genome revealed a viral ortholog
to the cellular D-type cyclins. The presence of D-type cyclin orthologs in KSHV
and HVS and modulation of cellular cyclin D expression by EBV indicate a common
and important theme in gammaherpesvirus pathogenesis (Chang et al. 1996; Nicholas
et al. 1992; Palmero et al. 1993; Sinclair et al. 1995). The overall genomic position
of the gene encoding the gammaherpesvirus cyclin homolog is largely conserved, as
are many key residues dictating interactions with host cyclin-dependent kinases
(CDKs) (Virgin et al. 1997). Despite its location in a region associated with latent
gene transcription, v-cyclin is not considered a latency-associated gene. Based on a
study using conservative criteria to define latency MHV68 candidates, v-cyclin was
excluded based on the abundant expression of v-cyclin transcripts during lytic replication (Virgin et al. 1999). V-cyclin transcripts were not detected in latently infected
PECs or splenocytes in this study, and studies of lytic infection in fibroblasts had
originally defined MHV68 v-cyclin as a lytic gene with leaky-late expression kinetics (van Dyk et al. 2000). However, subsequent analyses using latently infected
animals and cells have suggested that v-cyclin is more likely a latency-associated
gene with immediate-early kinetics during lytic replication or reactivation (Allen
et al. 2007; Forrest and Speck 2008; Martinez-Guzman et al. 2003).
The original classification of v-cyclin as a lytic gene is not surprising given the
proposed role of the protein during MHV68 infection. Despite sharing 25 and 21%
sequence identity with the KSHV and HVS cyclins, respectively, MHV68 v-cyclin
has been shown to preferentially interact with CDK2 and cdc2, rather than CDK6,
a characteristic that distinguishes it from the other gammaherpesvirus cyclins (Card
et al. 2000; Upton et al. 2005). However, like k-cyclin, MHV68 v-cyclin complexes
perpetuate phosphorylation of several targets in the G1 to S transition (discussed
further below), which presumably facilitates cell-cycle progression and DNA replication in infected cells (Upton et al. 2005). Also, crystal structure analyses of
MHV68 v-cyclin bound to cellular CDKs suggest that despite the sequence divergence among gammaherpesvirus D-type cyclins, interactions with respective CDK
binding partners are mediated by key conserved residues (Card et al. 2000).
MHV68 v-cyclin null mutants have profound reactivation defects as assessed by
ex vivo analyses. While the virus establishes latency in splenocytes and peritoneal
exudate cells (PECs) at a similar frequency to wild-type or a genetic marker-rescue
virus in C57BL6 mice, it is impaired for virus reactivation in both cellular compartments (Hoge et al. 2000; van Dyk et al. 2000). The defect is more apparent in PECs,
and B-cell-deficient mice infected with a cyclin-LacZ insertion virus exhibit an
exaggerated reactivation defect (van Dyk et al. 2003). This reactivation defect most
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likely contributes to the deterioration in the frequency of infected cells by 6 months

postinfection. These data suggest that, in addition to B lymphocytes, v-cyclin plays
a critical role in launching MHV68 lytic replication/reactivation and maintaining
latent infection in the non-B cell reservoir (e.g., macrophages). This supports a role
for v-cyclin in promoting lytic viral gene transcription and cell-cycle progression in
resting lymphocytes, perhaps simultaneously driving cellular proliferation and reactivation from latency. Interestingly, viral mutants containing only mutations in key
residues important for CDK-interaction and activation display a significant decrease
in reactivation from splenocytes, but do not appear compromised for reactivation in
PECs (Upton and Speck 2006). This defect, however, may be explained by the replication defects seen in the lung of infected animals, which may then result in
decreased establishment and reactivation in the spleen. This indicates that although
the overall result of v-cyclin expression may be cell-cycle progression, the mechanism for overcoming growth barriers in infected cells varies according to cell type
and ultimately requires both CDK-dependent and independent functions for a successful in vivo infection.
The role of v-cyclin in permissive lytic infection is not fully elucidated, but also
supports the possibility of cell-type dependent mechanisms. While high-dose intranasal inoculation of C57BL/6 mice with v-cyclin mutants produces lung titers similar to those with a marker-rescue virus, inoculation with a lower dose of virus reveals
a requirement for an intact v-cyclin including the amino acids essential for CDKbinding and activation (Upton and Speck 2006; van Dyk et al. 2000). This characteristic does not appear to be essential for all lytic replication, as v-cyclin is
dispensable for lytic replication in vitro in NIH 3T12 fibroblasts and some lung
epithelial cell lines, as well as for virus replication in the spleen following intraperitoneal infection (Hoge et al. 2000; Upton and Speck 2006; van Dyk et al. 2000).
However, the in vitro analyses are complicated by the fact that cells used in these
experiments were either actively cycling (in vivo), or serum-starved but capable of
reentering the cell cycle (in vitro), thereby potentially masking the requirement for
v-cyclin-CDK-interactions (via the action of host cyclin-CDK complexes). Thus, it
appears that the MHV68 v-cyclin plays a specific role in driving cell-cycle progression and lytic replication in terminally differentiated cells in the lung in vivo.
Evidence for v-cyclin as an oncogene. Much evidence exists to support the classification of gammaherpesvirus v-cyclins as oncogenes (Mittnacht and Boshoff 2000).
In the case of MHV68, some of the most convincing data are those demonstrating
T-cell lymphomagenesis in transgenic mice expressing v-cyclin under the control of
the lck promoter (van Dyk et al. 1999). These studies support a proproliferative role
for v-cyclin, as T lymphocytes exhibited a marked increase in BrdU-incorporation
and the percentage of S/G2/M cells compared to total splenocytes. However, despite
the increased proliferation of thymocytes in these animals, there was only a slight
increase in total T cell number, a disconnect attributed to increased apoptosis of
v-cyclin-expressing cells in the thymus. Although several factors may contribute to
cell death in the transgenic animals, one possible conclusion is that v-cyclin is sufficient to support cell cycle progression and proliferation yet insufficient to inhibit
apoptosis in the context of v-cyclin-driven thymocyte expansion.
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The effects of v-cyclin-CDK complexes on the activation status of key cell-cycle

regulatory proteins support its classification as an oncogene. The tumor suppressors
Rb, p21Cip1, p53, and p27Kip1, as well as the antiapoptotic protein bcl-2, have been
shown to be phosphorylation targets of MHV68 v-cyclin in a host-CDK-dependent
fashion (Card et al. 2000; Upton et al. 2005). This observation is in apparent contrast to those made in the context of MHV68 infection of endothelial cells. While
infection of epithelial cells results in productive viral replication followed by cell
lysis, infection of endothelial cells appears to support persistent infection wherein
cells produce titers equivalent to epithelial cell cultures but retain viability for weeks
following infection (Suarez and van Dyk 2008). This prolonged survival appears to
be dependent on v-cyclin, as cells infected with a v-cyclin-null virus produce infectious virus but exhibit greatly reduced viability. This seems to argue for a prosurvival role for v-cyclin during productive replication in endothelial cells. It may,
therefore, be that v-cyclin is not sufficient to rescue cells forced into cycling from
apoptosis (as in the transgenic mouse system) but is, nonetheless, required for protection against cell death in a persistently infected cell.
A particularly notable aspect of endothelial cell infection was the distinct downregulation of cellular adhesion molecules on infected cells, accompanied by uniform changes in cell size and morphology. These changes could be ameliorated by
treatment with PAA, which blocks late viral gene expression and, therefore, were
most likely a direct result of viral infection. Infected cells displayed markedly
reduced expression of ICAM-1 and VCAM-1, two molecules involved in cellular
adhesion. This complements the observation that a significant number of endothelial cells continued to survive and produce virus even after detaching from the tissue
culture matrix. It was, therefore, concluded that MHV68 infection contributes to the
acquisition of anchorage-independent growth characteristics via the down regulation of cellular adhesion molecules, a process with strong implications for both
trafficking of infected cells as well as tumorigenesis. V-cyclin is not required for
these changes in cell surface marker expression, so it, therefore seems as if its role
in endothelial cell infection is primarily to promote sustained viability of cells
undergoing active viral replication. Though not the same mechanism for tumorigenesis as cell-cycle promotion, this function of v-cyclin also has implications for
the preferential survival and outgrowth of infected cells.
v-cyclin and tumorigenesis. Although BALB/c b2m/ mice are predisposed to
developing tumors, MHV68 infection both increases the incidence and accelerates
the development of lymphomas in these mice (Tarakanova et al. 2005). In particular, MHV68 exacerbated the development of what the authors termed atypical lymphoid hyperplasia (ALH) in BALB/c b2m/ mice, a lesion of hyperproliferation
characterized by MHV68-positive cells and high numbers of plasmacytic CD138+
cells (Tarakanova et al. 2005). ALH lesions preceded the development of B cell
lymphoma and shared similarities with a subset of PTLD. ALH lesions were, therefore, used as a measure of MHV68-induced tumorigenesis. A subsequent study
found that mice infected with a v-cyclin-null virus displayed significantly less
splenic ALH than mice infected with wild-type virus, despite the presence of latent
viral genomes (Tarakanova et al. 2008). Mice infected with the v-cyclin null virus did,
274
however, exhibit plasmacytosis at a frequency similar to wild-type virus-infected

mice. This observation highlights the complexity of events leading to MHV68mediated tumorigenesis. While individual viral oncogenes, such as v-cyclin, may
contribute directly to cellular transformation and lymphomagenesis by promoting
replication or cell survival, it is important to consider that MHV68 infection as a
whole may also contribute to this process by promoting a proinflammatory environment, which leads to cell damage and the acquisition of transforming mutations.
v-bcl2
General characteristics. A key component of successful virus replication is the
override of host-derived cell death signals in response to viral infection. To this
end, many viruses have evolved specific mechanisms to allow survival of an
infected cell by suppressing multiple cell death pathways, such as apoptosis,
necrosis, or autophagy. Prosurvival genes are an especially important component of oncogenic virus infection for obvious reasons; pathogen-aided viability
of a cell with compromised genomic integrity can have deleterious consequences
for the host organism should this cell sustain further insult and acquire growthpromoting characteristics. However, given the hostile environment created by
the likely induction of interferon production and the antiviral state during the
initial stages of viral replication, it is essential that host-cell death signals be
suppressed for infection to progress and latency to be established, particularly
in B cells and macrophages.
The apoptotic pathway has been long-studied as a mechanism of programmed
host-cell death and is regulated by homo- and heterotypic interactions amongst a
family of pro- or antiapoptotic proteins. Very simplistically, the proapoptotic proteins Bad, Bid, and Bax are antagonized by their association with antiapoptotic
proteins Bcl-2 and BclXL. Cells respond to numerous insults, including viral infection, by undergoing programmed cell death. Premature cell death is likely deleterious to the long-term pattern of viral replication, latent persistence and reactivation.
Therefore, many viruses have evolved proteins that inhibit apoptosis by blocking
the actions of host proteins involved in cellular suicide. KSHV, EBV, HVS, and
MHV68 all encode molecules with homology to a cellular protein, bcl-2, that prevents cell death elicited by mitochondrial insults. (Virgin et al. 1997). M11/v-Bcl2
and MHV68 v-cyclin are encoded on opposite strands in close proximity (Virgin
et al. 1997). Despite the immediate juxtaposition of M11 and v-cyclin, the expression profiles of their transcripts are quite distinct with regard to acute replication.
The original characterization of M11 transcription in B-cell deficient mice suggested that M11/v-Bcl2 is expressed at low levels during lytic replication, but it is
easily detected in latently infected peritoneal cells and is, therefore designated a
latency-associated gene (Virgin et al. 1999). Subsequent studies in wild-type animals indicated that M11 is actively transcribed during both acute lytic and persistent
infection (Roy et al. 2000).
275
The gammaherpesvirus Bcl-2 orthologs bear less than 25% sequence homology
to their cellular counterparts and do not appear to contain distinct BH3 or BH4
domains (Tschopp et al. 1998). MHV68 M11, in particular, is also divergent from
the other gammaherpesvirus proteins in that it contains only a BH1 domain and
lacks an obvious BH2 domain consistently found in other v-Bcl2s. BH3 and BH4
domains are required for interaction of cellular Bcl-2 and Bcl-xl proteins to potentially block antiapoptotic function. Therefore, the lack of these domains may make
the M11 protein refractory to regulation by proapoptotic host proteins. Earlier studies demonstrated that M11 localizes to the cytoplasm and that overexpression was
sufficient to protect cells from TNF and Fas-induced apoptosis (Roy et al. 2000;
Wang et al. 1999), but no comprehensive analysis of cellular interacting partners has
been performed to verify M11s involvement in the host apoptotic pathway. More
recent work has demonstrated that despite the absence of domains required for association with BH3-domain containing proteins, M11 tightly associates with the
proapoptotic Bim, Bak, Bid, Bmf, Puma, and Noxa (Ku et al. 2008). In addition,
these and other studies revealed an additional prosurvival role for M11 in the inhibition of autophagy via inhibition of Beclin-1 (Xiaofei et al. 2009; Ku et al. 2008;
Sinha et al. 2008). Beclin-1 is a proautophagic BH3-domain containing protein
whose action is also inhibited by host Bcl-2. It was shown not only that M11 tightly
associates with Beclin-1 by isothermal titration calorimetric analyses but also that
this interaction was significantly stronger than Beclin-1 with host Bcl-2. It was subsequently shown that in addition to protection from apoptosis M11 could not only
protect against rapamycin-induced autophagy but could also do so more efficiently
than cellular Bcl-2 (Xiaofei et al. 2009). These analyses suggest that sequence
divergence from host Bcl-2 proteins has not compromised the effectiveness of viral
Bcl-2 homologs, but rather conferred upon them enhanced prosurvival functions
capable of protection against multiple host-cell-induced death pathways.
It would seem that survival of the host cell in the midst of death signals triggered
by lytic viral infection would require the function of virus-encoded antiapoptotic/
autophagic proteins, yet studies using viruses lacking the Bcl2 homolog demonstrated that M11, like BHRF1 of EBV, is dispensable for acute replication in vitro.
Similarly, M11-null MHV68 replicates equivalent to wild-type virus in the lung and
other sites in vivo (de Lima et al. 2005; Gangappa et al. 2002b). However, while
M11 is not required for the establishment of latent infection, it appears to contribute
to the efficiency of virus reactivation, a characteristic shared with the v-cyclin-null
virus as discussed above and made more interesting by the demonstration that both
v-cyclin and M11 appear to be involved in persistent replication and the associated
severity of inflammatory disease observed in IFNg/ and IFNgR/ mice (Gangappa
et al. 2002b; Tarakanova et al. 2008).
The fact that two viral proteins involved in forcing cell-cycle progression and
survival would be dispensable for acute replication, yet important for reactivation,
provides important clues about the pathogenesis of latent viral infection. It has been
hypothesized that low-level, spontaneous reactivation in vivo is one of the mechanisms by which herpesviruses maintain long-term latency. Given the observations
above, it seems that reactivation from a latently infected, perhaps resting, lymphocyte
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requires viral proteins to not only stimulate the cell cycle, but to preserve viability
in the cell driven into a cycling state. How this compromises the functional and
genetic integrity of the infected cell is not entirely clear, but may set the stage for
transforming events to occur. As discussed below, the involvement of the host
immune system in this process further highlights the complexity of these
interactions.
Evidence for v-bcl2 as an oncogene. As mentioned above, several studies have demonstrated the potent antiapoptotic activity of the M11 protein. When overexpressed,
M11 can protect against Sindbis virus-induced apoptosis in BHK cells, as well as
TNF-a and Fas-induced apoptosis in HeLa cells (Bellows et al. 2000; Roy et al. 2000;
Wang et al. 1999). The cell death pathways triggered by intrinsic factors (such as virus
infection) and extrinsic factors (such as TNF/Fas-ligation) have overlapping yet distinct pathways. It, therefore seems that the M11 can protect against both intrinsic and
extrinsic insults that may otherwise result in cell death in the context of these studies.
M11 likely supports survival during MHV68 infection and, given the data regarding
M11-mediated protection from Fas-ligation, may also protect against CTL-mediated
killing during an immune response. M11 may, therefore, enhance survival of infected
cells that have sustained transforming mutations potentially facilitating tumorigenesis. Since autophagy also mediates tumor-suppressive functions in vivo, subversion of
autophagy by M11 (Xiaofei et al. 2009; Ku et al. 2008; Sinha et al. 2008) via suppression of Beclin-1-mediated autophagosome formation may also result in viruspromoted survival of a cell that would otherwise be eliminated.
Similar to v-cyclin-null MHV68, M11-null virus does not induce ALH as efficiently as M11 competent MHV68 following infection of BALB/c 2m/ mice.
Since the autophagic pathway is important in tumor suppression (reviewed in Bialik
and Kimchi 2008; Gozuacik and Kimchi 2004), it may be that v-bcl2-mediated inhibition of this process contributes to the development of ALH in the absence of a
CD8+ T cell response. In the coincident absence of an optimal immune response and
antiautophagic or antiapoptotic v-bcl2, cell death may be induced by other extrinsic
or intrinsic signals, thus limiting the amount of cellular proliferation and controlling
lymphoid hyperplasia, limiting the initiating events of lymphomagenesis.
vGPCR
General characteristics. A third conserved gammaherpesvirus protein is that
encoded by MHV68 Orf74, giving rise to a product orthologous to host G-proteincoupled-receptors (GPCRs). These viral GPCRs (v-GPCR) share characteristics
reminiscent of host receptor proteins and are either activated by similar ligands or
signal through common pathways (Nicholas 2005) Like the KSHV vGPCR, the
MHV68 GPCR bears significant sequence homology to host CXCR2 receptors,
which binds several chemokines and has particularly high affinity for IL-8 (Virgin
et al. 1997; Wakeling et al. 2001). The KSHV vGPCR is highly transforming, which
is thought to be due in part to its ability to induce prosurvival and proangiogenic
277
signaling in response to IL-8 and other ligands (Arvanitakis et al. 1997; Sodhi et al.
2000; Yang et al. 2000). Similarly, MHV68 vGPCR confers on NIH 3T3 fibroblasts
the ability to form foci in culture and in soft agar, suggesting that the MHV68 protein may also be transforming (Wakeling et al. 2001). As a delayed-early gene,
Orf74 is transcribed weakly and variably during lytic replication, but is relatively
abundant in latently infected PECs, and it has, therefore been classified as a latencyassociated gene (Virgin et al. 1999). This classification would support a potential
role in tumorigenesis, as the majority of gammaherpesvirus-associated malignancies are associated with latent infection. Indeed, Orf74 appears to be dispensable for
lytic replication in vitro; however, Orf74 is required for augmented viral production
in response to CXCR2 agonists (Lee et al. 2003; Verzijl et al. 2004). One of these
studies also demonstrated the involvement of MEK and PI3K in Orf74-mediated
effects (Lee et al. 2003). These data suggest that Orf74 plays a specific role in
manipulating cell signaling in infected cells to exaggerate or induce a response to
extracellular stimuli during latent infection. This has several potential outcomes,
including prolonged survival, angiogenesis, chemotaxis, or viral reactivation from
the infected cell. In support of this hypothesis, Orf74 mutant viruses display normal
acute replication but are slightly compromised for reactivation. Separate studies
have demonstrated a requirement for Orf74 in efficient reactivation from splenocytes (Lee et al. 2003; Moorman et al. 2003a).
Evidence for vGPCR as an oncogene. Although the in vivo evidence for the MHV68
vGPCR is not as compelling as that for v-cyclin or v-bcl2 in terms of transforming
capabilities, it is important to consider its potential, more subtle effects on MHV68
pathogenesis. The vGPCR of KSHV is thought to contribute more to the growth and
migration of KSHV-positive tumors, facilitating survival of a transformed cell
rather than directly inducing its transformation (Arvanitakis et al. 1997). The
MHV68 vGPCR may have a limited role or, an as yet undefined function in the
context of normal infection, yet confer tumor-promoting properties in a cell already
compromised by a previous transforming event (a promoter rather than an initiator). That the viral protein bears resemblance to a host chemokine receptor, specifically one involved in inflammatory responses and angiogenesis, raises the possibility
that the vGPCR is involved in the trafficking of infected cells during latency. The
observation that infection with a vGPCR-deletion mutant results in an increased
frequency of infected peritoneal cells may support this possibility (Moorman et al.
2003a). In the same infections, however, the increase in genome positivity did not
correlate with an increase in reactivation frequency, suggesting at least two possible
explanations (1) the vGPCR is involved in reactivation from latency, or (2) absence
of the vGPCR results in altered trafficking of cells into the peritoneum that are
genome-positive but do not efficiently reactivate at this stage of latency (reminiscent of splenocytes at day 42). Should the latter be true, it can be extrapolated that
a viral protein capable of altering cell migration patterns, in the presence or absence
of an inflammatory response, might contribute significantly to dissemination of
MHV68-infected cells within the host. These changes may have effects on invasion
and metastasis in the MHV68-mouse model system, or in natural infection, that
have yet to be appreciated.
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mLANA
General characteristics. MHV68 Orf73 encodes a protein with homology to the
latency-associated nuclear antigen (LANA) of KSHV, which also shares limited
homology with HVS LANA (Virgin et al. 1997). Although significantly variant by
sequence from the EpsteinBarr nuclear antigen 1 (EBNA1), the gamma-2-herpesvirus
(KSHV, HVS, MHV68) LANA proteins function similarly to EBNA1. The best
studied of these functions is tethering of the viral genome to host chromatin during
latency. During latency, the viral genome is maintained as a circular episome; during
host cell division, EBNA1 and LANA are thought to be essential for the faithful segregation of the viral genome to host daughter cells, although this notion has not been
directly tested in EBV or KSHV-infected cells. From studies using an MHV68 LANA
(mLANA)-null virus, mLANA also appears to be critical in the establishment of
MHV68 latency in the spleen following intranasal infection (Moorman et al. 2003b).
This phenotype is complicated, however, by the observation that the mutant virus has
a lytic replication defect in the lung, which may compromise the process by which
cells traffic from the lung to the spleen where they seed the primary latency reservoir
(Moorman et al. 2003b). Importantly, recent data demonstrate that the splenic establishment defect following intranasal infection can be partially ameliorated by infecting mice intraperitoneally (Paden et al. 2010). While mLANA-null genome-positive
cells persist, the viral genome does not appear to be episomal in Orf73-null infected
cells and reactivation is entirely compromised. These data emphasize that initial events
taking place in the lung following intranasal infection are key to establishing splenic
latency, inextricably linking early lytic infection and long-term latent infection with
the host immune response.
mLANA as an oncogene. In addition to its role in episomal maintenance, gamma-2herpesvirus LANA is also a potent transcriptional regulator of both host and viral
gene transcription. A dual role for LANA in lytic and latent infection is evidenced
by the kinetics of LANA transcription. Orf73 was originally classified as a latencyassociated gene based on the specific and ready detection of LANA protein and
transcript in KSHV-associated malignancies (Courville et al. 2002; Si et al. 2005).
However, Orf73 transcription has immediately early kinetics, with protein detected
as early as 4 h postinfection and at low levels throughout lytic infection (Forrest
et al. 2007). In studies with KSHV and HVS, LANA has been reported to directly
interact with or regulate the activity of several host proteins with known roles in
tumorigenesis, such as HIF1a, DNMT3A, p53, Rb, and beta-catenin (Borah et al.
2004; Cai et al. 2006; Forrest et al. 2007; Liu et al. 2007; Shamay et al. 2006; Verma
et al. 2007). LANAs involvement with these proteins could influence lytic replication from both the virus and host cell prospective. For example, KSHV LANA
interacts with HIF1a to activate transcription of the primary lytic transactivator,
Rta, under hypoxic conditions, yet acts as a transcriptional repressor of Rta during
normoxia interactions with a potentially strong impact on initial infection and
reactivation (Cai et al. 2006). With respect to host proteins, KSHV LANA promotes
Rb phosphorylation and inactivation of p53 to facilitate cell-cycle progression and
override DNA damage signals during infection (Friborg et al. 1999).
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Recently, MHV68 LANA was shown to influence cell survival via these host
pathways. A common cellular defense in response to viral infection is via activation
of p53, followed by subsequent cell cycle arrest and induction of apoptosis. MHV68
induces DNA damage signals upon infection, including phosphorylation of gH2AX
(Tarakanova et al. 2007) and, as discussed above, also encodes proteins to promote
cell-cycle progression despite this arrested state. Subversion of p53 activity during
this process is, therefore critical to productive viral replication, and mLANA has
been shown to be a central mediator by coordinating viral gene expression and destabilizing p53. In these experiments, cells infected with an mLANA-null virus demonstrated increased p53 activation and reduced viability, while mLANA overexpression
afforded protection from etoposide-induced cell death (Forrest et al. 2007).
The implications of a p53-inactivating viral protein in oncogenic transformation
are clear, as has been extensively demonstrated for a number of viral proteins (e.g.,
HPV E6 and SV40 large T-antigen). A more subtle contribution of mLANA to
MHV68-induced tumorigenesis may be through its role as a transcriptional modulator. KSHV LANA expressed from its endogenous promoter in transgenic mice has
been shown to increase the incidence of lymphoma (Fakhari et al. 2006), a result
most likely linked to a combination of p53 subversion and regulation of host transcription. Although mLANA appears to be essential for reactivation, a separate role
for mLANA in viral gene transcription during latency has yet to be clearly defined.
It is possible that constitutive LANA expression during latency results in compromised p53 function and virus-controlled host and viral gene transcription, thereby
contributing to tumorigenesis by increasing an infected cells vulnerability to secondary transforming mutations.
Prooncogenic Alterations in Cell Signaling Associated

with MHV68 Infection
Many cellular alterations induced upon viral infection have been ascribed to specific
MHV68 proteins. However, there are signaling pathways affected by viral infection
that are most likely influenced by not-yet-identified viral proteins that act either
individually or in concert. This section highlights examples of host-cell signaling
networks altered upon infection that are likely targeted by viral proteins. Moreover,
these pathways are often altered in transformed cells, supporting the hypothesis that
perturbations incurred during MHV68 infection may facilitate tumorigenesis.
NF-kB
NF-kB is a key transcriptional regulator with diverse roles in numerous cellular
processes. It has been extensively studied in the context of inflammation due to its
role in regulating a myriad of functions including differentiation, metabolism, and
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the development and execution of immune responses. It has also been a subject of
intense focus in tumorigenesis, particularly blood-based lymphomas and leukemias.
NF-kB has potent prosurvival, proproliferative effects and is often constitutively
active in tumors of this origin. It is widely believed that prolonged inflammation
promotes tumorigenesis thus, in addition to its direct association with survival and
proliferation, constitutive NF-kB activation has also been implicated as a central
factor linking inflammation and cancer.
The NF-kB pathway is of central importance in normal lymphocyte biology and
is the converging point for several extrinsic and intrinsic B cell signals. Several
studies have demonstrated that gammaherpesviruses exploit NF-kB signaling to
achieve and maintain latent infection, promoting activation and translocation of
NF-kB complexes in infected cells (Brown et al. 2003; Krug et al. 2009; Krug et al.
2007; Sgarbanti et al. 2004; Thornburg et al. 2006; Yao et al. 1995). Indeed, studies
have shown that NF-kB is dispensable for lytic replication, but plays a clear role in
the establishment and, interestingly, the control of latent MHV68 infection (Krug
et al. 2009). Gel-shift analyses demonstrated activated p50 during both latent (S11E
lymphoma cells) and lytic infection. It, therefore, seems likely that NF-kB plays a
role in both promoting cell survival and proliferation during lytic replication and
reactivation, but also in controlling reactivation in a latently infected cell. This
implies that NF-kB is constitutively active throughout the duration of MHV68
infection, acting both on viral and host genes at each stage of the viral life cycle.
Several roles for NF-kB in inflammation-associated tumorigenesis have been proposed with regard to the development of Hodgkins lymphoma (for a review see
Khan 2006). The effects of constitutive NF-kB activation on the state of host gene
transcription during MHV68 infection are unknown, but it seems plausible that
constitutive activation of NF-kB as an inflammatory mediator contributes to oncogenic transformation through a combination of environmental (cytokines, chemokines)
and intrinsic (antiapoptotic/proproliferative signals) alterations in infected cells.
PI3K/Akt
The PI3K/Akt signaling axis is implicated in numerous cancers such as prostate,
thyroid, lung, and breast. It is also affected by MHV68 infection. As mentioned
previously, PI3K signals are perpetuated by the v-GPCR. Further, inhibiting PI3K/
Akt activation during MHV68 infection results in increased lytic replication, presumably mediated by increased Rta activity (Peng et al. 2010). This implies a mechanism by which MHV68 infection stimulates PI3-K/Akt pathways to facilitate the
establishment of latent infection by limiting lytic replication. This is similar to the
proposed mechanism of NF-kB, perhaps because the PI3K pathway is upstream and
feeds into the NF-kB pathway in some signaling pathways. Given potential involvement of PI3K/Akt signaling in tumorigenesis, MHV68-mediated activation of PI3K/
Akt may also play a role in the promotion of tumorigenesis in the context of MHV68
infection.
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Twist/Snail
As mentioned above, MHV68 infection of endothelial cells results in downregulation of cellular adhesion molecules and the subsequent loss of anchoragedependent growth. The loss of adhesion molecules on tumor cells has been
extensively studied in the epithelial-to-mesenchymal transition (EMT), the process by which tumor cells are thought to detach from the initial tumor site, enter
the blood or lymphatic system, and metastasize to distant anatomical locations.
Lymphocytes are circulating cells by nature, but their role, as well as the role of
non-B cells, in virus trafficking and the dissemination of infection is not defined.
The transcription factor Twist has been shown to be a positive regulator of EMT
by contributing to the downregulation of several epithelial markers and acquisition of mesenchymal cell markers. Recent studies indicate a role for Twist in
promoting cell-cycle progression via inactivation of pRb and p53. Indeed, high
Twist expression may reflect a poor prognosis in phenotyping for certain cancers
(Ansieau et al. 2010).
A recent study using MHV68 in a mouse model for idiopathic pulmonary fibrosis demonstrated that MHV68 infection enhances Twist expression in lung epithelial cells (Pozharskaya et al. 2009). Increased Twist expression is accompanied by
the expression of mesenchymal and epithelial markers on the same cell, such that
infected cells exhibit a mixed phenotype. Analyses of Twist expression have not
been performed in MHV68-infected lymphocytes, but it is possible that this pathway is involved in the migration of infected cells to and from sites of infection.
MHV68 Immunomodulation
Gammaherpesviruses establish latent infection in immune cells. The immune
response is, therefore key to shaping and promoting a successful latent infection
in terms of (1) successful host immune evasion by the virus; and (2) the contribution of the affected cell during long-term infection. As a result, these viruses have
evolved mechanisms to modulate the immune response in favor of establishing
and maintaining latency in these specialized cell populations. Several viral proteins have been shown to promote and maintain latent infection by directly manipulating infected cells, while others indirectly affect surrounding cells to facilitate
immune evasion and create an environment conducive to latency establishment.
Many of these proteins are powerful modulators whose expression is sustained
throughout chronic infection, which is perhaps why the majority of gammaherpesvirus-related tumors are associated with latent infection. In combination with
immunosuppression, these proteins may create the idea environment for tumorigenesis. Below, we describe a few MHV68 proteins with known immunomodulatory functions and discuss their possible contribution to MHV68-mediated
tumorigenesis.
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M2
General characteristics. MHV68, like the other characterized gamma-herpesviruses,
harbors several genes that encode proteins with no known host or viral homologues
(Virgin et al. 1997). However, despite the lack of obvious homologues in the other
gammaherpesviruses, frequently these novel gammaherpesvirus gene products
share common functions. One key example is the M2 protein of MHV68. Both
KSHV and EBV encode proteins that manipulate host cytokine production, in particularly the IL-10 and IL-6 signaling pathways which have significant effects on B
lymphocyte proliferation, differentiation, and survival. In humans, IL-10 influences
B cell proliferation and differentiation, a link with implications for gammaherpesvirusrelated lymphomagenesis. The BCRF1 gene of EBV encodes an IL-10 homolog
(vIL-10), while KSHV encodes an IL-6 homolog (vIL-6), both of which have been
linked to increased proliferation of infected cells in vitro and in virus-positive tumor
cells (Nicholas 2005). Although M2 is not a cytokine homolog, it induces the production of several cytokines pivotal to B cell physiology, most notably IL-10, and to
a lesser extent, IL-6 (Siegel et al. 2008). In addition, M2 induces expression of both
the high-affinity IL-2 receptor and IL-2 in primary murine B cells (Siegel et al.
2008) which may also play a role in driving B cell proliferation. How M2 manipulates cytokine signaling pathways remains unclear; however, M2 does contain
several motifs capable of interacting with proteins involved in lymphocyte signal
transduction (discussed below).
M2 expression is primarily limited to latent infection, and it is, therefore considered a latency-associated gene (Virgin et al. 1999). The phenotype of an
M2-null virus is complex and is dependent on both dose and route of infection.
M2 is critical for the efficient establishment of and reactivation from latency following low-dose (100 pfu) intranasal infection (Herskowitz et al. 2005; Jacoby
et al. 2002). Increasing the size of the intranasal dose (106 pfu) partially ameliorates the defect in the establishment of latency but the reactivation defect remains.
Infection via the intraperitoneal route largely overcomes the establishment of
latency defect, with low-dose intraperitoneal infection largely recapitulating the
high-dose intranasal phenotype (Herskowitz et al. 2005). Interestingly, under
infection conditions where the frequency of infected cells is largely similar in
M2-null and marker rescue-infected animals, the profile of M2-null-infected cells
is altered; while the majority of viral genome-positive cells in wild-type infection
bear hallmarks of class-switched, germinal-center experienced B cells (sIgD-),
mice infected with M2-null virus harbor significantly increased frequencies of
viral genome-positive nave B cells (sIgD+). This phenotype is resolved by 6
months postinfection, when the majority of viral genome-positive cells for both
mutant and marker rescue virus are sIgD-.
Immunomodulatory functions of M2 may promote lymphomagenesis. The significance of the M2-null phenotype in long-term latent infection increases given recent
data demonstrating that M2 drives B-cell differentiation and class-switching (Liang
et al. 2009; Siegel et al. 2008), a mechanism integral to the proposed model for
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EBV-driven B cell differentiation via viral proteins such as LMP1 and LMP2A.
These studies collectively showed that M2 overexpression in nave primary B cells
or a pregerminal center B cell line resulted in partial loss of B-cell and acquisition
of plasma-cell-surface phenotypic markers. M2-expressing cells also exhibited a
blast-like phenotype and enhanced proliferation, further supporting the hypothesis
that M2 expression in infected cells serves to promote the differentiation of nave B
cells in vivo.
How M2 drives this transition is not completely understood. The M2 protein
contains two tyrosines which, when phosphorylated, may provide targets for SH2domain containing proteins (Herskowitz et al. 2008; Madureira et al. 2005; Pires
de Miranda et al. 2008; Rodrigues et al. 2006). It also has several PxxP motifs,
which function as docking sites for SH3-domain containing proteins. SH2 and
SH3 domains are critical components of lymphocyte signaling cascades, allowing
proteinprotein interactions that perpetuate signals from the cell surface to the
nucleus to modulate transcription. Studies have reported the interaction of M2
with several proteins involved in signal transduction, such as Vav, Grb2, and Fyn,
as well as Ras GTPase-activating protein 1, and Rho GTPase-activating protein 4
(Herskowitz et al. 2008; Madureira et al. 2005; Pires de Miranda et al. 2008;
Rodrigues et al. 2006).
As previously mentioned, constitutively active or deregulated signaling, for
example that resulting from mutated proteins or gene fusions, has been implicated
in innumerable cancers there is, therefore, a possibility that M2 function may have
implications for tumorigenesis in MHV68-infected animals. The relationship
between M2-host protein interactions and M2-mediated effects has not been clearly
established, but it is hypothesized that the greatly augmented IL-10 produced in
M2-transduced cultures is a result of a sequence of signaling events initiated by M2
at the plasma membrane. IL-10 has pleiotropic effects on murine lymphocytes; it
enhances B cell survival while suppressing proinflammatory Th1-type immune
responses, including T cell and NK cell function. Mice infected with M2-null virus
have decreased serum IL-10 and increased MHV68-specific CD8+ T cells (Siegel
et al. 2008). We later discuss the potential role of CD8+ T and NK cells in tumor
surveillance with regard to MHV68 infection. By simultaneously promoting B cell
viability and suppressing anti-MHV68 immunity, one can imagine a scenario in
which M2 function serves as the fulcrum in the balance between successful longterm latency and the advent of malignant disease in infected animals. Further
studies delineating the direct effects of M2 in host cells will provide more clues
as to how this balance is maintained in healthy animals.
Overexpression of M2 in nave B cells results in cells displaying a preplasmablast phenotype; they downregulate hallmark B lineage markers such as B220,
MHC class II, and sIgD, while retaining CD19 and IgG expression and only modestly upregulating expression of Syndecan-1, a marker for differentiated plasma
cells (Siegel et al. 2008). Yet M2 seems to be crucial for class-switching in infected
primary B cells and induces IgM secretion in BCL-1 cells, accompanied by transcriptional changes associated with plasma cell differentiation as well as acquisition of a large, granular, blast-like appearance (Liang et al. 2009). In other words,
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M2 seems to drive nave cells to a gray zone of differentiation, too differentiated

to be called a true B cell, but not yet a full-fledged plasma cell. Importantly, this
partially differentiated phenotype has been observed in several human lymphomas,
some of which are associated with gammaherpesvirus infection. Hodgkin Reed
Sternberg cells, the diagnostic marker for classical Hodgkins lymphoma, contain
rearranged immunoglobulin loci and are, therefore, presumably derived from
germinal center-experienced B lymphocytes. However, many HD tumors do not
express CD19, CD20, or a functional BCR (Kuppers 2009; Schwering et al. 2003).
Accumulating genetic evidence supports the B-cell origin of these cells it has,
therefore, been hypothesized that HRS cells are B cells that have undergone a
crippling mutation, but are rescued from apoptosis by an alternative transforming event (e.g., bcl-2 translocation). These cells then survive the germinal
center reaction and persist as genetically and phenotypically altered cells of B cell
origin that function neither as a plasma cell nor a traditional differentiated memory
B cell (Brauninger et al. 2003; Kanzler et al. 1996; Kuppers 1999). EBV-positive
HRS cells exhibit a type II latency program and express LMP1 and LMP2A. LMP1,
via CD40-like signal transduction, has been shown to generate cells with a post-GC,
preplasma cell phenotype (Rastelli et al. 2008; Uchida et al. 1999; Vockerodt et al.
2008), while LMP2A has been shown to compete for binding of Src-family kinases
in EBV-transformed cells (Fruehling et al. 1996; Fruehling and Longnecker 1997;
Miller et al. 1995; Miller et al. 1994). The consequences of M2 overexpression in
nave B cells share remarkable similarities with these particular LMP1 and
LMP2A-mediated effects, and support the possibility that M2 may serve a hybrid
LMP1/LMP2A function in MHV68 infection. The possibility that M2 can also
rescue cells from apoptotic cell death in vivo remains to be tested, but evidence
shows that in addition to its role in driving proliferation and differentiation, as well
as cytokine production, M2 also interacts with the DDB1/COP9/cullin repair
complex and the ATM DNA damage signal transducer to protect cells from
DNA-damage-induced apoptosis, such as that which is sustained by B cells during
the germinal center reaction (Liang et al. 2006). The phenotype resulting from M2
overexpression in B cells is also reminiscent of that observed in several KSHVassociated malignancies, including primary effusion lymphomas and a plasmablastic variant of multicentric Castlemans disease (MCD). Cells from these tumors
share important characteristics with M2-expressing cells in that they retain several
features to indicate a B cell origin such as immunoglobulin gene rearrangement, yet
acquire several characteristics of plasma cells, such as CD38 cell surface expression
and transcription of plasma-cell specific genes (Du et al. 2007, 2001; Dupin et al.
2000; Jenner et al. 2003). It, therefore seems that partial differentiation of B
lymphocytes into plasma cells is an associated consequence of gammaherpes
virus infection. The fact that M2, an MHV68 protein lacking any known cellular or
viral homologs, can induce similar phenotypic changes in B cell lines and primary
splenocytes is quite intriguing and may provide an opportunity to better understand
mechanisms involved in the transformation in human lymphocytes.
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mK3
A critical aspect to successful herpesvirus infection is the ability to escape immune
clearance by CD8+ cytotoxic T cells (CTL). Many viruses have achieved this by
encoding proteins specifically designed to reduce MHC class I expression on
infected cells, thereby hindering the cells ability to present viral antigen to activated CTLs. The mechanisms by which viral proteins downregulate MHC class I
expression vary. Like KSHV, MHV68 encodes a zinc finger protein, MK3, which
functions as a ubiquitin ligase to target newly synthesized MHC class I for degradation (Boname and Stevenson 2001; Lybarger et al. 2003). MK3 is localized to
the ER membrane, where it binds to and ubiquitinates the cytoplasmic tails of
nascent MHC class I molecules, leading to rapid proteosomal degradation (Boname
and Stevenson 2001). However, this mechanism does not seem to be the sole process responsible for MHV68 immune evasion. First, infected B cells can also present to CD8+ T cells via the nonclassical class I glycoproteins Qa-1 and Qa-2, which
are not susceptible to ubiquitination. Second, sufficient levels of IFNg, such as
those present as a result of MHV68 infection, have been shown to overcome MHC
class I virus-mediated degradation. Further studies have demonstrated that in addition to targeting classical MHC class I, MK3 also facilitates the degradation of
TAP, a major component of the class I peptide loading complex (Boname et al.
2004; Boname et al. 2005). This mechanism inhibits both classical and nonclassical MHC class I presentation, as well as conferring increased resistance to the
effects of IFN.
The consequences of reduced MHC class I expression on infected cells are
advantageous from the viral standpoint of CD8+ T cell immune evasion. However,
cells lacking MHC class I are susceptible to clearance by host natural killer (NK)
cells (missing-self hypothesis). Many viruses circumvent this by encoding inhibitory receptors that block NK cytolytic activity. Alternatively, while the synthesis of
many types of MHC class I molecules is affected by the action of viral proteins,
there is evidence from KSHV studies that some HLA subtypes are spared, thus still
allowing for inhibitory receptor ligation on NK cells (Ishido et al. 2000; Means
et al. 2002).
It has been suggested that mK3 serves different roles during lytic and latent
infection; the gene is transcribed in proliferating, latently infected germinal center
B cells, as well as in lung tissue during lytic infection (Marques et al. 2003;
Stevenson et al. 2002). Studies characterizing the pathogenesis of mK3 mutant
viruses demonstrated reduced splenomegaly and infectious centers compared to
wild-type virus (Stevenson et al. 2002). This reduction in latency establishment was
attributed to an increased antigen-specific effector CD8+ T cell response, as depletion of CD8+ T cells resulted in the restoration of wild-type latency. Interestingly,
mice infected with mutant mK3 virus exhibited a marked reduction in Vb4+ CD8+ T
cells (discussed below), most likely a result of the decreased frequency of latently
infected cells (Stevenson et al. 2002). These studies indicate that MHV68 K3
specifically inhibits the cytolytic activity of MHV68-specific CD8+ T cells, thus
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preventing them from clearing their infected target cells following infection. This
may allow an infected cell to escape CTL tumor surveillance, and whether that cell
is transformed by MHV68 or by a secondary mechanism, this escape may have
implications for the development of MHV68-positive tumors.
M1
Another unique MHV68 protein is that encoded by the M1 ORF. Original sequence
analysis noted that M1 shared sequence homology to a poxvirus serpin, but the
absence of key residues required for serpin function make it unlikely that M1 is a
functional serpin (Virgin et al. 1997). Subsequent experiments have demonstrated
that M1, whose transcripts can be detected during lytic replication, encodes a
secreted protein required for the massive clonal expansion of Vb4+ CD8+ T cells
observed following the initial establishment of MHV68 latent infection (Evans et al.
2008). In C57Bl/6 mice infected with wild-type MHV68, CD8+ T cells bearing this
isoform of the TCR can comprise nearly half of the entire CD8+ T cell population
(Flano et al. 2004; Tripp et al. 1997). The frequency of these cells remains high
throughout the lifetime of the infected host, and contrary to the functional exhaustion seen in other models of chronic viral infection, the MHV68-induced Vb4+ T
cells retain the ability to produce inflammatory cytokines upon ex vivo stimulation
and do not upregulate expression of the cell surface marker PD-1 (Evans et al.
2008). A similarly disproportionate expansion of CD8+ T cells bearing a particular
Vb chain is also seen following infection with EBV in human peripheral blood.
Studies using large deletion mutants, and subsequently an M1-specific null virus,
have demonstrated that M1 is required to induce the Vb4+ expansion in vivo
(Clambey et al. 2002, 2000; Evans et al. 2008). Notably, evidence also indicates that
M1 is able to induce T cell activation in the absence of MHC class I, and it has,
therefore, been proposed as a superantigen-like molecule, presumably capable of
bridging the TCR and coreceptor molecules to induce activation and negating the
need for presentation via cognate MHC (Evans et al. 2008).
Infection of IFN / mice with wild-type MHV68 infection results in severe
pathology, characterized by multiorgan fibrotic disease, arteritis in the great elastic
vessels and hyper-reactivation of MHV68 from peritoneal exudate cells (PECs)
(Ebrahimi et al. 2001; Tibbetts et al. 2002; Weck et al. 1997). Identification of M1s
causal role in the Vb4+ CD8+ T cell expansion was facilitated by observations made
following infection of IFN / mice with an MHV68 virus containing a large deletion within the M1 open reading frame (Clambey et al. 2000; Evans et al. 2008).
Infection with this mutant virus ameliorated disease, providing the first evidence
that the viral component contributing to the extreme pathology in IFN / may be
encoded by the M1 open reading frame. It was subsequently demonstrated that
M1-null viruses not only failed to induce the Vb4+ CD8+ T cell expansion in
C57Bl/6 or IFN / mice, but also did not induce fibrotic disease, thereby linking
these effector-memory-like CD8+ T cells to the severe pathology observed in
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MHV68-infected immunocompromised mice (Evans et al. 2008). The resulting

model from these infections implies a role for Vb4+ CD8+ T cells in controlling
MHV68 reactivation via IFNg-mediated repression, with M1 playing a pivotal role
in their expansion. IFN has been shown to suppress reactivation from macrophages,
perhaps by inhibiting the Rta promoters (Goodwin et al. 2010; Steed et al. 2007;
Steed et al. 2006), and it was further proposed that reactivation specifically from
alveolar macrophages is a primary target for Vb4+ CD8+ T cell effector function.
Implications of Vb4 CD8 T cells in tumorigenesis. IFNg is a potent proinflammatory cytokine with multiple functions, produced by multiple cell types. The frequency
of IFNg-producing cells increases during the initial stages of MHV68 infection and
these cells most likely play dual roles in controlling lytic replication and shaping the
immune environment in the infected host (Stevenson and Doherty 1998). Although
the overall frequency of effector CD8+ T cells wanes during later infection, the frequency and effector functions of the expanded Vb4+ population are maintained
(Evans et al. 2008; Tripp et al. 1997). Countless studies have documented the effects
of IFNg on various players in the global immune response, including T cells, macrophages, and NK cells. An expanded, functionally active population of IFNproducing cells persisting throughout the duration of MHV68 infection most likely
has implications not only for subsequent host immune responses to pathogens, but
also for tumorigenesis. CD8+ T cells and NK cells involved in tumor surveillance
may be affected by the Vb4+ population, and long-term maintenance of a proinflammatory environment by constitutive IFN production may recapitulate certain events
now documented as contributors to inflammation-associated tumorigenesis, including constitutive activation of NF-kB, macrophage infiltration, and angiogenesis. In
addition to IFN, the Vb4+ T cells also produce high levels of TNFa, which may also
contribute to inflammation-mediated tumorigenesis (Evans et al. 2008).
Role of Immune System in Control of MHV68

Lymphomagenesis
Viral infection typically induces a Th1-biased immune response, yet gammaherpesviruses encode proteins promoting Th2-polarization and the production of Th2-type
cytokines. MHV68 infection results in a strong B cell and antibody response, but
also induces nonspecific polyclonal and MHV68-specific CD4+ and CD8+ T cell
expansions. As discussed above, a subset of CD8+ T cells is specifically induced by
an MHV68 protein and actively involved in controlling reactivation. This suggests
that a fine balance in the immune response exists throughout the course of the
immune response to gammaherpesvirus infection. When this balance is perturbed
such as during immunosuppressive therapy, immune senescence, or genetic immunodeficiency it may greatly increase the risk for developing gammaherpesvirusrelated disease. Below, we describe the general features of the MHV68 immune
response as a model for gammaherpesvirus pathogenesis and discuss its potential
role in limiting MHV68-related lymphomagenesis.
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Kinetics of MHV68 Immune Response

Although the natural route of infection is not known, the immune response to MHV68
in inbred mice has been best characterized following intranasal infection. Studies
using bioluminescent viruses have verified the previously proposed course of infection and localization of lytic virus during the early stages of infection. Following
intranasal inoculation, some degree of lytic replication occurs in the nasal epithelium
before the virus trafficks to the lung where the majority of lytic replication occurs.
Viral particles are detectable in the lung by day 4, with lytic titers peaking by day 9
following infection. Based on the poor establishment of latency in the spleens of B
cell-deficient mice following intranasal inoculation (Weck et al. 1999a), it is hypothesized that B cells are infected in the lung during this period of lytic replication and
serve to traffic virus to the spleen to seed latent infection. Lytic virus is detectable in
the spleen by day 9 and reaches peak titer by day 12 postinfection. It is possible that
the secondary wave of acute replication observed in distal organs is initiated by reactivation of virus from latently infected B cells that have trafficked to those sites.
Indeed, this mechanism may link the M2-associated reactivation defect with the failure to efficiently establish latency in the spleen following intranasal inoculation (see
discussion of M2 null virus phenotype above). By day 16, the infection is largely
latent, with the majority of viral load accounted for by infected B cells bearing markers of either active participation in or transit from the germinal center.
MHV68 can establish latency in macrophages and dendritic cells, but the majority of long-term latent infection is maintained in the memory B cell population
(CD19+sIgD-) (Flano et al. 2000; Willer and Speck 2003). By 3 months postinfection, a steady-state frequency of MHV68-infected cells has been reached and persists largely unchanged for the lifetime of the infected animal. How this latency
reservoir is maintained is a subject of great interest. It has been hypothesized that
while there may be long-lived cells that carry the viral genome and persist throughout the lifetime of the host, there is most likely an active cycle of reactivation and
reinfection a dynamic process referred to as reseeding the latency reservoir.
Recent studies have demonstrated that during early infection, the majority of reactivation from splenocytes can be accounted for by plasma cells nearly 100% of
sorted CD138 + B220low cells reactivate upon explant into culture, while the plasmacell-depleted population contains very few reactivating cells (Liang et al. 2009).
Previous studies with KSHV and EBV have indicated that plasma cell differentiation is one of several mechanisms to elicit virus reactivation from infected B cells
(Bhende et al. 2007; Jenner et al. 2003; Laichalk and Thorley-Lawson 2005; Yu
et al. 2007). It is, therefore, likely that throughout the lifetime of the infected host,
low-level episodic reactivation and lytic replication occurs. How this changes the
immune landscape is not yet well-defined, but evidence exists to suggest an ongoing
effort on the part of the host to control chronic MHV68 infection (discussed further
below). Also, as mentioned above, the Vb4+ CD8+ T cell population expands around
the same time serum antibody is readily detected, and remains constant and functional long after initial infection most likely serving as another safeguard to control long-term latency.
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Innate response. The importance of NK cells in controlling herpesvirus infections has

been demonstrated both in humans with genetic NK cell disorders, as well as NK-deficient
transgenic mouse models of herpesvirus infection. However, the role of NK cells in
limiting MHV68 infection is less obvious initial studies using IL15-deficient (which
lack NK and NKT cells) and NK-cell depleted mice revealed no direct requirement for
this cell population in controlling lytic replication in the lung or spleen (Usherwood
et al. 2005). The authors did note an increase in the total number of NK cells in infected
mice versus nave controls, an observation also made in a subsequent study (Thomson
et al. 2008). In addition to the expanded NK population, this second study also demonstrated that these cells were activated, IFNg-producing cells with MHV68-infected
cytolytic activity. However, depletion studies carried out to 5 days postinfection failed
to reveal a requirement for NK cells in controlling lytic replication in the lung, mesenteric lymph nodes, or spleen. The role of NK cells in persistent or latent infection has not
been addressed, nor has the contribution of NKT cells. Both these cell types are capable
of producing IFNg and, therefore, have the potential to contribute to both long-term
control of MHV68 reactivation and pathology related to constitutively high levels of
inflammatory cytokines. A recent study demonstrated that mice infected with latent
MHV68 harbor a population of armed NK cells capable of rapid effector functions
upon ex vivo stimulation, including cytotoxicity and IFNg production (White et al.
2010). These cells also appear to protect against a lethal MHV68 infection and, therefore, most likely contribute to the control of latent MHV68 infection, at least in part via
production of IFNg. Although these innate cytolytic cell populations do not appear to be
essential to control acute viral replication, they may in fact contribute to MHV68 latency
and, therefore, impact MHV68-mediated tumorigenesis.
More obvious is the requirement of an innate cytokine response for control of
MHV68 lytic replication. While mice deficient in type II interferon responses
(IFNg/ or IFNgR/) display pathology associated with unresolved, persistent infection, interferon ab-receptor knockout mice (IFNabR/) succumb to MVH68 infection within 12 days following inoculation with an intermediate-level dose of virus
(Barton et al. 2005). Stat-1 deficient mice, like IFNabg-receptor knockout mice, are
even more susceptible, exhibiting 100% lethality even at doses as low as 10 pfu,
suggesting a nonredundant role for type I and type II interferons and further supporting an undefined role for NK cells during acute infection. This study also
revealed perturbations in reactivation and viral gene expression in latently infected
IFNabR/ mice and mice in which IFNa and IFNb had been depleted in vivo. This
supports a role for type I interferons in regulating MHV68 latency and further
emphasizes the importance of the intersecting of innate and adaptive immunity for
the control of long-term infection.
Adaptive Immune Response

Antibody response. B cells are an important aspect of MHV68 infection due to the
intimate association of viral pathogenesis and B cell biology, but the humoral
immune response is also involved in shaping the course of infection. As previously
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mentioned, B cells are primary MHV68 long-term latency reservoir, but latency is
still established in B-cell-deficient mice (Weck et al. 1996). Subsequent studies
demonstrated that macrophages and DCs can also support MHV68 latent infection
(Weck et al. 1999b). Several lines of evidence have indicated that the phenotype of
the infected cell influences the nature of MHV68 latency (Gray et al. 2009; Steed
et al. 2007; Weck et al. 1999b). Not surprisingly then, latent infection in B-cell
deficient mice is inherently different from wild-type latency; at six weeks postinfection, mMT mice display a higher frequency of infected and reactivating splenocytes,
a phenotype which may indicate a greater propensity toward reactivation in non-B
cell reservoirs and/or a requirement for antibody-mediated control of virus reactivation (Weck et al. 1999a). Despite the contraction in the frequency of infected cells
that occurs following the initial establishment of latent infection, levels of antiMHV68 serum IgG are first detectable at around 2 weeks and continue to rise following infection (Stevenson and Doherty 1999). Previous studies have demonstrated
that MHV68-immune serum from infected animals confers protection from infection in nave animals (Gangappa et al. 2002a; Gargano et al. 2008); this would suggest that serum anti-MHV68 IgG may have some role in limiting lytic replication
during long-term latent infection. However, the role of antibody in controlling
MHV68 replication is perhaps a phenomenon only relevant in vivo, as mixing B
cells from infected wild-type mice with those from mMT mice in ex vivo reactivation assays does not ameliorate the mMT hyperreactivation phenotype (Weck et al.
1999a). Interestingly, recent data have demonstrated that although MHV68-specific
IgG titers remain constant in aged mice (>18 months) relative to younger controls,
sera from aged mice provides less-efficient virus neutralization and reduced protection during passive immunization of nave mice against de novo MHV68 infection
(Yager et al. 2010). This reduction in neutralizing ability does not translate into
reduced control of latent infection, however, as aged mice exhibit no obvious signs
of viral recrudescence or other pathology associated with aberrant virus reactivation. This is in apparent contrast to the increased frequency of gammaherpesvirusassociated malignancies reported in elderly humans, yet the authors argue that (1)
the direct evidence supporting a link between aging and most gammaherpesvirusrelated tumors is not strong; and (2) the increased incidence in the elderly is negligible considering >90% of the world population harbors gammaherpesvirus
infection. Therefore, the role of anti-MHV68 humoral immunity in long-term
latency and tumorigenesis is still only partially understood.
CD8+ T cells. Cell-mediated immune functions are key in the host response to viral
pathogens. As discussed above in the context of the mK3 protein, the classic antiviral adaptive immune response centers around the recognition of viral antigens
derived from intracellular processes and presented to cytotoxic CD8+ T lymphocytes (CTLs) via MHC class I molecules. The requirement for a strong CTL response
in controlling MHV68 infection has been demonstrated by several groups using
both transgenic mice lacking mature CD8+ T cells, as well as CD8-depleted animals. For the latter, BALB/c mice whose CD4+ T cells were depleted prior to and
during infection exhibit delayed but eventual clearance of virus from the lung, while
CD8-depleted mice fail to clear virus from the lung or spleen and eventually succumb
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to infection (Ehtisham et al. 1993). Similarly, C57B1/6b2m/ mice deficient in

CD8+ T cells exhibit exaggerated, unresolved splenomegaly and ongoing lytic replication in the spleen, which persists even at 6 weeks postinfection (Coppola et al.
1999). These studies clearly indicate the importance of CD8+ CTLs in limiting lytic
viral replication, a critical barrier to the establishment of a normal latent infection.
Ex vivo depletion analyses have further demonstrated that the majority of cell cytotoxicity results from the action of CD8+ T cells rather than CD4+ T cells (Topham
et al. 2001) (although CD4+ CTLs most likely play a role in controlling latent infection as discussed in the following section). Alternatively, another study showed that
C57BL/6 CD8+ T-cell-deficient mice remain healthy after infection, while concomitant CD4+ T-cell-deficiency consistently led to mortality (Stevenson et al. 1999b).
Therefore, the role of CD8+ T cells in limiting MHV68 infection seems to be strain
dependent and relies in part on the presence of CD4+ T cell help.
Characterizations of MHV68-specific CD8+ T cell populations were initially performed during the establishment of latency and have been subsequently completed
for mice infected long-term as well. The kinetics and nature of both CD8+ and CD4+
T cell expansions during the acute phase of infection bear strong similarities to
those observed during the initial stages of infectious mononucleosis in humans
newly infected with EBV. Infected mice exhibit a massive expansion of CD8+ T
cells during the first few weeks of infection. The number of MHV68-specific CD8s
in the lung begins to decline by day 15, but the spleen and MLN still experience a
massive influx of lymphocytes (Stevenson and Doherty 1998). Many of these cells
bear markers of activation, but a significant portion are nave (CD62Lhi), a cellular
profile similar to that seen in the blood of IM patients and attributed to activation of
both MHV68-specific effector cells as well as bystander activation of non-MHV68specific lymphocytes. Concomitant with the resolution of lytic replication, total
numbers of CD8+ T cells decline after 4 weeks.
The exception to this pattern is the Vb4+ CD8+ T cell population discussed previously. Cells in this population begin to expand around the third week of infection and
persist at a disproportionately high frequency (as high as 50% of all CD8s) throughout the duration of infection. This expanded population is entirely distinct from the
virus-specific CD8s dominating the early stages of latency as they are clonal, not
specific for MHV68-derived antigens, and exhibit an effector-memory phenotype
even long after the presence of detectable lytic replication (Evans et al. 2008; Flano
et al. 2004; Stevenson et al. 1999a; Tripp et al. 1997). When both Vb4+ and Vb8+
populations were transferred into MHV68-infected mice, both populations proliferated in 2-week-infected, while only Vb4s proliferated in 10-week infected mice
(Flano et al. 2004). This suggests that the Vb4+ CD8+ T cell expansion is induced and
maintained by a factor specifically produced during latent infection. B cells and reactivation competence also appear to play a role, as mMT mice and mice infected with
reactivation-incompetent viruses fail to develop robust Vb4+ CD8+ T cell populations
(Brooks et al. 1999). In further support of an association with latency, the presence
or absence of Vb4s does not affect lytic replication, as depleting Vb4s before adoptive transfer does not alter frequency of infected cells in spleen, nor does transfer of
Vb4s into infected animals (Flano et al. 2004). However, the latter results must be
interpreted with some caution since a subsequent study showed that the sole available
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anti-Vb4+ antibody is incapable of depleting the expanded Vb4+ CD8+ T cell population
(but does mask their subsequent detection) (Evans et al. 2008).
CD4+ T cells. One of the most dramatic phenotypes associated with MHV68 infection is the development of marked splenomegaly that is coincident with the resolution of lytic infection in the lung and establishment of latent infection in the spleen.
As discussed above, this aspect of infection is shared with acute EBV infection and
is most likely a reflection of polyclonal B and T cell activation in response to viral
infection. In the above T cell-depletion experiments, mice depleted of CD8+ T cells
still develop splenomegaly comparable to undepleted controls upon infection, while
spleens from CD4+ T cell-depleted mice do not increase in size or cell number
(Brooks et al. 1999; Ehtisham et al. 1993; Usherwood et al. 1996a). Mice depleted of
CD4+ T cells exhibit a delayed kinetics in resolving acute virus replication in the
lung clearing virus by day 13 postinfeciton while CD8-depleted mice fail to clear
lytic virus by this time (Ehtisham et al. 1993). This suggests CD8+ T cells play the
primary role in controlling early infection and become activated effector cells even in
the absence of CD4+ T cell help. However, separate CD4+ T-cell depletion analyses
reveal the absence of an expanded Vb4+ CD8+ T cell population following the establishment of latency in mice depleted of CD4+ T cells (Brooks et al. 1999). It is
possible that although they are marginally involved in the direct resolution of early
lytic infection in the lung, CD4+ T cells, and perhaps mechanisms underlying CD4+
T cell-induced splenomegaly, are critical in setting the stage for particular cellmediated and humoral immune responses observed during latent MHV68 infection.
This notion is corroborated by analyses of MHV68 infection in mice lacking
molecules required for receiving CD4+ T cell costimulation. Ligation of CD40 on B
cells by CD40-ligand (CD40L)-expressing T cells is important for development,
survival, and differentiation of normal B cells. Experiments using mixed bone marrow
chimeras demonstrated that MHV68 long-term latency is preferentially established
in CD40-sufficient B cells, while it is progressively lost from CD40/ B cells
suggesting that this interaction between T and B cells is important for maintaining
the latently infected B cell population (Kim et al. 2003). CD40CD40L interactions
are also required for the development of splenomegaly (Brooks et al. 1999), further
highlighting the role of CD40L-expressing CD4s in normal MHV68 pathogenesis.
By contrast, infection of CD40/ mice is largely unaltered from infection of wildtype mice, with viral genomes detectable in IgD-negative memory B cells even at 3
months postinfection (Willer and Speck 2005). EBV LMP1 mimics many aspects of
CD40 signaling in B cells; although MHV68 does not encode a direct homolog of
LMP1, it is interesting to note that CD40/ mice still harbor MHV68 genomes in
IgD- negative B cells, suggesting that the virus may in fact be able to overcome the
requirement for CD40 to establish latency in class-switched memory B cells. In
these studies, however, CD40/ mice displayed evidence of persistent infection in
the lung long after initial infection. Nascent infection resulting from ongoing lytic
replication, therefore, likely contributes to the high frequency of infection in CD40/
animals and may explain the apparent discrepancy of these results with those
obtained using mixed bone marrow chimeric mice. Conflicting evidence exists
regarding the role of CD40-mediated CD8+ cytolytic activity, but given the delayed
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clearance of virus from the lung in CD4-depleted mice, there is a possibility that
CD40L supplied by CD4+ T cells is required for CD8+ effector functions during the
initial adaptive immune response to MHV68 in the lung.
Ligation of CD28 on B cells and CD8+ T cells by CD4+ T cells is also important
for humoral and cellular immune responses. However, in contrast to CD40-deficient
mice, CD28/ mice develop wild-type splenomegaly and are able to control MHV68
infection (Lee et al. 2002; Lyon and Sarawar 2006). Splenocytes from CD28/ mice
exhibited attenuated IFNg responses when stimulated ex vivo at day 7 postinfection,
but produced levels comparable to wild type by day 15 (Lee et al. 2002). Perturbations
in the humoral immune response were also observed in CD28/ with respect to antibody isotypes present in infected animals. As mentioned above, these data collectively
indicate that costimulatory signals provided by CD4+ T cells are important in shaping
normal cellular and humoral immune responses and creating the ideal environment
for establishment and maintenance of long-term latency in an infected host.
Adaptive Immune Response in Controlling Malignant Disease

Numerous studies have documented the characteristics of MHV68 infection in
immunocompromised mice. Not surprisingly, many immune defects result in
increased pathology, such as the multifibrotic organ disease observed in IFNgR/
mice or the uncontrolled, systemic viremia and subsequent mortality associated
with loss of a type I interferon response in IFNabR/ mice (Barton et al. 2005).
While these results are not unexpected in virally challenged mice lacking either arm
of an interferon response, what is more remarkable is the ability of mice with
humoral immune defects to effectively control viral infection and achieve latent
infection without significant pathology. For example, despite the involvement of B
cells in the maintenance of long-term latency, mMT mice (B cell-deficient) are still
able to support latent infection (Weck et al. 1996, 1999a). These mice display a
defect in acute splenic infection, yet the frequency of infected splenocytes during
long-term latency is higher than that in wild-type controls. Reactivation is also
enhanced at later time postinfection, suggesting a role for the humoral immune
response in controlling virus reactivation, a notion supported by the perpetual rise
in serum MHV68-specific antibody in infected mice. Although these mice lack the
gene required to generate a pre-B cell receptor to allow B cell maturation, it is possible that some mature B lymphocytes are circulating in the animal. However, the
clearest indication of the host immune components involved in controlling MHV68associated tumorigenesis comes from studies involving mice with T cell defects, not
only RAG/ and nude mice, which are deficient in mature T cell populations, but
also BALB/c b2m/ mice which lack functional CD8+ T cells. MHV68 has been
implicated in the causation of lymphoproliferative disease in these models, as well
as in IFNgR/ mice, providing strong evidence that T cells and their effector functions play an integral role in controlling MHV68-associated tumorigenesis.
b2m/ mice (LPD and ALH). As discussed above, MHV68 infection of BALB/c
b2m/ mice results in atypical lymphoid hyperplasia (ALH) and lymphoproliferative
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disease (LPD) (Tarakanova et al. 2005). While these mice are CD8+ T-cell-deficient
and prone to developing tumors, MHV68 infection both exacerbates this propensity
and reduces the time to develop disease (relative to mock-infected controls).
Development of proliferative disease appears to require an intact v-cyclin and
v-bcl2, supporting the hypothesis that ALH and LPD are either directly or indirectly
induced by wild-type MHV68 infection (Tarakanova et al. 2008). Importantly,
BALB/c mice did not develop disease following infection, strongly suggesting that
cells absent in BALB/c b2m/ mice are important for limiting lymphomagenesis in
a wild-type infection. BALB/c mice do not develop a robust Vb4+ CD8+ population,
suggesting that background and, therefore T cell repertoire may be involved in
preventing malignant disease (Tripp et al. 1997). Interestingly, while the incidence
of LPD in BALB/c b2m/ mice infected with an M1-null virus was similar to wildtype virus, the severity of the disease was increased in mice infected with mutant
virus (Tarakanova et al. 2008). As discussed above, M1 is required for the expansion of an effector-memory-like Vb4+ CD8+ T cell population hypothesized to produce IFNg that suppresses virus reactivation. The Vb4+ expansion requires both
CD4+ T cells, B cells, and MHC class II (Flano et al. 2000), yet MHC is not required
for M1 to activate Vb4+ CD8+ hybridomas (Coppola et al. 1999; Evans et al. 2008).
This suggests that the requirement for MHC class II seen in an earlier study is due
more to its importance for the development of a full CD4+ T cell repertoire which
can facilitate the maturation of B cells that support latent infection and elicit the
Vb4+ CD8+ expansion via production of secreted M1 protein. Despite defective
positive T cell selection in the thymus, a majority of C57BL/6 b2m/ mice still
develop an expanded Vb4+ CD8+ T cell population following MHV68 infection
(Coppola et al. 1999). Along these lines, B2/ 129/Pas mice in this study did not
develop disease. Other strain-specific differences most likely play a role in regulation aspects of infection in these animals, but the exaggerated pathology in M1-null
infected BALB/c b2m/ mice indirectly implies the involvement of an M1-induced
cell population capable of limiting lymphoproliferative disease.
IFNgR/ mice (lymphoid granulomatosis). Most pathology in immune-compromised
mice has been shown to require v-cyclin, presumably reflecting a requirement for
viral replication in the onset of disease. Yet a recent study carefully examining lung
specimens from IFNgR/ mice demonstrated that although ongoing viral replication
is only detected in the lungs of mice infected with wild-type virus, mice infected
with either wild-type or v-cyclin-null virus develop angiocentric infiltrates, exhibit
hallmarks of prolonged inflammation, and develop pulmonary B cell lymphomas
(Lee et al. 2009). Cells within these tumors were shown to be MHV68-positive, and
latent viral transcripts were detected in lung tissue from mice infected with either
virus, suggesting that this pathology, unlike MHV68-mediated fibrosis or arteritis,
is associated with latent, not lytic, infection in IFNgR/ mice. This provides evidence
to support an association of latent gammaherpesvirus infection with tumorigenesis
in immunocompromised hosts as a specific consequence of a deregulated immune
response in the context of MHV68 infection.
Although CD8+ T cells are typically the population to which most cytolytic activity is ascribed, the existence of cytolytic (CTL) CD4+ T cells has been reported for
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many years (Brown 2010). The contribution of CD4+ CTLs in the control of viral
infection and tumorigenesis is becoming increasingly well appreciated. In particular,
EBV-specific CD4+ CTLs isolated from infected individuals have the ability to lyse
EBV transformed B cells, and CTL CD4+ T cells generated in vitro have been used
therapeutically to treat EBV+ malignancies or transplant recipients infected with
EBV (Adhikary et al. 2006; Heller et al. 2006). A recent report demonstrated that
MHV68-infected mice harbor CD4+ T cells bearing a cytolytic phenotype (reduced
CD27 expression) (Stuller and Flano 2009). When isolated from infected C57BL/6
mice, this population degranulated upon stimulation with MHV68 gp150 on isotypematched MHC class I. Furthermore, these cells could induce cytolysis of virus-pulsed
IAb expressing fibroblasts, while cells from BALB/c mice could not. CTL CD4+
T cells were also demonstrated to have cytolytic activity in vivo. The implications of
this study are significant given the well-established association of gammaherpesvirus-associated lymphoproliferative disease and immunosuppression. In particular,
EBV and KSHV-associated lymphomas occur at much higher incidence in AIDS
patients whose CD4+ T cells are greatly compromised. It is, therefore, entirely possible that CTL CD4+ T cells specific for gammaherpesvirus-infected cells are essential for controlling lymphomagenesis in infected hosts. When this population is
diminished, cytolytic control of gammaherpesvirus-antigen-expressing cells is
affected, and growth and expansion of these cells may proceed unchecked.
The antigen specificity of both CD8+ and CD4+ CTL T cells is also a subject of
interest. Tetramer analyses of MHV68-specific CD8+ T cells have been performed to
identify the kinetics of virus-specific CD8+ T cell responses during various phases of
MHV68 infection (Freeman et al. 2010; Husain et al. 1999; Usherwood et al. 2000).
During acute EBV infection, a high percentage of CD8+ T cells are specific for
epitopes derived from EBV lytic antigens, while CD8s specific for latency-associated
antigens are less prolific (Khanna et al. 1992; Murray et al. 1992). This balance shifts
during latent infection, however, as lytic cycle-specific cells diminish while the frequency of latency-specific cells remains constant. These cells most likely play a role
in controlling virus reactivation in EBV+ individuals, as studies with MHV68 have
demonstrated that CD8+ T cells specific for the latency-associated M2 antigen can
reduce the latent viral load during the initial establishment of latent infection in
BALB/c mice (Usherwood et al. 2000). Subsequent studies have demonstrated that
vaccination with M2, as well as other latent and lytic antigens, can reduce latent viral
load during early infection (Hoegh-Petersen et al. 2009; Stewart et al. 1999;
Usherwood et al. 2001) but do not affect long-term latent viral load. One of these
studies identified CD4+ CTLs that can respond to stimulation with a lytic epitope
(gp150) (Stewart et al. 1999) and other studies of MHV68-specific CD8+ T cells have
also demonstrated the existence of CD8+ T cells specific for lytic antigens (Freeman
et al. 2010; Stewart et al. 1999). Studies characterizing the CD8+ T cell response to
two previously identified epitopes in C57BL/6 were elaborated upon in this recent
study identifying several new MHV68 epitopes (Freeman et al. 2010). The CD8+
T cell responses to MHV68 antigens largely follow two distinct kinetic patterns
defined most likely by the major stages of MHV68 infection: acute lytic replication,
latency amplification, and reactivation from latency.
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The results of these studies strongly suggest that the host immune response is
exquisitely sensitive to alterations in antigen expression in latently infected cells
throughout the lifetime of the infected host. Gammaherpesviruses have most certainly evolved to exploit the B cell arm of the immune system to perpetuate longterm latent infection. The broad repertoire of CD8+ T cells, and most likely CD4+
T cells, specific for MHV68 antigens is most likely a strategic mechanism on the
part of the virus to use a healthy host immune system to control the waxing and
waning of latent infection that is an essential part of latency maintenance. When this
finely tuned system is perturbed by coinfection or immune dysfunction, the result
may be unchecked latency-promotion, a transcriptional program demonstrated to
have transforming potential in EBV-infected lymphocytes. Further studies addressing the role of the immune system in controlling MHV68 infection and tumorigenesis will be important to address this issue.
Implications and Application of MHV68 System

for Study of Human Gammaherpesvirus-Related Tumorigenesis
In line with key pathogenic features shared with its human gammaherpesvirus
family members, evidence discussed in the above sections supports a role for
MHV68 in tumorigenesis in mice. However, as mentioned in the introduction, the
area of gammaherpesvirus-mediated tumorigenesis has not yet experienced the
full benefit of the MHV68 model system because of (1) the inability to transform
primary murine B cells, and (2) the absence of a robust MHV68 tumor model.
However, there are promising developments on the horizon that appear likely to
remove these barriers. Once these systems are in place, key aspects of gammaherpesvirus-related disease can be addressed. Some of the outstanding questions
include the following:
What is the precise role of individual viral genes in lymphomagenesis? Are there
complementary or combinatorial effects?
What is the role of host proteins in controlling gammaherpesvirus-related
oncogenesis?
What is the role of the host immune system in controlling gammaherpesvirusrelated oncogenesis?
What are the consequences of age-related immune senescence on control of
lymphomagenesis?
How do repeated pathogenic assaults shape/alter the lymphocyte repertoire?
Do these changes affect gammaherpesvirus-related tumorigenesis?
How does immunosuppression, particularly loss of CD4+ T cell function, facilitate malignant disease?
What therapies will be most effective to treat/prevent gammaherpesvirus-associated
disease?
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The MHV68 system is a readily manipulable animal model; the entire viral
genome has been cloned into a bacterial artificial chromosome, thereby allowing the
easy generation of recombinant viruses to study the functions of viral genes.
Similarly, the availability of numerous transgenic mice, coupled with the faithful
recapitulation of key features of gammaherpesvirus infection in these animals, provide opportunity to study host aspects in the development of gammaherpesvirusassociated malignancies including, but not limited to, known tumor suppressors
and promoters and components of the host immune system. In addition, several
mouse models have been developed to study the effects of aging and senescence in
mice, and one can envision how these systems may be used to answer questions
regarding the increased incidence of lymphoma in elderly individuals. Studies of
infectious disease in mice are moving into the realm of coinfection, trying to establish relationships between the carriage of chronic pathogens and acute infection.
Since MHV68 infection is maintained for the lifetime of the infected host, it provides an attractive model to examine the effects of subsequent or preexisting infection on gammaherpesvirus-related disease. Murine systems have long been used in
the study of transplant medicine with respect to gaining insights into compatibility
and rejection, and MHV68 has been used in these models to understand the pathogenesis of PTLD. As this field advances, the MHV68 system provides a rich resource
for the evaluation of new therapies and their consequences with regard to disease in
immunosuppressed patients. Finally, with an MHV68 tumor model in place, chemoor immune-therapeutic treatments for gammaherpesvirus-associated lymphomas
can be developed and tested in tumor-bearing mice. Humanized mice bearing human
CD20 recapitulate the cytolytic effects of rituximab treatment, used to manage
PTLD in transplant recipients, and M3, a chemokine-binding protein not discussed
in this chapter, has been used to enhance the efficacy of oncolytic therapy in a rat
model of hepatocellular carcinoma (Wu et al. 2008).
In conclusion, MHV68 is a well-established system to study aspects of gammaherpesvirus pathogenesis. Its contribution to the study of gammaherpesvirus-associated
tumorigenesis is in its infancy but has the potential to grow substantially upon the
development of appropriate model systems. Once in place, the ready genetic manipulation of both the virus and the host, as well as the ability to study salient features of
host cell biology, should allow important new discoveries regarding the intimate relationship between viral pathogenesis, host immune responses, and the delicate balance
between successful long-term latency and the genesis of malignant disease.
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3:13461353
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581:34853488
Chapter 13
Mareks Disease Virus-Induced

T-Cell Lymphomas
Mark S. Parcells, Joan Burnside, and Robin W. Morgan
Mareks Disease
Mareks disease (MD) describes pathologies induced by an avian alphaherpesvirus,
subfamily Mardivirus, called Mareks disease virus (MDV) (Osterrieder et al. 2006).
MD presents as a combination of immunosuppressive, inflammatory, and lymphoproliferative lesions in chickens. Common signs include paralysis, skin leukosis,
dermatitis, cachexia, neurological signs (ataxia, torticollis, etc.), stunting, and
death. Common lesions of MD are inflammatory nerve and brain lesions, bursal
and thymic atrophy, gross visceral lymphomas, and skin leukosis.
MD is caused by infection with MDV1 strains, and signs are observed in chickens
typically between 10 and 49 days postinfection depending on the challenge strain,
the challenge dose, breed susceptibility, and vaccination status. Because of its ubiquitous presence in commercial chicken operations, its stability, the large numbers of
chickens produced annually for consumption, and the fact that vaccination is not
sterilizing, MD is considered to be the most prevalent clinically diagnosed cancer in
the animal kingdom.
M.S. Parcells (*)

Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA
Department of Biological Sciences, University of Delaware, 052 Townsend Hall,
531 South College Avenue, Newark, DE 19716, USA
e-mail: Parcells@UDel.edu
J. Burnside R.W. Morgan
Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA

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308
M.S. Parcells et al.
History
MD was initially described as paralysis that was associated with inflammation of
the major nerves in egg-laying chickens (Marek 1907). This condition, also referred
to as range paralysis, was found to be associated with the formation of lymphomas
in 1929, and was termed Neurolymphomatosis gallinarum (Pappenheimer et al.
1929a, b). MD was described sporadically for the next thirty years, but a fundamental change in virulence of field strains noted in the early 1960s coupled with the
expansion of commercial poultry husbandry catalyzed more aggressive studies on
the pathogenesis of MDV (Biggs et al. 1966) as well as isolation of the causative
agent in cell culture (Churchill 1968; Churchill and Biggs 1968; Solomon et al.
1968; Witter et al. 1969). Since the identification of acute MDV strains, at least two
notable increases in virulence of MDV1 field strains have occurred (Witter 1997).
The evolution of MDV1 field strains of increased virulence has been apparently
mediated through vaccine use. In the early 1980s, following the near ubiquitous use
of herpesvirus of turkeys (HVT) as the vaccine of choice in the USA (Okazaki et al.
1970), very virulent MDVs (vvMDVs) evolved (Schat et al. 1982a; Witter 1983).
Strains of this pathotype (MD5, RB-1B) cause rapid lymphoma formation and
overcome the vaccinal protection elicited by HVT. Isolation of a nonpathogenic
herpesvirus from chickens, termed MDV2 (type strain SB-1), allowed the formulation of a bivalent vaccine (HVT in combination with MDV2), which showed
increased efficacy against vvMDV challenge (Calnek et al. 1983; Chang et al. 1983,
1984; Witter et al. 1984). This bivalent vaccine remains in widespread use throughout much of the world.
In the early 1990s, field strains began to emerge that could overcome the protection
elicited by bivalent vaccination (Rosenberger et al. 1997; Witter 1997). These
strains, termed very virulent plus MDVs (vv + MDVs) cause more severe lesions
within a shorter time frame. These lesions include rapid and severe neurological
disorders (Gimeno et al. 1999, 2002), acute dermatitis or red-leg, stunting, and
tumors with necrotic centers (Montiel 1995; Olmeda-Miro 1996; Olmeda-Miro
et al. 1996).
Continued ubiquitous vaccination, the anticipation of further evolution of MDV1
virulence, and the rapidity and reproducibility of T-cell lymphoma induction make
MDV a very attractive model for the study of herpesvirus lymphomagenesis, virulence evolution, and anticancer vaccines.
Pathogenesis
Historically, the pathogenesis of MDV1 strains has been characterized according to
four phases; namely, early cytolytic infection, latency, secondary cytolytic infection, and transformation (Calnek 1986). In this model, entry is via inhalation of
infectious dander, and early virus replication takes place in B-cells and macrophages
recruited to the lung epithelium (Baigent and Davison 2004). Replication takes
13
Mareks Disease Virus-Induced T-Cell Lymphomas
309
place in the primary lymphoid organs (bursa of Fabricius, thymus), which undergo
lymphoid depletion and atrophy due to apoptosis of infected cells (Morimura et al.
1995, 1996, 1997). The spleen then becomes a major site of virus replication and
undergoes hyperplasia followed by necrosis. As the host encounters this early
cytolytic infection, an induced innate response begins to impair virus replication,
and the virus enters latency primarily in CD4+ T-cells (Shek et al. 1983). Interferons
and other soluble factors yet to be defined mediate the induction of latency (Buscaglia
and Calnek 1988; Buscaglia et al. 1988).
Several lines of evidence implicate macrophages in the early response to MDV
lytic infection. First, cytokine expression associated with early lytic replication
includes proinflammatory cytokines IL-1a, IL-1b, IL-6, IL-8, as well as type I and
II IFNs and iNOS (Davison and Kaiser 2004; Jarosinski et al. 2005a; Kaiser et al.
2003). Second, induction of iNOS expression in macrophages positively correlates
with both genetic resistance and vaccine-induced protection (Djeraba et al. 2002b,
d). Third, myelomonocytic growth factor (MGF) provides some level of protection
against MD in the absence of vaccination (Djeraba et al. 2002a, c). Fourth, chickens
infected with highly virulent strains of MDV undergo transient paralysis and brain
edema, which are directly linked to monocytic perivascular cuffing and high levels
of proinflammatory cytokine expression (Abdul-Careem et al. 2006; Jarosinski
et al. 2005a; Swayne et al. 1988, 1989).
During the latent phase of infection, MDV lytic expression is suppressed and
latently infected cells proliferate and disseminate virus to peripheral sites. As the
innate response maintaining latency wanes, MDV begins to reactivate at secondary
sites (Schwann cells, feather follicle epithelium, etc.). During this time, transformed
T-cells begin to accumulate within the host.
This distinction of separable phases varies according to host and virus strain factors. Chickens challenged after 12 weeks of age typically undergo rapid early
cytolytic infection within a few days followed by latency by 710 days and secondary cytolytic infection by 1421 days postinfection. Lymphomagenesis usually
occurs 46 weeks postinfection using vvMDV challenge, but may vary according to
the inherent susceptibility or resistance of chickens to MD.
Oncogenes Encoded by MDV

The efficiency of MDV-mediated tumor formation (8095% in naturally exposed,
unvaccinated chickens), suggested that MDV encodes a potent oncogene or oncogenes. In MDV-transformed cells, a cluster of transcripts mapping to the repeats
flanking the unique long region of the virus (TRL and IRL) were implicated due
to their continuous expression in latency and transformed cells (Fig. 13.1). Of genes
expressed in MDV-induced lymphomas and cell lines established from tumors, only
one gene, meq (for Mareks EcoRI-Q-encoded protein) fulfills several of the criteria
consistent with being an oncogene (Kung et al. 2001). First, Meq is consistently
expressed in tumors and cell lines derived from tumors (Jones et al. 1992), and
310
UL
TRL
US
IRS
IRL
TRS
UL36 (MTP)
~ 13 kbp
ORIL
HEP
LORF12
IRES
IRES
B
pp38
miRNAs
RLORF9
1
3
Meq
RLORF5a RLORF4
RLORF6
RLORF1
23 kd
vTR
A-C = pp14
B-C = splice variant
vIL8
d
Meq
Meq
Meq
Meq/vIL8
(212 aa, 24 kd)
Meq/vIL8 exon 3
(149 aa, 17 kd)
Meq
Meq/ORF-30
(150 aa, 17 kd)
Meq
Meq/ORF-20
(140 aa, 16 kd)
RLORF4
Fig. 13.1 Transformation-associated genes of Mareks disease virus. Panel (a) depicts the MDV
genome and location of the unique and repeated sequences. The approximate location and orientation of the UL36 (major tegument protein, MTP) gene is shown. Panel (b) shows an amplified map
of the internal repeat long (IRL) with pathogenicity-associated genes LORF12, pp38, HEP, pp14,
RLORF9, Meq, RLORF5a, RLORF4, vIL8 and vTR. The lytic origin of replication (ORIL), internal ribosomal entry sites (IRES) for the expression of pp14 and RLORF9 gene products and
miRNA coding regions (blue box) are shown. Color-coding of ORFs depicts an association with
pathogenicity. Meq is shown in red, as it is essential for MDV-mediated transformation. ORFs in
pink are associated with pathogenicity or oncogenicity as their deletion partially attenuates MDV
but does not prevent transformation. Panel (c) shows the pattern of Meq protein expression during
in vivo infection of spleen cells over four weeks (VR1, -2, -3, and -4), and MD-induced tumor and
cell line established from an MD-induced tumor (UD35, transformed by RB1B). As controls for
full-length and spliced products, macrophage cell line HTC (Rath et al. 2003) was transfected with
Meq, Meq/vIL8 (M8), and Meq/vIL8exon3 (M83) expression vectors. Protein samples were
separated by SDS-PAGE, electroblotted, and probed with a rabbit polyclonal antibody to the amino
terminus of Meq (Lee et al. 2003). As a control for protein loading, the blot was probed with a
murine monoclonal antibody to GAPDH (Sigma). As controls for lytic infection, the blot was
subsequently probed with a rabbit anti-US1 protein polyclonal antibody (Parcells et al. 1994), and
viremia data for each sample is given in PFU/million spleen cells. Panel (d) depicts the full length
and variant forms of Meq reported in the literature. The sizes of each protein in terms of number
of amino acids and calculated molecular sizes are given at the right
inhibition of its expression decreases proliferation of these cell lines (Levy et al.
2005; Xie et al. 1996). Second, expression of Meq in cell lines (rat and chicken
fibroblast lines) induces many aspects of transformation, including proliferation,
apoptosis resistance, colony formation in soft agar (Levy et al. 2005; Liu et al. 1998)
13
311
as well as microtumors in a chick embryo-based model (Levy et al. 2005). Third,

deletion of Meq from the MDV genome, and even mutation of one particular binding motif, is sufficient to block tumorigenesis (Brown et al. 2006; Lupiani et al.
2004). Although Meq has not been shown to induce IL-2 independence in a chicken
T-cell model (as no IL-2 dependent chicken T-cell lines currently exist), it has been
shown to bind the IL-2 promoter in vivo (Levy et al. 2003).
Although necessary, Meq is not sufficient for MDV transformation, since Meq is
also encoded and expressed by attenuated MDV1 strains that are nononcogenic
(Ajithdoss et al. 2009). Consequently, discussion of MDV1 transformation includes
a major focus on Meq, but also includes other gene products that contribute to lymphoma development and progression.
Meq
Meq is a basic leucine zipper (b-ZIP) protein having characteristics of several viral
oncoproteins including v-Jun (Levy et al. 2005), HBZ of HTLV-I (Reinke et al.
2010), Tat of HIV (Kung et al. 2001; Liu and Kung 2000), and EBNA-3C of EpsteinBarr virus (EBV) (Brown et al. 2006; Hickabottom et al. 2002). Meq is encoded as
a 339-amino-acid unspliced open reading frame in very virulent and very virulent
plus pathotypes of MDV (vv, vv+MDV). Mild and virulent MDVs (m/vMDVs)
from the 1960s and 1970s encode a larger form of Meq (398-amino acids) having
reiterations of a C-terminal, proline-rich repeat (PRR) domain (Shamblin et al.
2004). Most of the biochemical, localization, and functional analyses of Meq have
concentrated on the 339-amino acid form, and this will be the focus of the present
discussion.
Meq Localization and Dynamics

The full-length, unspliced Meq protein functions as a transcription factor (Qian et al.
1995) having rapid nuclear mobility indicative of this function (Anobile et al. 2006).
Meq partitions to nucleolar and coiled body (CB) subnuclear domains as well as the
nucleoplasm in transfected and infected cells (Liu et al. 1997). Its nuclear and nucleolar localizations are mediated by basic regions 1 and 2 (BR1 and BR2) in the amino
terminus of the protein (Fig. 13.2), with BR2 being most efficient in mediating nucleolar localization (Liu et al. 1997). Meq also encodes a nuclear export signal and
shuttles out of the nucleus during cell cycle progression (Anobile et al. 2006; Kung
et al. 2001). The functional relevance of the nuclear exclusion is unknown but is
mediated by phosphorylation of serine 42 (S42), which lies between basic regions 1
and 2 in the amino terminus of Meq (Liu et al. 1999b). S42 is a substrate of cyclindependent kinase 2 (CDK-2), and while the functional significance of these interactions is not clear, it is possible that Meq mediates interactions of CDK-2 with a
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Activities
Transrepression
Induced Proliferation
Blocked Apoptosis
Transformation (?)
120
basic
ZIP
78
basic
30
100
ZIP
78
basic
252
LACHE
78
30
PLDLS
1
Transrepression
Transformation (?)
30
PLDLS
PLDLS
Transactivation
Transrepression
Blocked Apoptosis
Transformation
Proline rich repeats
100
ZIP
TA
Meq
soluble fraction, high mobility

212
145
exon 2
339
exon 3
Meq/vIL8 (Meq-sp)
149
exon 2 Meq/vIL8exon3
insoluble fraction, low mobility
BR1 BR2
Transcriptional
Activation
Localization
DNA-binding
Transcriptional Repression
Dimerization
Fig. 13.2 Comparative domains of Meq proteins. The picture above shows comparative domain
maps of Meq, Meq/vIL8, and Meq/vIL8 exon3 proteins. The activities associated with each of the
proteins are provided in the list at left, with the relative prominence of the activity with the protein
shown in bold. Activities for each domain are also depicted by bars, below. Domain abbreviations
are PLDLS canonical CtBP-1-binding site, BR1 and BR2 basic regions 1 and 2, ZIP leucine zipper
domain, LACHE Rb-binding domain (consensus sequence LxCxE), TA transactivation domain.
Light green bars in exon 2 depict putative secondary CtBP-1-binding domains. Dark green bars
show putative SUMOylation sites in exon 3
number of its cellular substrates (i.e., retinoblastoma, Rb) or in targeted degradation

of these factors through recruitment of SKP-2 and the Skp, Cullin, F-box containing
complex (SCF complex) (Kung et al. 2001; Liu et al. 1999a).
Meq Dimerization Partners

Meq can form homo- and heterodimers, and apparently both dimerization forms are
essential for MDV-mediated transformation (Brown et al. 2009; Suchodolski et al.
2009, 2010). Meq has been shown to heterodimerize with c-Jun, CREB, ATF-1, -2,
-3, and Fra-2 (Levy et al. 2005). Using a coiled-coil array, a number of other b-ZIP
proteins were found to dimerize with Meq, including ATF-2, Jun-B, Jun-D, and
NFIL3 (Reinke et al. 2010). Among these interactions, the association of Meq and
c-Jun has been thoroughly characterized. When associated with cJun, Meq appears
to stabilize the c-Jun protein and potentiate its signaling (Levy et al. 2005), which
was shown through the upregulation of known v-Jun targets, including Hb-EGF,
JTAP-1 and JAC. The importance of Meq-c-Jun interactions was further demonstrated
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313
through the interference of Meq transformation via downregulation of c-Jun expression

(Levy et al. 2005). c-Jun not only affects cellular proliferation and antiapoptotic
pathways (see Meq target genes, below) but also mediates the migration of CD4+
T-cells via the upregulation of stem cell factor (SCF) (Katiyar et al. 2007).
Consequently, the interaction of Meq and c-Jun could be important not only for the
suppression of apoptosis and induction of proliferation, but for increased mobility
of latently infected CD4+ T-cells. This increased mobility/metastasis expression has
also been suggested by proteomic data obtained using an MDV-transformed cell
line, MDCC-UA01 (Buza and Burgess 2007).
Meq-Binding Proteins
In addition to its ability to dimerize through its coiled-coil, leucine zipper domain,
Meq has been found to bind numerous cellular proteins, including cell cycle regulators CDK-2, p53 and Rb (Kung et al. 2001) as well as repression complex protein
CtBP-1 (Brown et al. 2006) and HSP-70 (Zhao et al. 2009a). Reports at recent meetings have implicated Meq binding to p300/CBP (H.-J. Kung, personal communication), prostate apoptosis response 4 (Par-4) (V. Nair, personal communication), and
SKP-2 (Kim et al. 2010).
The most defined of these interactions is the binding to CtBP-1, which is essential for MDV oncogenicity (Brown et al. 2006). CtBP-1 is the founding member of
a family of proteins involved in transcriptional repression, lipid metabolism, and
Golgi vesicle fission (Chen et al. 2009). In animal cells, CtBP-1 functions in both
the nucleus and nucleolus as a dimer and recruits a large complex of proteins to
repress genetic loci (Chinnadurai 2007) based on the DNA-binding proteins with
which it interacts. CtBP-1 does not bind to DNA directly, but is recruited to loci
through DNA-binding proteins that contain the motif PLDLS (prolineleucine
aspartic acidleucineserine). Proteins recruited to repress transcription include
histone deacetylases (HDACs), small ubiquitin-like modifier (SUMO),
E2-conjugating (UBC9), and E3-ligase (HPC2) enzymes, H3-K9 histone methyltransferases (G9a/HMTase1), and H3-K4 histone demethylases (LSD1) (Chinnadurai
2007). Consequently, CtBP-1 represses transcription of select genes through alteration of local chromatin structure.
CtBP-1 shares homology with NAD/NADH-dependent, D-isomer-specific
2-hydroxy acid dehydrogenases (D2-HDHs), and has been shown to possess this
activity in vitro. The substrate for CtBP-1 in vivo appears to be 2-keto-4-methylthiobutyrate, an intermediate in the methionine salvage pathway (Chinnadurai 2007).
Binding of NADH mediates dimerization of CtBP-1 and binding to PLDLS proteins. Therefore, CtBP-1 has the ability to serve as a sensor for the redox potential
within the cell and binds PLDLS-containing proteins when in the NAD(H)-bound
form (Chen et al. 2009). Several of the genes targeted for CtBP-1 repression are
adhesion molecules including E-cadherin (Chinnadurai 2009). The expression of
CtBP-1 is induced by hypoxia, a condition associated with the high metabolic activity
314
and NAD(H) levels within tumor cells. Therefore, repression of cellular adhesion
molecules contributes to tumor cell motility and metastatic potential during tumorigenesis (Chinnadurai 2009). In the case of the adenovirus E1A protein, a loss in
CtBP-1-binding through mutation of the PLDLS domain resulted in greater E1Amediated malignant transformation, including the ability to cause metastatic tumors
in vivo. The binding of a CtBP-1 by E1A, therefore, actually decreases the transforming potential of E1A.
As a repressor, CtBP-1 downregulates E-cadherin and contributes to the epithelialmesenchymal transition (EMT) associated with metastasis (Chinnadurai 2009).
In addition, proapoptotic genes p21, Bax, Noxa, and PTEN are targets of CtBP-1
repression. PTEN negatively regulates AKT (PKB) and, therefore, repression of
PTEN results in sustained phosphorylation of AKT, a kinase directly involved in
cell survival. Additionally, the inhibitor of cyclin kinase p16 (p16/Ink4a) and alternative reading frame (ARF) pathway, which modulate Rb function, are repressed by
CtBP-1. CtBP-mediated repression of these factors permits cell cycle progression;
however, in response to UV damage, CtBP is phosphorylated and targeted for proteosomal degradation, thus relieving its repression of tumor suppressor and apoptotic pathways.
In addition to repressing adhesion and proapoptotic gene loci, CtBP-1 is a key
regulator of cellular differentiation. In CD4+ T-cells, IL-4 expression is downregulated by CtBP-1, directly affecting the differentiation of nave CD4+ T cells into TH1
cells (Kitamura et al. 2009). Thus, CtBP-1 can regulate proliferative, apoptotic, and
developmental signaling within cells through the selective repression of targeted
genetic loci.
Finally, in Caenorhabditis elegans, genetic deletion or targeted downregulation
of CtBP-1 increased worm lifespan (Chen et al. 2009). Genes selectively repressed
by CtBP-1 are included or are part of the insulin-like growth factor (IGF)/lipid
metabolism axis and repression resulted in lower levels of lipid metabolism.
Interestingly, alteration of lipid metabolism is a hallmark of MD and has been
described since the early 1970s (Fabricant et al. 1981; Fujii et al. 1988; Haider and
Ringen 1970; Hajjar 1986). The molecular basis of the observed changes in lipid
metabolism, therefore, may be due to the effects of Meq on selective CtBP-1 binding in MDV-transformed cells.
Meq binds CtBP-1 through a PLDLS domain in its amino terminus (Brown
et al. 2006; Hickabottom et al. 2002). Since Meq binds to different DNA
sequences based on dimerization partner usage (Qian et al. 1996), it likely
recruits CtBP-1 to specific loci during latency and transformation. Indeed,
proapoptotic genes downregulated by Meq include Fas and DAP5 (Levy et al.
2005). Moreover, cells transformed by MDV show a distinct regulatory T-cell
(Treg) immunophenotype (Buza and Burgess 2007; Shack et al. 2008), suggesting that this expression profile contributes to lymphoma progression. Since the
Meq-CtBP-1 interaction is essential for MDV-mediated tumorigenesis (Brown
et al. 2006), it is likely that this interaction is directly involved in repressing
the sensitivity of the latently infected T-cell to apoptosis, while also causing the
differentiation of the T-cell to a Treg expression pattern.
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Meq Target Genes

Genes upregulated by Meq in a fibroblast cell line model include several proliferation-associated genes (JAC, JTAP-1, HB-EGF), but also antiprotein Bcl-2 and
TGFb-signaling regulatory protein c-Ski (Levy et al. 2005). The effect of Meq on
DF-1 cell growth was directly related to its interaction with c-Jun, as downregulation of either Meq or c-Jun using gene-specific siRNAs decreased cellular proliferation. Another major class of Meq-responsive genes in this fibroblast model, as well
as a class of proteins upregulated in an MDV-transformed T-cell line, are cell adhesion/cytoskeleton regulators (Buza and Burgess 2007; Levy et al. 2005). This alteration in cell shape and movement-associated gene expression was interpreted to
indicate increased mobility and metastatic potential and differed from superantigenactivated T-cells (Buza and Burgess 2008).
In MDV-induced tumors and cells lines, Mareks tumor-associated surface antigens (MATSAs) have been described since the 1970s as being indicative of malignant transformation by MDV (Horie et al. 1991; Ikuta et al. 1980, 1981, 1984; Lee
et al. 1983; Matsuda et al. 1977; McColl et al. 1987; Murthy and Calnek 1979;
Witter et al. 1975). Several of these were found to be activation-associated lymphocyte antigens (McColl et al. 1987), but to date, only one has been cloned and identified, CD30, the chicken homolog of the ReedSternberg antigen of EBV-associated
Hodgkins lymphoma (Burgess et al. 2004). The CD30 promoter was directly
upregulated by Meq, which directly linked Meq to expression of at least one MATSA
(Burgess et al. 2004). The importance of CD30 expression to the immunophenotype
of MDV-transformed cells and its contribution to disease progression are described
in recent articles elsewhere (Buza and Burgess 2007, 2008; Shack et al. 2008).
Splice Variants of Meq

In addition to the full-length version of the Meq protein, there are at least two spliced
gene products that result in the fusing the first 100 amino acids of Meq to exons 2 and
3 of viral interleukin 8 (vIL8) (Figs. 13.1 and 13.2) (Kumar et al. 2010; Peng and
Shirazi 1996; Peng et al. 1995). These spliced forms, termed Meq/vIL8 and Meq/
vIL8exon3 generate smaller proteins (212 and 149 amino acids, respectively) and
have starkly different nuclear mobility than full-length Meq (Anobile et al. 2006).
The smaller variants also localize to the nucleus, nucleolus, and CBs (Anobile et al.
2006) and were originally thought to serve as negative regulators of Meq (Peng and
Shirazi 1996), since they lack transcriptional activation functions. However, the
spliced forms do not form heterodimers with Meq as determined by fluorescence
resonance energy transfer (FRET) and colocalization studies (Anobile et al. 2006).
The spliced forms are expressed in vivo, primarily during latency, in primary lymphomas and derived cell lines (Fig. 13.1) (Kumar et al. 2010). Based on chromatin IP
(ChIP) studies, the smaller variants were found to bind to the MDV genome and exert
316
potent transcriptional repression activity. Despite their lack of transactivation, both

variants induced proliferation of fibroblast and macrophage cell lines, while only one
form could block staurosporine-induced apoptosis (Kumar et al. 2010). Examination
of exons 2 and 3 of MDV vIL8 shows that two putative secondary binding sites exist
for CtBP-1 (NRPTGLPII and RRTEIIFA/SL) bearing homology to the E1A conserved region 3 of adenovirus (consensus RXNTGDXEXL) (Bruton et al. 2008).
Consequently, variants of Meq may contribute to the transformation of T-cells
through interactions with CtBP-1 and repression of select cellular loci as well as
silencing of lytic promoters within the latent MDV genome. The multiple forms of
Meq observed in the MDV-induced lymphoma and cell line (Fig. 13.1c) may also
indicate differential modification of the spliced forms, compared to full-length Meq.
Examination of Meq, Meq/vIL8, and Meq/vIL8exon3 using modification prediction software shows two putative type II sumoylation motifs (IKKLER and
RTRKENL, where K denotes the target lysine for sumoylation) in exon 3 of Meq/
vIL8 (http://sumosp.biocuckoo.org/), which could account for one or more of the
protein species seen between Meq/vIL8 and Meq (Fig. 13.1c). Since both Meq/vIL8
and Meq/vIL8exon3 maintain the CtBP-1-binding domain of Meq (PLDLS), and
CtBP-1 serves as a recruitment platform for sumoylation factors (Kuppuswamy et al.
2008), these Meq splice variants may very well serve as substrates for sumoylation.
Alternatively, these proteins may bind sumoylated proteins via a sumoylation interaction motif (SIM) in a manner similar to EBNA3C of EBV. The full transforming
properties of EBNA3C require the both CtBP- and SIM domains (Lee et al. 2009).
Phenotype of a Meq Deletion Mutant

Deletion of Meq from the genome of Md5, a vvMDV strain, resulted in a marked
decrease in virus replication in vivo and the complete attenuation of tumorigenicity
(Lupiani et al. 2004). This deletion virus, MD5DMeq, replicated within lymphoid
organs (spleen, bursa, and thymus) to near wild-type levels for approximately one
week and then rapidly declined in titer to being barely detectable by four weeks
postinoculation (Lupiani et al. 2004) and (Parcells et al., unpublished). Several
reports on the use of this virus, Md5Meq, as a vaccine have demonstrated it to be
highly protective, despite this limited replication (Lee et al. 2010; Su et al. 2010).
We have found that in SPF leghorn chickens, Md5Meq induces an acute inflammatory response and profound thymic atrophy, suggesting that protection is mediated by a loss of target cells (T-cells) during infection (Parcells et al., unpublished).
Target cell loss was also found to be the means by which another candidate vaccine,
RM-1, which has an LTR-insertion in the MDV genome, reduced tumor incidence
upon subsequent challenge (Liu et al. 2001; Witter et al. 1997). RM-1 has an
insertion of ~600 bp of the LTR from reticuloendotheliosis virus (REV) located
between the ICP4 and SORF2 genes (Jones et al. 1996). Insertion at this locus
upregulated the expression of SORF2, but may also affect the regulation of ICP4
during latency. Since Meq binds to and represses the expression of MDV lytic
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promoters (Levy et al. 2003), including the ICP4 promoter, the similarity in replication
and thymic atrophy mediated by both MD5Meq and RM-1 suggests that Meq may
serve as an important switch between lytic and latent infections. In summary, Meq
appears to have an important function during the lytic phase of infection in which it
appears to block the massive lytic replication within the thymus and mitigate the
associated proinflammatory response to MDV lytic replication.
Viral Telomerase RNA

A noncoding RNA having structure and function homologous to a telomerase RNA
(TR) is encoded in the RL segments of the MDV genome adjacent to the L and S
component repeats (IRLIRS and TRLTRS) (Fragnet et al. 2003). This virus-encoded
TR (vTR) is expressed during MDV lytic and latent infections as well as in MDVtransformed cell lines (Trapp et al. 2006). Biochemically, Viral Telomerase RNA
(vTR) functions as a TR in telomerase in vitro assays and out-competes the chicken
cellular TR for telomerase reverse transcriptase (cTERT) (Fragnet et al. 2003; Fragnet
et al. 2005). Deletion of vTR from the MDV genome does not abolish oncogenicity,
but does affect lymphoma frequency, size, and progression (Trapp et al. 2006).
Over-expression of vTR induced cellular proliferation, decreased anchorage dependence, increased colony formation in soft agar, and increased expression of integrin
alpha V, suggesting that it functions above and beyond its telomerase activity. The
promoter regulating the expression of vTR in the MDV genome has recently been
found to be three times more active than the chicken cellular TR promoter (Chbab
et al. 2010) as measured by pRT-PCR. This increased expression of vTR was found
to be important to efficient MDV transformation (Chbab et al. 2010). In a recent
study, mutations that abolished the ability of vTR to complement telomerase activity
did not affect the transformation efficiency of MDV, suggesting function(s) independent of telomerase activity (Osterrieder, personal communication). Since increases
in cellular telomerase activity accompany T-cell activation (Bodnar et al. 1996), the
sustained high expression of MDV vTR apparently not only serves to maintain
telomere length in proliferating, latently infected T-cells but also contributes other
functions (i.e., migration, metastasis) to developing lymphomas.
Viral Interleukin 8
Within the block of genes unique to MDV1 strains, an interleukin 8 homolog (vIL8)
was identified that affects MDV pathogenicity (Cortes and Cardona 2004; Cui
et al. 2004, 2005; Parcells et al. 2001). An interesting aspect of vIL-8 is that its
intronexon junctions are very similar to those of the chicken cellular IL-8 proteins
(9E3/CEF4, aka cCAF, and K60), suggesting that it was captured by MDV1 from
the chicken genome (Parcells et al. 2001). Nevertheless, vIL-8 has several features
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that distinguish it from chicken cellular IL-8 proteins. vIL-8 lacks the glutamic
acidleucinearginine (ELR) motif important for heterophil (neutrophil in mammals) chemoattraction, which is replaced with an aspartic acidlysinearginine
(DKR) motif. In addition, vIL-8 also contains a larger third exon that is markedly
divergent from the cellular homologs (Parcells et al. 2001).
In addition, vIL-8 is a chemo-attractant for chicken PBMCs and macrophages
(Parcells et al. 2001; Parcells, unpublished), but lacks angiogenic activity (Cui and
Lee, unpublished). Deletion of vIL-8 in its entirety (Cortes and Cardona 2004; Cui
et al. 2004; Parcells et al. 2001) or exon I only (which contains the signal peptide for
secretion) (Jarosinski and Schat 2007) results in an MDV that has decreased lytic
infection and an accompanying decrease in oncogenicity. A vIL-8 knockout virus
was found to protect against vv+MDV challenge, but retained a low level of inherent oncogencity, making this virus unsuitable as an MD vaccine (Cui et al. 2005).
A cell line established from an RB1BvIL8smGFP-induced tumor, MDCC-UA20,
showed constitutive expression of the marker gene smGFP, was notably lymphocytoid (as opposed to lymphoblastoid), and replicated poorly in culture (Parcells et al.
2001), suggesting a putative role for vIL-8 or portions of vIL-8 in maintaining T-cell
transformation (see Spliced forms of Meq, above). The vIL-8 gene of a BAC version
of the vaccine strain CVI988 is truncated compared to the vIL-8 genes of virulent
MDVs (CVI988 vIL-8 is 121 vs. 134 amino acids for Md5, RB1B), further suggesting a role in MDV pathogenicity (Spatz et al. 2007); however, this truncated version
was not identified in CVI988 vaccine strains.
Viral Ubiquitin-Specific Protease

Recently, MDV, like herpes simplex virus (HSV), has been found to encode a ubiquitin-specific protease domain within the major tegument protein (MTP) encoded
by the UL36 gene (Jarosinski et al. 2007). MTP is a very large (3,348 amino acids
for MD5), multidomain protein expressed late during infection that acts in an
immediate-early fashion during subsequent rounds of infection. Ubiquitin ligases
and proteases are key components of important regulatory networks within cells in
which the abundance of signaling intermediates, enzymes, chaperones, and sensing
proteins, etc., is regulated through their targeted degradation. During the course of
viral infection, cells may become less permissive to viral replication through the
targeted degradation of proteins, and hence, some viruses, notably herpesviruses,
have evolved enzymes for the targeted removal of ubiquitin. Through alignment of
MTPs encoded by other herpesviruses, a Ubiquitin-Specific Protease (USP) enzymatic signature was identified within a 322-amino-acid segment of the MTP of
MDV1 (Jarosinski et al. 2007). This signature contained cysteine, aspartic acid, histidine, and glutamic acid residues essential for function as a USP. Herpesvirus USPs
fall into the USP7 family of deubiquitinases (DUBs) and have a cysteine essential
to catalysis (Komander et al. 2009). Mutation of this cysteine to alanine at position
98 resulted in complete loss of USP activity (Jarosinski et al. 2007). Mutant viruses
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having this mutation (C98A-1 and -2) showed a slight replication deficiency in cell
culture and replicated at near wild-type levels for one week postinfection in vivo but
showed a marked decrease in replication after this time, and both mutant viruses
induced tumors in only 1020% of the infected chickens (Jarosinski et al. 2007). Of
the tumors induced by in the mutant-infected groups (C98A-1 and -2), several were
found to have reversion mutations, reestablishing the cysteine at position 98.
Consistent with a role in transformation, UL36 was found to be transcribed in MDVtransformed cell lines as well as during lytic replication.
Additional Gene Products Contributing to Oncogenesis

RLORF4
Between Meq and the vIL-8 coding sequences of the MDV genome, there are two
open reading frames RLORF4 and RLORF5a (aka L1) encoded in the same orientation as both Meq and vIL-8. RLORF4 encodes a methionine and cysteine-rich ORF of
142 amino acids predicted to localize to the ER and the mitochondrial and/or plasma
membrane based on PSORT-II prediction (http://psort.ims.u-tokyo.ac.jp/form2.html).
Several attenuated strains of MDV were found to have deletions of this ORF (Jarosinski
et al. 2003), and targeted deletion of RLORF4 resulted in a marked attenuation of the
BAC-based RB1B strain of MDV (Jarosinski et al. 2005b). The RLORF4 deletion
mutant also showed decreased plaque size in cell culture, and although no protein
product has been demonstrated for this ORF, the gene is transcribed during infection
(Jarosinski et al. 2005b). Based on the high degree of splicing within this region of the
genome (Jarosinski and Schat 2007), RLORF4 or portions of RLORF4 may represent
an as yet undescribed splice variant of Meq (see Western blot, Fig. 13.1c).
Upstream and adjacent to RLORF4, RLORF5a also appears to be expressed, at
least transcriptionally (Jarosinski et al. 2005b). Originally identified in cDNAs cloned
from the MDV-transformed cell line MDCC-CU41, RLORF5a (aka L1) encodes a
107-amino acid open reading frame, with no homology to known proteins, that is
predicted to localize to mitochondrial membranes (http://psort.ims.u-tokyo.ac.jp/
form2.html). A small portion of RLORF5a was found to be among several splice
variants cloned by 3-RACE that included exons II and III of vIL-8 (Jarosinski and
Schat 2007). Deletion of RLORF5a did not affect virus replication in cell culture or
in vivo and did not result in attenuation of the virus (Jarosinski et al. 2005b).
Phosphoprotein 14 and RLORF9

In the initial screening of the MDV genome for oncogenes, several research groups
searched for differences in gene expression between oncogenic strains and their
attenuated derivatives (Bradley et al. 1989a, b; Ross et al. 1993). A cluster of RNAs
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encoded at the MDV lytic origin of replication were found to change with serial
passage in cell culture, and several cDNAs were cloned from this region (Hong and
Coussens 1994; Hong et al. 1995; Peng et al. 1992). Transcripts encoding phosphoprotein 14 (pp14) are expressed with immediate early kinetics and differentially
spliced yielding 86- and 94-amino acid open reading frames, respectively, that share
a common 71-amino acid second exon (Hong and Coussens 1994; Hong et al. 1995).
The two ORFs are predicted to have both nuclear and cytoplasmic localization but
the proteins appear to be primarily cytoplasmic (Hong et al. 1995). Both proteins
have numerous putative serine/threonine phosphorylation motifs and a prominent
PEST (prolineglutamic acidserine/threonine) domain in the common C-terminus,
suggesting that they undergo rapid turnover. The discrepancy between the predicted
size of the proteins (9 and 10 kDa, predicted) and 14 kDa (observed) could not be
linked to phosphorylation or glycosylation (Hong and Coussens 1994; Hong et al.
1995). Consequently, these proteins are likely to have additional, yet uncharacterized, posttranslational modifications (Fig. 13.3).
Immediately downstream of pp14 and encoded in a bicistronic transcript is
RLORF9, which predicts an unspliced open reading frame of 108 amino acids
(Tahiri-Alaoui et al. 2009b). Expression of RLORF9 is regulated posttranscriptionally via an internal ribosomal entry site (IRES) between the second exon of pp14
and RLORF9. Deletion of this IRES abolishes translation of RLORF9 but was
found to not affect MDV pathogenesis (Tahiri-Alaoui et al. 2009b). In more recent
work, a second IRES has been identified in the 5 leader of the longer pp14 transcript, and that this form also could regulate RLORF9 expression (Tahiri-Alaoui
et al. 2009a).
Several lines of evidence suggest that pp14 is important to MDV replication and/
or pathogenicity. First, pp14 species are expressed with immediately-early kinetics
to very high levels during lytic infection (Liu et al. 2006), suggesting important
regulatory and/or host cell interaction functions (Hong and Coussens 1994; Hong
et al. 1995). Second, serial passage with attenuation affects expression of pp14, suggesting a role in pathogenicity. Finally, the encoding of two IRES sequences within
pp14 transcripts suggests that translation of these proteins is essential in the face of
cellular interferon responses. The combination of IRES sequences, cytoplasmic
localization, and the prominent PEST domains in their C-termini, in fact, suggest
that these proteins may be involved in the regulation of the intracellular host immune
response through the targeted degradation of specific proteins. The functions of
these proteins await further characterization.
MDV-Encoded MicroRNAs
MDV microRNAs (miRNAs) were first described following 454 Life Sciences
pyrosequencing of MDV1 strain RB1B-infected CEF (Burnside et al. 2006). These
miRNAs were found to cluster to two distinct regions of the MDV genome. One
cluster lies near the meq gene, and another maps in the LAT/ICP4 region. These
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Fig. 13.3 Colocalization of Meq proteins with CtBP-1. Cell line HTC (Rath et al. 2003) was
cotransfected with Meq protein eYFP (Meq-eYFP, top; Meq/vIL8-eYFP, middle; Meq/vIL8exon3-eYFP, bottom) and chicken CtBP-1-eCFP expression vectors (Anobile et al. 2006).
Transfected cells were fixed with 2% paraformaldehyde at 24 h posttransfection and stained with
DAPI prior to imaging. Images were acquired using a Nikon TE-2000 microscope with epifluorescence, a Roper CCD camera and NIS-Elements image acquisition and analysis software (http://
www.nis-elements.com/). Representative fields are shown for colocalization of proteins. Arrows of
the same color (white, yellow, blue, green, and pink) denote common cells imaged with respective
filters. Overlays at right show colocalization of proteins within nuclei of transfected cells. Note
that Meq spliced proteins show very dense nuclear localization with CtBP-1
results were confirmed and extended following sequencing of a small RNA library
prepared from MSB-1 cells (Waidner et al. 2009; Yao et al. 2007, 2008). Since the
MSB-1 cells used also harbored MDV2, this study included identification of MDV2
miRNAs. MDV2 miRNAs were found to cluster in a 4.2-kb repeat region encoding
R-LORF2 to R-LORF3, with one additional miRNA positioned in the 3 end of the
ICP4 gene. MicroRNAs of herpesvirus of turkeys (HVT) have been reported based
on analysis of deep-sequencing libraries of HVT-infected CEF (Waidner et al.
2009). While there is no sequence conservation among the miRNAs of MDV1,
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MDV1
14 miRs
UL
TRL
MDV2
164,270 bp
IRL
HVT
159,160 bp
UL
US
IRS
TRS
18 miRs
UL
TRL
TRL
177,874 bp
IRL
IRS
US
TRS
17 miRs
IRL IRS
US
TRS
Fig. 13.4 Comparative genomic locations of MDV miRNAs. Genome maps of MDV1, MDV2,
and HVT are shown with the approximate genomic locations of their respective miRNAs. The
number of miRNAs reflects data found in miRBase Release 15 (April 2010) (http://mirbase.org)
and does not include passenger or star strands as separate miRNAs
MDV2, and HVT, for all of these herpesviruses, the general genomic location of the
miRNAs is conserved (Waidner et al. 2009) (see Fig. 13.4). At present, mirBase lists
14 MDV1, 18 MDV2, and 17 HVT miRNAs.
Expression of miRNAs has been confirmed, in general, by Northern blot analysis
or qPCR (Burnside et al. 2006; Xu et al. 2008; Yao et al. 2007). By and large, patterns of expression reflect meq and LAT expression; however, expression during
lytic infection of CEF is also detected (Burnside et al. 2006; Yao et al. 2007).
Expression of mdv1-miR-M4 was particularly high in MDV-induced tumors and
represented approximately 72% of all sequences matching the MDV genome from
deep sequencing analysis (Morgan et al. 2008). In some cases, the miRNA precursors were detected. The relative expression of the 3p and 5p arms of mdv1-miR-M2
and mdv1-miR-M8 differed between MSB-1 lymphoblastoid cells and lytically
infected CEF, but the significance of this differential processing remains to be
understood (Burnside et al. 2006).
Since all of the Gallid (Infectious Laryngotracheitis [ILTV], MDV1, MDV2) and
Meleagrid (HVT) herpesviruses encode miRNAs, their presence in and of itself
does not signal oncogenic potential. However, because the targets and functions of
miRNAs are unknown, a role in the manifestation of oncogenicity remains possible,
and even likely, given that they are present in many undifferentiated cells and that
their expression has, in some cases, been linked to oncogenicity (Esquela-Kerscher
and Slack 2006; Garzon et al. 2009).
One approach to determining if MDV1 miRNAs contribute to oncogenic potential has been to determine if expression of various MDV1 miRNAs is correlated
with relative oncogenic potential of a variety of MDV1 strains that have been characterized over the years (Morgan et al. 2008). Sequencing of PCR products derived
from the meq and LAT miRNA cluster regions and comparing the sequence data to
the Md5 reference sequence (AF243438) revealed that no SNPs within miRNA
coding sequences are associated with relative oncogenicity (Morgan et al. 2008).
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Sequencing of the putative promoter for the meq miRNAs did reveal at least one
potentially interesting SNP, but its significance awaits more detailed promoter characterization (Lagasse, preliminary results). Overall, miRNAs appear to be under
stringent selective pressure for sequence conservation.
A second approach to examining oncogenic potential of MDV1 miRNAs has
been to compare miRNA expression among tumors induced by vv versus vv+
viruses or among various lymphoblastoid cells. When small RNA samples from
MDV-induced tumors were examined, meq miRNAs were found to clearly accumulate to a greater degree in tumors induced by vv+ MDV (615K, aka T. King) compared to tumors induced by the vv strain RB1B (Morgan et al. 2008). This differential
expression was specific for the meq miRNA cluster and was not observed for LAT
miRNAs. In addition, this difference could not be explained by general differential
transcription of the meq gene between tumors induced by 615K or RB1B, since
qPCR revealed that meq mRNA levels were similar in these samples.
Seven MDV1-induced lymphoblastoid cell lines have been compared to an
MDV1-negative REV-T-transformed CD4+ cell line (AVOL-1) and an MDV1negative avian leukosis virus (ALV) HPRS F42 strain transformed B-cell line
(HP45) with regard to micRNA expression signatures (Yao et al. 2009). All MDV1
lymphoblastoid cell lines showed MDV1 miRNA expression as expected, although
some variation in relative expression of specific miRNAs was noted. With regard to
host miRNAs, downregulation of miR-155, miR-150, and miR223 was noted, with
downregulation of miR-150 and miR-223 also being observed in AVOL-1 (MDV1negative) cells.
A third approach to determining if MDV1 miRNAs contribute to oncogenic
potential has been to look for orthologs among other oncogenic viruses. MDV1miR-M4 shares a seed sequence (TAATGCT) with gga-miR-155, which is considered an oncomir (Morgan et al. 2008; Zhao et al. 2009b). In addition, Kaposis
sarcoma herpesvirus (KSHV) encodes a miRNA, kshv-miR-K12-11, which shares
an identical seed sequence (Gottwein et al. 2007; Skalsky et al. 2007). In addition,
mdv1-miR-M32 shares a seed sequence with gga-miR-221 (Morgan et al. 2008).
miR-155 is interesting in that it is expressed in activated B and T cells, and it is
important for B-cell development and germinal center development (Rodriguez
et al. 2007). A key role for miR-155 is the downregulation of proinflammatory protein expression during the immune response (Ceppi et al. 2009). miR-155 is
expressed in many human malignancies, including B-cell lymphomas (Eis et al.
2005), breast, lung, and colon cancers (Volinia et al. 2006), and it is expressed in
avian leukosis virus (ALV)-induced B-cell lymphomas (Tam et al. 2002). In humans,
miR-155 is processed from exon 3 of BIC (B-cell integration cluster) RNA, an RNA
that enhances c-Myc-associated lymphomagenesis. Overexpression of miR-155 in
transgenic mice induces B-cell lymphoma formation (Costinean et al. 2006).
However, in MDV-induced T-cell lymphomas, gga-miR-155 is not overexpressed,
but mdv1-miR-M4 accumulates to a greater degree than any other MDV1 miRNA.
Thus, it appears that in MDV-induced T-cell lymphomas, mdv1-miR-M4 may alleviate the need for gga-miR-155 overexpression in inducing and/or maintaining the
transformed phenotype (Morgan et al. 2008).
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The search for mdv-miR-M4/miR-155 targets has revealed a long list of potential
targets based on results obtained using a variety of target prediction software (Zhao
et al. 2009b). A few targets have been validated using luciferase-based reporter assays,
including PU.1, CEBP, HIVEP2, BCL2L13, and PDCD6 (Lambeth et al. 2009). In
human dendritic cells (DC), an important set of targets for miR-155 are those induced
through Toll-like receptor signaling (Ceppi et al. 2009). Treatment of DCs with IL-1,
TNF, IL-6, etc. resulted in a rapid increase miR-155 expression and concomitant
downregulation of signaling molecules Pellino-1 and TAB2. Consequently, miR-155
is part of a negative feedback loop in the downregulation of proinflammatory signaling. For the PU.1 target, evidence has been presented that chicken macrophage cell
line HD11 shows reduced expression of endogenous PU.1 when transfected with
miR-M4 or miR-155 (Lambeth et al. 2009). Although not shown directly for mdv1miR-M4, BACH1/Brip1 is likely to be a target since it is recognized by both miR-155
and KSHV miR-K12-11 (Gottwein et al. 2007; Skalsky et al. 2007). BACH1 is a basic
leucine zipper transcription factor that participates in a variety of key cellular functions such as hypoxia and DNA repair (Cantor et al. 2001; Reichard et al. 2008).
miR-221, which shares a seed sequence with mdv1-miR-M32 (Morgan et al.
2008), has been shown to target p27Kip1, a negative regulator of the cell cycle that,
when downregulated, promotes cancer cell proliferation (Fornari et al. 2008). GgamiR-221 and gga-miR-222 have been shown by Northern blotting to be upregulated
in MSB-1 cells compared to CEF or splenocytes (Lambeth et al. 2009). In vitro
luciferase reporter assays have indicated that p27Kip1 has the potential to be specifically targeted by gga-miR-221 and gga-miR-222 in CEF and MSB-1 cells (Lambeth
et al. 2009). In addition, Western blotting and retroviral mediated expression of antagomirs to gga-miR-221 and gga-miR-222 were used to demonstrate an inverse relationship between p27Kip1 protein levels and gga-miR-221 and gga-miR-222 levels, with
the gga-miR-221 effect being the stronger of the two (Lambeth et al. 2009).
A fourth approach to associating MDV1 miRNA expression with oncogenicity
has been to construct mutants that fail to express miRNAs and/or to construct
recombinant HVT strains that express MDV1 miRNAs. These mutants are works in
progress, and their phenotypes in cell culture and in vivo will be interesting to
dissect.
In our laboratory, a recombinant HVT expressing mdv-miR-M4 shows improved
growth characteristics in vitro and in vivo suggesting that miR-M4 can provide replicative assistance. Since this recombinant displays no tumorigenicity or pathogenicity, mdv1-miR-M4 should not be considered an oncomir in and of itself (Anderson
et al., unpublished results).
A Developing Model of MDV-Mediated Lymphomagenesis

Based on our current understanding of the expression patterns, molecular and biochemical functions of MDV gene products, we have developed a model describing
important events in MD-lymphomagenesis (Fig. 13.5). Latency and transformation
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Full Latency/
MDV-Transformed
CD4+ T-cell
Early Latency
Activated
CD4+ T-cell
CD30Lo
UL36-USP+
CD30Hi
CD4Hi
TCR-2 (V1)
or TCR-3 (V2)
Meq
MHC-II
CD25+
CD28+
UL36-USP+
mdv1-M4-miR+
Hi
Meq
Meq/vIL8Hi
Meq/vIL8exon3+
CD4Hi
TCR-2 (V1)
or TCR-3 (V2)
MHC-IIHi
CD25+
vTR+
Genome State
(Integrated?)
Genome Integrated
10 14 dpi
~ 15 + dpi
Latently-infected TH cell
Transformed TREG cell
Fig. 13.5 A developing model of MDV-mediated Lymphomagenesis. The diagram above comprises our relative understanding of MDV-mediated transformation. T-cells become activated following early cytolytic infection and latently infected at 1014 dpi. Factors affecting the transition
of latently infected to transformed are currently unknown but are correlated with increased expression of Meq splice variants, viral miRNA expression, as well as sustained expression of vTR,
UL36 and other products. Multiple MDV genome integration events may serve as a catalyst for
sustained, high-level expression of these gene products, as integration is common to MD lymphomas and derived cell lines. Sustained expression of spliced Meq products likely affects the T-cell
immunophenotype through CtBP-1-mediated repression of specific loci giving rise to the transformed Treg cell. Expression of factors from these cells shapes the tumor microenvironment as
well as suppresses antitumor responses. Transformed cells are also latently infected from which
virus can reactivate. Although highly efficient at transformation, MDV is believed to induce multiple-monoclonal tumors as opposed to polyclonal transformation events (Delecluse and
Hammerschmidt 1993; Delecluse et al. 1993)
are closely related, as depicted in Fig. 13.5. Events distinguishing latency from
transformation are not known, but likely reflect differences in expression level and
the accumulation of secondary mutations in proliferating lymphocytes.
Initiating Events
One of the key initiating events in MD lymphomagenesis is the effect of lytic replication on immune surveillance. MDVs that have limited lytic replication (e.g., pp38
and vIL-8 deletion mutants) are capable of retaining oncogenicity, but at a much
reduced level (Parcells et al. 2001; Reddy et al. 2002). It is, therefore, likely that the
primary immunological damage elicited by early MDV replication is important to
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tumor development. In the Cornell model for MD, early replication in B-cells activates
T-cells and allows them to become infected (Calnek 1986, 1992). Expression of
vIL-8 elicits macrophages to sites of lytic replication and drives the virus latent
through expression of interferons, NO, etc.
Establishment of latency is required, but not sufficient, for transformation. MDV
establishes latency and transforms CD4+, TCR-2+, and TCR-3+ T-cells exclusively. No
MDV-associated B-cell or TCR-1+ (gd) T-cell tumors have been identified (Parcells
and Burgess 2008; Schat et al. 1982b). In our findings regarding Meq and Meq splice
variant expression (Fig. 13.1c), we see that Meq variants are detected at three weeks
(VR3), but not at 2 weeks postinfection (VR2), a time frame consistent with latency.
Stages of latency have been described previously for MDV (Volpini et al. 1995), with
cells latently infected at one week being more sensitive to induced viral antigen
expression than after two weeks. Coupled with our observation of Meq expression
(Fig. 13.1c), we hypothesize that the later, more repressed stage of latency reflects
increased expression of spliced forms of Meq. In our model, therefore, Meq and Meq
variants repress viral lytic gene expression (ICP4, pp38, etc.) through recruitment of
repression complexes by CtBP-1. These complexes block apoptosis and maintain cellular proliferation through interaction with cell cycle regulators Rb and p53 and
repression of proapoptotic signals via interaction with CtBP-1 (and other factors, i.e.,
Par-4). Additional latent gene products vTR, USP (UL36), and pp14 maintain telomerase activity and alter cellular signal transduction. During latency, Meq induces the
upregulation of MATSAs, including CD30, which likely have important consequences
to lymphoma progression. In addition, host differences in cytokine responses likely
contribute to initial tumor promotion events through the TH2 polarization of the
immune response (Heidari et al. 2008; Kaiser et al. 2003). MDV-encoded miRNAs,
notably mdv1-miR- M4, likely modulates proinflammatory signaling in latently
infected T-cells and its sustained expression likely contributes to further lymphoma
progression. Accessory roles of MDV1 miRNAs would be in the balance between
lytic replication and latency, as MDV lytic gene mRNAs (ICP4, ICP27, pp38, etc.) are
likely targets for these micros, but these targets have yet to be demonstrated.
Lymphoma Progression
A key step in tumor progression is the sustained Meq-mediated blocking of T-cell
anergy/apoptosis by downregulation of proapoptotic gene expression (through
interactions with CtBP-1) and alteration of TGF signaling (via upregulation of
c-Ski). Since Meq unspliced and spliced forms are highly expressed in tumors and
lymphomas, it is likely that the spliced forms code for proteins that are involved
primarily in repression complexes, while Meq homo- and heterodimers function in
the targeted transactivation of cellular genes as well as in a positive feedback loop
on expression of itself and its spliced forms. This model is supported by the lack of
colocalization of Meq and the spliced Meq proteins (Anobile et al. 2006) and the
inherent activities of the proteins (Kumar et al. 2010).
13
327
During lymphoma progression, Meq-cJun heterodimers increase proliferation,

mobility and apoptosis resistance of cells that form initial tumor masses, perhaps
through upregulation of adhesion molecules via vTR etc. Meq-induced CD30hiexpressing cells begin to shape the tumor microenvironment and begin to shift to a
Treg immunophenotype. This is likely accomplished through the signaling mediated by CD30 engagement and the selective repression of T-cell expression to differentiate latently infected and transformed cells to a Treg immunophenotype.
Alternatively, MDV may selectively target Treg cells for transformation as these
are localized to distinct thymic regions and undergo different thymic programming.
In either case, cells expressing high levels of CD30 shape the tumor microenviroment to block cell-mediated responses to tumor cells. MD tumor cells are somewhat
resistant to CTL killing, as noted in the selection of REV-based cell lines for studying CTL responses to MDV (Omar and Schat 1997; Omar et al. 1998; Weinstock
and Schat 1987). As lymphomas become larger and begin to become hypoxic,
increased CtBP-1 expression and dimerization would drive further MeqCtBP complex formation, repressing intracellular adhesion molecule expression and thus driving increased mobility/metastasis. vTR contributes to progression through
upregulation and perhaps increased turnover of integrins, contributing to increased
migration and also increased resistance to anoikis. Sustained MDV1 miRNA expression likely contributes to progression through the targeting of cell cycle regulatory
and immune signaling pathways within transformed cells, based on their homology
to human and murine counterparts. Although MDV is highly efficient at transformation, MDV-induced tumors are monoclonal, as determined through integration site
analysis (Delecluse and Hammerschmidt 1993; Delecluse et al. 1993). The efficiency of MDV-mediated transformation, however, suggests that multiple monoclonal events may be present per chicken, and those that progress most rapidly give rise
to the large frank lymphomas associated with MDV.
Acknowledgments The authors would like to thank Drs. Venugopal Nair, Klaus Osterrieder, and
Hsing-Jien Kung for the sharing of unpublished data and critical reading of select sections.
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Chapter 14
Polyomaviruses and Cancer

Ole Gjoerup
Introduction
Polyomaviruses are deceptively simple because of their small genomes and limited
coding potential, yet they provide us with highly sophisticated model systems for
understanding basic cellular processes. The power of studying them lies in their targeting of the most central players and mechanisms for proliferation control. Studies of
polyomaviruses have allowed us to make significant advances in deciphering the
molecular basis of cancer. Thus, critical signaling elements and pathways that have
been elucidated with the help of these viruses include the p53 tumor suppressor, pRB
(retinoblastoma) function in cell cycle control, protein phosphatase 2A (PP2A) function in regulation of cell proliferation, as well as discovery of tyrosine kinases and
phosphoinositide 3-kinase (PI3K) (Linzer and Levine 1979; Lane and Crawford 1979;
DeCaprio et al. 1988, 1989; Pallas et al. 1990; Eckhart et al. 1979; Whitman et al.
1985). These seminal discoveries were made by direct biochemical analysis of immunoprecipitates of the polyomaviral T antigens. In summary, the polyomaviruses have
provided us insight to cellular signaling of general applicability, extending far beyond
the realm of viruses. Moreover, studies of polyomavirus gene products have been
instrumental not just for the understanding of molecular mechanisms related to cancer
development, but also for understanding of enhancers and transcriptional regulation,
splicing and polyadenylation, and DNA replication and nuclear translocation.
A fundamental principle of polyomaviruses is that they operate by encoding
proteins that bind and alter the key host proteins. Their interaction with a cellular
protein might lead to its functional inactivation, activation, or redirection toward a
different target (for example, an associated kinase or ubiquitin ligase). As is
discussed, there appears to be a convergence on a small number of cellular target
O. Gjoerup (*)
Cancer Virology Program, University of Pittsburgh Cancer Institute,
Research Pavilion Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA 15213, USA
e-mail: ovg27@pitt.edu
337
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O. Gjoerup
proteins, notably pRB, p53, Hsc70, p300/CBP, and PP2A, that nearly all of the
polyomaviruses encode one or more gene products to interact with. In addition,
there are multiple other binding proteins, where functionality of the interaction is
not fully established, and their role may not be universal for all the polyomaviruses
but perhaps allowing more specialized functions.
The intention of this chapter is to introduce the reader to this important family of
viruses and to emphasize the defining characteristics of the polyomaviruses, including
their conserved as well as unique features. Since most of the viruses discussed
here are reviewed individually in the following chapters, the focus is on the larger
picture and how they compare with each other in mechanisms and biology.
History and Discovery

The founding member of the polyomaviruses, murine polyomavirus (MPyV), was
identified by Ludwik Gross in 1953 when, searching for cell-free transmission of
leukemia, he found a filterable agent capable of inducing salivary gland tumors in
newborn mice (Gross 1953). This new virus became the archetypal member of the
Polyomaviridae family. Polyomavirus is so named because of the ability of MPyV
to induce solid tumors at many different sites.
Shortly thereafter, Sweet and Hilleman discovered simian vacuolating virus 40
(also known as simian virus 40 or SV40) as a contaminant in monkey kidney
cells used to grow the poliovirus vaccine (Sweet and Hilleman 1960). Subsequent
studies estimate that as many as 100 million people may have been inadvertently
exposed to vaccine possibly containing live SV40 virus, mainly between 1955 and
1963, as part of the poliovirus vaccination program (Garcea and Imperiale 2003).
Due to the lack of convincing epidemiological and serological evidence, SV40 has
not been conclusively linked to human malignancies, although it has been proposed
to be causally associated with mesotheliomas, osteosarcomas, choroid plexus brain
tumors, and non-Hodgkins lymphoma (Poulin and DeCaprio 2006a). Nevertheless,
a lot of questions remain. Most recent surveys indicate that SV40 is not a human
polyomavirus, but it might be present at low prevalence in the population, and the
behavior of SV40 in humans has not been clarified (Garcea and Imperiale 2003).
In 1971, the first human polyomaviruses, BKV and JCV, were discovered in
urine from a renal transplant patient and brain tissue of a patient with progressive
multifocal leukoencephalopathy (PML), respectively (Padgett et al. 1971; Gardner
et al. 1971). BK and JC refer to patient initials. Amazingly, technological advances
have allowed the identification of six additional human polyomaviruses within the
last 3 years. In 2007, Karolinska Institute virus (KIV) and Washington University
virus (WUV) were discovered in symptomatic pediatric respiratory specimens
(Gaynor et al. 2007; Allander et al. 2007). These viruses were detected by treatment
of the specimens with endonuclease, which digests host DNA, but not viral DNA
protected within virions, followed by high-throughput shotgun sequencing of all
DNAs present. There is currently no evidence linking KIV and WUV to cancer or
non-neoplastic disease (Dalianis et al. 2009).
14 Polyomaviruses and Cancer
339
In 2008, Merkel cell polyomavirus (MCV) was detected by digital transcriptome

subtraction (DTS) analysis of Merkel cell carcinoma (MCC) samples (Feng et al.
2008). MCC is a rare but highly aggressive type of skin cancer of neuroendocrine
origin. The incidence of MCC is highly elevated in immunosuppressed patients
suggesting an infectious etiology. DTS involves preparing a library of sequences of
the entire transcriptome from a sample, followed by comparing and subtracting
in silico known human sequences from all available databases (Feng et al. 2007).
The remaining sequences after rigorous subtraction potentially belong to novel viruses
and may be identified as such by shared homology with known families of viruses.
Accumulating evidence strongly supports a causal role for MCV in about 80% of
MCC patients, and this is now becoming widely accepted, thus providing the most
conclusive direct link so far between a polyomavirus and human malignancy.
In 2010, rolling circle amplification of DNA from normal human skin samples
identified an additional human polyomavirus-designated HPyV6 (Schowalter et al.
2010). Several isolates of variant MCV genomes were identified in the same search.
A PCR-based search with degenerate primers for additional polyomaviruses in skin
samples further led to discovery of HPyV7.
Finally, in 2010, another polyomavirus, trichodysplasia spinulosa-associated
polyomavirus (TSV), was discovered by rolling circle amplification from facial
spicules of a heart transplant patient with the extremely rare disease trichodysplasia
spinulosa (van der Meijden et al. 2010). Phylogenetic analysis revealed that it is
most closely related with orangutan polyomavirus and MCV. Taken together, BKV,
JCV, KIV, WUV, MCV, HPyV6, HPyV7, and TSV constitute the eight currently
known human polyomaviruses.
Moreover, lymphotropic polyomavirus (LPV), also known as African green
monkey polyomavirus, which was discovered in 1979, could also be of relevance to
human cancer (zur Hausen and Gissmann 1979; Pawlita et al. 1985). There is
long-standing evidence that a human counterpart to LPV exists, but it has not been
identified yet. Thus, different groups have found serological evidence that an LPVlike human virus circulates in 1520% of the human population (Brade et al. 1981;
Kean et al. 2009).
Phylogenetic Comparison of Polyomaviruses

At least 23 different polyomaviruses have been found so far with hosts ranging from
birds, bats, rabbits, and rodents to primates (see Dalianis and Garcea (2009) for a
comprehensive list). Phylogenetic analysis, based on an early gene product, LT, or a
late gene product, VP1, divides polyomaviruses into separate clades (Fig. 14.1).
Within the SV40 group, there is close homology among SV40, BKV, JCV, and
SA12 when comparing either LT or VP1. However, KIV and WUV, while very similar
to each other, are more distantly related to the rest of the SV40 group. Especially the
VP1 proteins of KIV and WUV exhibit significant sequence divergence from
the remainder of the SV40 group and cluster more closely with HPyV6 and HPyV7.
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O. Gjoerup
Fig. 14.1 Phylogenetic analysis of polyomavirus LT and VP1 protein sequences. Phylogenetic
trees were generated for the LT and VP1 protein sequences from 19 polyomaviruses: crow, goose,
finch, budgerigar fledgling disease polyomavirus (BFPyV), bovine polyomavirus (BPV), bat, LPV,
MCV, hamster polyomavirus (HaPyV), murine pneumotropic virus (MptV), MPyV, BKV, simian
agent 12 (SA12), JCV, SV40, KIV, WUV, HPyV6, and HPyV7. Sequences were aligned with the
ClustalX program, and the alignment was imported into TreeView for generation of phylograms
Interestingly, MCV is belonging to a separate clade of MPyV and is most similar

with LPV (Feng et al. 2008). Generally, the avian polyomaviruses cluster together
and form their own clade. Bovine polyomavirus (BPV), murine pneumotropic virus
(MptV, also known as Kilham strain), and little brown bat polyomavirus are outliers
and do not readily fit into the clades of MPyV, SV40, or avian polyomaviruses.
Genome Organization
Genomes of the polyomaviruses range in size from 5 to 5.4 kb of circular, doublestranded, covalently closed DNA. The DNA is present in complex with cellular
histones, with the exception of histone H1, thus resembling cellular genomes
(minichromosomes). Viral particles consist of nonenveloped icosahedral capsids
with a 4045-nm diameter. Viral genomes are divided into three parts: the early
region encodes the early transcript, which is expressed before the onset of viral
DNA replication (see Fig. 14.2 for a schematic drawing of SV40 and MPyV genomes).
Conversely, the late region produces the late transcript, which is predominantly
expressed after the onset of viral replication. The noncoding regulatory region (NCRR),
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Fig. 14.2 Overview of SV40 and MPyV genome organization. The genomes of SV40 (5,243 bp)
and MPyV (5,297 bp) are outlined, including the early region that encodes the T antigens and the
late region that encodes the capsid proteins VP1-3 (VP4 is unique to SV40). Shown are the open
reading frames that are generated through alternative splicing. An miRNA known to target the
early message is also included. The core origin of replication is indicated (ori). Some of the differences to be noted are the existence of an agnoprotein generated from the leader of the SV40 late
message, as well as the unique MT of MPyV. The tiny T antigen of MPyV was not included in the
drawing
consisting of the viral replication origin and transcriptional control elements, is

intertwined between these. Early and late promoters are in close proximity with
the origin of replication. The transcriptional control elements in the NCRR of
various polyomaviruses were recently reviewed (White et al. 2009). Early and late
transcripts are of approximately equal length but encoded on opposite strands of
the viral genomes.
The early transcript is differentially spliced to generate 25 early proteins
(Fig. 14.3). Large T antigen (LT) and small t antigen (ST) are expressed by all the
known polyomaviruses, although for some like KIV and WUV, these have only
been predicted by open reading frame analysis. These proteins are referred to as
T antigens because they were first identified using antibodies from tumor-bearing
animals. LT and ST have yielded profound insights to biological processes when
used in model systems.
Besides LT and ST, a variable number of accessory T antigens are expressed
from the early transcript. These include the 17k T antigen of SV40 (Zerrahn et al.
1993), the T165, T136, and T135 of JCV (Trowbridge and Frisque 1995), the
truncT of BKV (Abend et al. 2009b), the 57kT of MCV (Shuda et al. 2008), and the
tiny T of MPyV (Riley et al. 1997). Because of the splicing arrangement, all of the
early viral transcripts share exon 1 at the N-terminus, whereas the C-terminus is
unique. The role of the auxiliary T antigens is not well-understood, although in the
case of JCV they have been demonstrated to contribute to efficient viral replication
(Prins and Frisque 2001). SV40 17k exhibits minimal transforming activity (Zerrahn
et al. 1993), but its role during the viral life cycle remains to be demonstrated.
MPyV and hamster polyomavirus are unique in that they also encode a middle
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Fig. 14.3 The splicing patterns of various polyomavirus T antigens. The splicing patterns are
shown for all the T antigens from MCV, JCV, BKV, SV40, and MPyV. The different reading
frames are outlined by different color rectangles, whereas the broken line illustrates introns. The
N-terminus of each T antigen is shared (exon 1), but the C-terminus is most often unique (except
57kT and T165). TT refers to tiny T antigen
T antigen (MT), which is their principal transforming protein (reviewed in Fluck

and Schaffhausen 2009; Schaffhausen and Roberts 2009). How MT evolved during
evolution is an intriguing question.
The late message encodes at a minimum three capsid proteins, known as VP1,
VP2, and VP3. Only for SV40 has a VP4 been reported as well (Daniels et al. 2007).
Both alternative splicing and internal translation, the late message being bicistronic,
generate the various capsid proteins. While VP1 is the main capsid protein, the
minor components VP2/3 are also required for encapsidation. VP1 can spontaneously assemble into virus-like particles (VLPs) when expressed individually. Since
VP1 is the major capsid protein, it is frequently used to develop serologic assays
to detect the polyomaviruses and estimate their prevalence in the human population.
While we think of VP1 as being inert with regard to transformation-related signaling,
it is worth noting that interaction of VP1 with the cell is known to trigger activation
of c-myc and c-fos genes (Zullo et al. 1987), which have been associated with transformation, and also recently poly(ADP-Ribose) polymerase 1 (PARP-1), caspases,
phospholipase Cg1 (PLCg1), and Akt-1 (Butin-Israeli et al. 2010). VP4 is believed
to be important for egress of the viral genomes at the end of the lytic cycle (Daniels
et al. 2007). Leader sequences of the late transcripts from SV40, BKV, and JCV are
known to also encode an agnoprotein, which has not been detected for most of the other
polyomaviruses (Ng et al. 1985; Khalili et al. 2005). The agnoprotein contributes
343
to viral assembly and maturation but is not contained in the virions. Several
polyomaviruses (SV40, BK, JC, MCV, MPyV) encode a miRNA from the late transcript probably by a read-through mechanism (Sullivan et al. 2005, 2009; Seo et al.
2008). The general function of this miRNA is to cleave the early message late in
infection, thus downregulating its expression and escaping immune surveillance by
cytotoxic T cells (Sullivan et al. 2005).
Viral Life Cycle

Polyomaviruses are internalized by interaction of the VP1 capsid protein with sialic
acids on specific gangliosides acting as receptors. Known receptors are gangliosides
GD1b and GT1b for BKV, GT1b and the serotonin receptor 5HT2AR for JCV, GM1
for SV40, and GT1b for MCV (Tsai et al. 2003; Low et al. 2006; Elphick et al. 2004;
Erickson et al. 2009). The polyomaviruses mainly enter cells through caveolae that
are lipid rafts invaginating from the plasma membrane; however, JCV passes
through clathrin-dependent pathways (Eash et al. 2006). The viruses then traffic
through the perinuclear ER to the nucleus, where uncoating occurs, followed by
early transcription of the viral genome via host RNA polymerase II. After translation of LT, it subsequently binds GAGGC repeats within the origin and initiates
melting and unwinding of the core origin, followed by recruitment of host replication
factors, like polymerase a/primase. LT is a prototype initiator of replication with
intrinsic ability to bind DNA specifically, assemble into single or double hexamers,
and unwind DNA using its ATP-dependent helicase activity (reviewed in Fanning
and Knippers 1992; Simmons 2000). LT also has an ability to modulate transcription,
which is important during infection, where it represses early transcription while
enhancing late viral gene expression. After viral replication commences, the late genes
are expressed and their protein products assemble into capsids that encapsulate the
viral genome. A full lytic cycle, in the case of SV40, spans approximately 72 h.
Polyomaviruses normally have a narrow host range and restricted cell-type tropism.
SV40 only productively infects monkey cells and MPyV only murine cells. These
restrictions are believed to be in part mediated by the species-specific interactions
between LT and polymerase a that are critical for a productive initiation of viral
replication (Schneider et al. 1994). For example, JCV prefers to grow in fetal glial
cells (Feigenbaum et al. 1987); however, it is not strictly neurotropic, as it can replicate
in many other cell types, including human embryonic kidney (Major et al. 1992).
LPV preferentially grows in B-lymphoblastoid cells. Other polyomaviruses, like
SV40 and MPyV, in particular, replicate in a broad range of cell types.
Polyomaviruses productively infect their natural (permissive) host, resulting in
cell lysis and release of virions. Tumors are very rarely caused in the natural host
because the lytic cycle kills the cell. These viruses can also establish persistent
infections that rarely cause tumors or other diseases, except in immunocompromised or newborn animals. SV40 does not induce disease in rhesus monkeys, except
when these are immunosuppressed through simian immunodeficiency virus infection
(Horvath et al. 1992).
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Conversely, nonpermissive cells elicit an abortive infection, meaning that viral

entry and early gene expression occur normally, but somehow there is a block
in viral replication and late gene expression. At high multiplicities of infection,
the whole cell population can transiently become transformed, but as the cells
divide and the viral genome is diluted out, only rare transformants grow out in
which the viral genome is integrated. It is important to emphasize that oncogenic
transformation upon integration of these viruses can be viewed as a biological
accident, since it is a dead-end event preventing multiplication of the virus (Shuda
et al. 2008).
Human cells are considered to be semipermissive for SV40, meaning that low
levels of replication and virus production occur. Not just species dictates the outcome of a polyoma infection, but also the cell type. Thus, SV40 productively
infects human fibroblasts, whereas in mesothelial cells the outcome is a persistent
infection without cell lysis, thereby facilitating oncogenic transformation (Bocchetta
et al. 2000).
General Properties
Despite 50 years of intense studies, some fundamental questions of polyomavirus
biology continue to elude us. What cells are initially infected? How is the virus
disseminated between tissues? Is there a major virus reservoir in certain tissues that
are persistently infected? How is the virus transmitted between individuals? These
are all questions for which we have still incomplete answers.
Most of the human polyomaviruses are widespread in nature and cause lifelong
infections that are asymptomatic or subclinical, except in immunosuppressed
individuals. In other words, these viruses appear to be most often kept in check by
the immune system. Individuals contact BKV, JCV, and MCV in early childhood or
adolescence leading to seroconversion (Gjoerup and Chang 2010; Kean et al. 2009).
Approximately 5080% of the human population is seropositive for BKV and
JCV (Egli et al. 2009; Knowles et al. 2003; Carter et al. 2009). Preliminary tests
indicate that MCV, KIV, and WUV are equally prevalent (Kean et al. 2009; Carter
et al. 2009).
Transmission routes have not been definitively established (Gjoerup and Chang
2010). Fecal/oral and oral transmissions have been proposed for BKV and JCV,
whereas KIV, WUV, and MCV may occur through the respiratory system.
Interestingly, tonsillar DNA can be shown to contain genomic DNA from most of
the polyomaviruses, suggesting a potential entry point. JCV infects tonsillar cells
but is circulated for dissemination via B lymphocytes, which is how JCV is believed
to cross the bloodbrain barrier and reach the central nervous system. The kidney
is considered a key site of persistence for BKV and JCV, and these viruses are
consistently associated with viruria, especially upon reactivation. Another site of
persistent infection for BKV is the urinary tract. BKV and JCV can also be detected
in blood.
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LT Induces Cell Cycle Progression from G0/G1 S Phase

The polyomaviruses have very limited coding capacity that they have exploited
maximally by utilization of alternative splicing. Since these viruses depend extensively on the cellular replication machinery and nucleotide pool, they must
reprogram the host environment to support viral DNA replication by inducing cell
cycle progression from quiescence into S phase (Hatanaka and Dulbecco 1966;
Dickmanns et al. 1994). This is especially important, since viruses like SV40 are
believed to normally infect terminally differentiated kidney epithelial cells that are
withdrawn from the cell cycle. This bypass of normal proliferation control mechanisms associated with unscheduled DNA synthesis in a viral replication setting,
when uncoupled from a complete lytic cycle, sends a cell on the path to malignancy
(Dickmanns et al. 1994). Accordingly, activities of LT required for promotion of
cell cycle progression are overlapping or identical with those required for oncogenic
transformation. The LT mitogenic effect is believed, at a minimum, to be dependent
on binding of negative growth control elements, such as pRB family members and
p53. The DnaJ domain and an uncharacterized function in the origin-binding domain
(OBD) also appear to contribute to efficient induction of cell cycle progression
(Dickmanns et al. 1994).
Finally, based on lessons from the large DNA tumor viruses (EpsteinBarr virus
and Kaposis sarcoma-associated herpesvirus), it should be emphasized that targeting
of pRB and p53 to drive cell cycle progression may not be the only rationale from
the virus point of view. Evidence indicates that tumor suppressors like p53 play
key roles in innate immunity; for example, interferons a and b are known to boost
the p53 response (Moore and Chang 2003; Takaoka et al. 2003). Thus, it remains
possible that polyomaviruses also target these tumor-suppressor proteins to evade
antiviral signaling through innate immunity and as an unfortunate by-product,
breaks down our antitumor defenses. Currently, there is little known about the
polyomaviruses and interferon signaling.
Non-neoplastic Disease Association

BKV and JCV are linked to nonmalignant human disease with high morbidity and
mortality. BKV is, mainly in bone marrow transplants, associated with hemorrhagic
cystitis, which is an inflammation of the bladder, and also polyomavirus-associated
nephropathy, which is a primary cause of kidney transplant rejection (Imperiale
2000; Jiang et al. 2009). It is believed to be immune suppression that triggers
reactivation. PML is a fatal neurodegenerative disease caused when JCV, upon
reactivation, destroys the oligodendrocytes, which are the cells that produce myelin
(Imperiale 2000; Maginnis and Atwood 2009; Khalili et al. 2006; Tan and Koralnik
2010; Major et al. 1992). PML is predominant in patients with a compromised
immune system, for example AIDS patients, of which around 5% develop PML.
Recent attention to PML and JCV has come from the observation that treatment
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O. Gjoerup
with the multiple sclerosis drug Natalizumab apparently on rare occasion can cause
JCV reactivation and PML in patients (Kleinschmidt-DeMasters and Tyler 2005;
Langer-Gould et al. 2005). Natalizumab is a monoclonal antibody against a4
integrin. It may facilitate JCV reactivation and PML by blocking migration of
leukocytes across the bloodbrain barrier to the site of reactivation.
Assays for Transformation

There are multiple assays by which the transformed phenotype can be scored. Each
of these assays measures different, although often related, parameters. For each
assay, the general principle is that one measures the ability to overcome normal
restraints to cell proliferation, an ability, which is shared by most tumor cells. As is
discussed below, these restraints can be in the form of a limited life span, absence
of or limiting growth factors, and anchorage dependence or contact inhibition.
These are all restrictions that are believed to be imposed on nascent tumor cells
in vivo, and that they most overcome to further develop. There are additional properties relevant for a malignancy, but these are not easily measured outside of an
animal, for example invasiveness, metastasis, and impact of the immune system.
Immortalization
Primary cells grown in culture have a finite life span. After a certain number of
passages, cells become permanently growth arrested, also referred to as senescent.
However, both SV40 and MPyV LT have been demonstrated to efficiently immortalize
rodent cells in culture, meaning that the cells can be serially passaged indefinitely.
SV40 LT appears to require both its pRB and p53 binding sites for immortalization,
whereas MPyV LT immortalizes via binding of pRB family members (Tevethia
et al. 1998; Larose et al. 1991). Immortalization is generally considered to be the
first step toward tumorigenesis, although there may be instances where it is not a
prerequisite (Freund et al. 1992). Human cells are fundamentally different with
regard to cell immortalization perhaps in part because their life span in culture is
much longer than that of rodent cells. Whereas rodent cells can spontaneously
immortalize at some low frequency, this does not occur with human cells.
Furthermore, human cells do not have constitutive telomerase activity, which means
that every time cells divide, their telomeres shorten, unless the telomerase gene
(hTert) is turned on. At a certain point, the telomeres are shortened beyond a threshold,
and cell death occurs by a cellular process known as crisis. In most cell types,
SV40 LT does not upregulate telomerase expression. Accordingly, LT does not
immortalize human cells, but it does extend their life span (Neufeld et al. 1987).
After the extended life span, most cells undergo crisis, but extremely rarely clones
grow out that are immortal. Conversely, LT cooperates with hTert to efficiently
immortalize human cells (Counter et al. 1998; Zhu et al. 1999).
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Transformation
Besides immortalization, several assays that measure the transformed phenotype
are relevant for in vivo tumorigenesis. While normal cells require growth factors
that are present in serum for their proliferation, transformed cells can often grow
under low or no serum conditions, making this one important assay. Also, normal
cells are contact inhibited, meaning that cell proliferation is arrested when a confluent
monolayer is formed. However, transformed cells are able to overcome these restrictions, thus allowing dense foci of cells to grow out on top of the monolayer. The
focus formation assay, often performed in rodent cells, has been instrumental for the
genetic analysis of functions needed for T antigen-mediated transformation.
However, this assay is predominantly carried out with fibroblast, not epithelial,
cells, reflecting some intrinsic limitations. A related assay to focus formation is to
determine saturation density by establishing growth curves. Because transformed
cells are not contact inhibited, they continue proliferating on top of a packed
monolayer and thus reach higher saturation densities than normal cells. Another
important transformation assay relates to loss of anchorage dependence and is
referred to as the soft agar assay. Most normal cells need to adhere to a solid surface
of a culture vessel, and if they are suspended in semisolid medium like agar, they
will growth arrest but remain viable for weeks. However, many transformed cells
have acquired anchorage-independent growth. This assay is considered a more
stringent assay for transformation than focus formation and frequently correlates
well with tumor induction in animals. While focus formation assays are often
performed by direct transfection of T antigen expression vectors, the soft agar assay
most often employs cell lines that stably express T antigen(s). The best correlate for
tumorigenesis is not surprisingly considered to be a direct analysis of tumor formation
in susceptible animals.
SV40 Large T Antigen

Since SV40 LT is the most highly studied of the polyomavirus LTs, it is introduced
first as the prototype, followed by other family members. SV40 LT is a truly remarkable, highly multifunctional nuclear protein living a double life as both an initiator
of viral replication and an oncogene. Several reviews on LT have been previously
published (Ahuja et al. 2005; Cheng et al. 2009; Fanning and Knippers 1992; Pipas
1992, 2009; Manfredi and Prives 1994; Gjoerup and Chang 2010). The oncogenic
activities are a by-product of LT stimulation of S-phase entry in order to prepare
cells for viral replication and of blocking antiviral signaling through innate immunity. Replication and transformation activities of LT were shown early on to be
genetically separable from each other. Most of the functions of LT are mediated by
interaction with critical host proteins. Strikingly, most polyomaviral LT proteins
have converged on a small number of common targets (Hsc70, pRB family, p53,
and possibly p300/ CBP) that are discussed.
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O. Gjoerup
Fig. 14.4 Schematic diagram of SV40 LT. The main domains and binding sites contained in LT
are illustrated. The DnaJ domain at the N-terminus mediates binding of the Hsc70 chaperone
dependent on the canonical sequence HPDK. Downstream of the DnaJ domain, LT contains
separate binding sites for Cul7, Bub1, and IRS1. Pocket proteins (pRB, p107, p130) bind to LT
dependent on an LxCxE consensus sequence. The nuclear localization signal (NLS) is a requirement for nuclear translocation of LT. The OBD mediates not just binding to the viral origin of
replication, but also binding of a number of factors, including Nbs1, RPA, and components of the
transcriptional machinery. LT has a C2H2 Zn2+ binding element important for hexamer assembly.
The C-terminus consists of an AAA+ type ATP-dependent helicase (ATP outlines the minimal
ATPase domain), which is interspersed with a bipartite binding site for p53 that in turn bridges
binding of p300/CBP coactivators. At the extreme C-terminus, LT contains a host range (HR)
function and binding site for the F-box protein FBW7
LT is highly modular in structure, with discrete regions corresponding to the

various functions (see Fig. 14.4 for a diagram of LT with domains and major binding
proteins). At its N-terminus, within the first 70 amino acids, LT contains a DnaJ
domain, which serves to recruit chaperones like Hsc70 (Kelley and Landry 1994;
Campbell et al. 1997; Srinivasan et al. 1997). The consensus motif found in all DnaJ
domains is HPDK, which is conserved in nearly all polyomavirus LT and ST
proteins. LT binding to Hsc70 leads to stimulation of its ATPase activity, which is in
turn used to act on a chaperone substrate (Sullivan and Pipas 2002). For SV40 LT,
it is known that the DnaJ domain is required for disruption of repressive complexes
between p107/p130 pocket proteins and E2F family members, as well as p130 degradation (Srinivasan et al. 1997; Stubdal et al. 1996, 1997; Zalvide et al. 1998). The
DnaJ domain must be present in cis with the pRB family binding site. DnaJ mutants,
depending on the nature of the mutation, exhibit variable defects in different assays
for transformation. A role of the DnaJ domain in viral replication and virion production
has also been reported (Campbell et al. 1997; Spence and Pipas 1994).
At the N-terminus, following the DnaJ domain, LT contains binding sites in close
proximity for the Cul7 member of the SCF (skp1, cullin, F-Box)-type ubiquitin
ligase complex as well as the Bub1 mitotic checkpoint kinase (Kohrman and
Imperiale 1992; Ali et al. 2004; Cotsiki et al. 2004). Cul7 binds LT dependent on
residues 6983, and its binding has been linked to SV40 transformation of rodent
cells (Kasper et al. 2005). Bub1 requires amino acids 8997 to bind LT, which has
been shown to be important for rat-1 focus formation (Cotsiki et al. 2004). This
region of LT contains the sequence motif WExWW, which is highly conserved in
a subset of the polyomaviruses. LT binding to Bub1 furthermore elicits tetraploidy
and a DNA damage response (Hein et al. 2009). Bub1 is a prime candidate for LTs
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ability to cause chromosomal instability. For both Cul7 and Bub1, it is unclear
whether binding occurs with other polyomaviral LTs.
Downstream of the Cul7/Bub1 binding sites, the region from residues 101115,
and in particular the LxCxE motif contained within, is required for interaction
with pRB family members. The founding member of this family is the retinoblastoma protein, pRB, which is a key cellular tumor suppressor mutated in the pediatric
tumor of the same name. Adenovirus E1A was first shown to bind pRB (Whyte
et al. 1988), quickly followed by SV40 LT (DeCaprio et al. 1988). Now we know
that every polyomavirus LT is likely to bind pRB family members, given the absolute
conservation of the LxCxE motif. LT binding to the pRB family is linked to induction
of S-phase entry, which is likely why it is being targeted. For nearly all transformation
in vitro, as well as tumor induction in transgenic models, the pRB binding site is
essential (DeCaprio et al. 1988; Kalderon and Smith 1984; Chen and Paucha 1990;
Manfredi and Prives 1994).
Structurally, pRB is composed of two domains, A and B, that together form a
pocket conformation critical for tumor suppression (Burkhart and Sage 2008), hence
the name pocket proteins. There are two other family members, p107 and p130.
These share structural and functional properties but have not been shown to be bona
fide tumor suppressors like pRB. They are nevertheless very important for cell cycle
regulation. The ability of LT to transform depends not just on overcoming pRB, but
also on p107/p130 inactivation (Sullivan et al. 2000; Stubdal et al. 1997; Zalvide
et al. 1998). The primary function of the pRB family is to associate with and repress
E2F family members, which are important transcription factors (Morris and Dyson
2001). LT, by binding pRB family members, allows derepression of E2F target
genes (reviewed in DeCaprio 2009). The pRB family/E2F circuit regulates a broad
range of cellular target genes involved in cell cycle (DNA replication, mitosis),
DNA repair, development, and apoptosis. Normally, pRB family members are regulated by cyclin-dependent kinases that phosphorylate them in a cell cycle-dependent
manner, thus causing release from E2F at the appropriate phase of the cell cycle.
LT also binds to insulin receptor substrate 1 (IRS1), with the binding site overlapping the LxCxE (Fei et al. 1995; Yu and Alwine 2008). IRS1 binding studies
were prompted by initial observations that LT cannot transform insulin-like growth
factor I receptor (IGF-IR)-deficient cells (DAmbrosio et al. 1995; DeAngelis et al.
2006). IRS1 is a major downstream target of IGF-IR. LT causes relocalization of
IRS1 from the cytoplasm into the nucleus. Binding of IRS1 appears to be associated
with Akt activation and protection from apoptosis (Yu and Alwine 2008).
Immediately downstream of the LxCxE is located a classic nuclear localization
signal, the first one to be studied closely (Kalderon et al. 1984; Lanford and Butel
1984), and following it, the OBD, which resides in the next 130 amino acids.
The OBD is by itself highly multifunctional with roles in transcriptional activation,
an uncharacterized activity contributing to transformation (Dickmanns et al. 1994;
Kalderon and Smith 1984), binding sites for replication protein A (RPA) and Nbs1
(Weisshart et al. 1998; Wu et al. 2004), besides the major role in site-specific
DNA binding. The central part of LT contains a Zn2+ coordination element, which is
required for hexamer assembly (Loeber et al. 1991). This element is highly conserved
between polyomaviruses.
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The C-terminus, spanning approximately residues 350650, contains the

ATP-dependent helicase region interspersed with a bipartite p53 binding site (Kierstead
and Tevethia 1993). LT possesses key activities required for initiation of viral
replication (reviewed in Fanning and Knippers 1992; Simmons 2000). It assembles
as a single or double hexameric complex at the viral origin by specific binding to
GAGGC repeats contained within a palindrome. The ATP-dependent helicase
activity is then used to unwind the origin DNA, and subsequently LT recruits replication enzymes like polymerase a/primase, RPA, and topoisomerase I to initiate
viral DNA replication.
The p53 protein was first discovered as a LT binding protein in 1979 (Lane and
Crawford 1979; Linzer and Levine 1979). Soon, it was realized that the majority of
viral proteins target it for inactivation. p53 is a transcription factor that can turn on
the expression of genes involved in either cell cycle arrest or apoptosis (Levine and
Oren 2009; Vousden and Prives 2009). It is a master regulator that responds to genotoxic
stress of various kinds like DNA damage or oncogenic stress. A role in the interferon
response indicates contributions to innate immunity as well (Takaoka et al. 2003).
While initially perceived as an oncogene because mutant forms of the gene had
been studied, it is now clear that p53 is instead a tumor suppressor (Baker et al.
1990; Finlay et al. 1989; Eliyahu et al. 1989; Malkin et al. 1990; Donehower et al. 1992).
It has been dubbed guardian of the genome (Lane 1992). Strikingly, it is the most
mutated gene in human cancer, with greater than 50% of all tumors harboring p53
mutation or loss (Hollstein et al. 1994). LT binds the site-specific DNA-binding
domain of p53 and prevents it from contacting DNA, thereby blocking target gene
activation (Bargonetti et al. 1992; Jiang et al. 1993; Segawa et al. 1993; Mietz et al.
1992). It is generally believed that LT binding to pRB family members triggers
unscheduled DNA synthesis, which is sensed by the cell. To prevent an apoptotic
response to this, p53 must be inactivated. The binding of p53 is required for immortalization of rodent cells but not always strictly required for oncogenic transformation (Kierstead and Tevethia 1993; Tevethia et al. 1998; Zhu et al. 1991; Srinivasan
et al. 1997). Interestingly, and somewhat paradoxically, LT not only binds and
inactivates p53 but also potently stabilizes it (Oren et al. 1981; Reich et al. 1983).
Accumulating evidence indicates that the stabilized form of p53 has acquired a gain
of function in transformation possibly because LT in complex with p53 turns on a
unique set of genes (Deppert et al. 1989; Hermannstadter et al. 2009; Tiemann and
Deppert 1994a, b; Bocchetta et al. 2008). Several naturally occurring mutants of
p53 also exhibit a gain of function in transformation (Dittmer et al. 1993; Cadwell
and Zambetti 2001), making studies of the LT-induced gain of function relevant for
these nonviral oncogenic mechanisms.
As it was first shown for adenovirus E1A, LT also binds the transcriptional
coactivators/histone acetyltransferases p300 and CBP (Eckner et al. 1996). These
large scaffolds are considered potential tumor suppressors, function in a variety of
different biological processes, and are targeted by many viral oncoproteins (Gayther
et al. 2000; Goodman and Smolik 2000; Iyer et al. 2004). Binding is mainly
indirect, bridged by p53, but a direct contact between LT and p300/CBP has also
been demonstrated (Lill et al. 1997; Borger and DeCaprio 2006; Ahuja et al. 2009).
351
This mutant, defective in p300/CBP binding, is also defective in transformation

(Ahuja et al. 2009). Binding of p300/CBP has been suggested to underlie the p53
gain of function because LT effectively gains access to these via p53 (Borger and
DeCaprio 2006; Hermannstadter et al. 2009). Like E1A, LT also binds the p400
protein (Lill et al. 1997), which is a member of the SWI2/SNF2 family of chromatinremodeling factors. While for E1A this is required for transformation (Fuchs et al.
2001), in the case of LT this has not been elucidated.
The extreme C-terminus of LT contains two separate functions. The host range
function is conserved only among SV40, BKV, and JCV and allows SV40 to grow
in certain cell lines like CV-1 but is not required for growth in others like BSC40
(Pipas 1985). Host range function is likely mediated by binding to a cellular protein,
which has yet to be identified (Poulin and DeCaprio 2006b). The extreme C-terminus
also binds the F-box protein FBW7 via a phosphodegron that mimics the consensus
normally found in FBW7 substrates (Welcker and Clurman 2005). FBW7 is the
substrate-recognizing component of SCF complexes that target cellular proteins
for proteasomal degradation. Although LT was shown to mislocalize FBW7 from
nucleoli to the nucleoplasm and slightly increased cyclin E levels, this region of LT
is not required for transformation in the context of full-length LT. Therefore, its
function remains unknown.
Non-SV40 LT Proteins
While SV40 LT is a versatile transforming protein, which can potently induce focus
formation in cultured cells and a wide variety of tumors when expressed transgenically, other polyomavirus LT proteins appear less potent. MPyV LT, because it
strikingly lacks p53 binding, has no independent transforming activity, but it does
immortalize primary rodent cells dependent on pRB family binding (Larose et al.
1991). MPyV LT is a replication initiator with DnaJ domain, site-specific DNA
binding, and ATP-dependent helicase activities similar to SV40. MPyV LT has an
insert sequence of ~120 amino acids between the end of first exon and the beginning
of the OBD, which corresponds to the coding sequence for MT in a different reading
frame (Pipas 1992). Even though it does not bind p53, it appears to have evolved an
independent binding mode for p300/CBP (Cho et al. 2001).
BKV, and JCV in particular, have lower transformation potential in vitro than
SV40 (Abend et al. 2009a; Harris et al. 1996; Frisque et al. 2006). This is in part due
to the restricted tropism causing lower LT expression in many cultured cells, but
also because of reduced binding affinities for pRB and p53 (Bollag et al. 1989;
Harris et al. 1996, 1998b). For BKV, it has been demonstrated that there is not
enough LT to sequester pRB and the DnaJ domain plays a major role in pRB family
inactivation (Harris et al. 1996, 1998b). BKV early region can immortalize and
transform embryonic fibroblasts from mouse, rat, and hamster. Transformation of
human embryonic kidney cells is not efficient and often abortive, but coexpression
with c-myc or oncogenic Ras leads to full transformation.
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O. Gjoerup
JCV can transform human fetal glial cells and also primary hamster brain cells,
but the efficiency is not high (Frisque et al. 2006). Mainly JCV transforms cells of
neural origin. This is because the NCRR directs glial-specific transcription of the
early region. The host range is mainly restricted by the NCRR because the promoter/
enhancer region within the NCRR contains binding sites for transcription factors
that are expressed in a tissue-specific manner. The promoter/enhancer region is
known to undergo numerous rearrangements in the form of mutations, deletions,
and duplications. These rearrangements are more frequent in disease states, but their
exact relationship with pathogenesis and how they are generated remain unresolved
questions (Newman and Frisque 1999). JCV LT, apparently uniquely, binds to
b-catenin (Gan and Khalili 2004), a key protein in the Wnt pathway, and the putative
tumor-suppressor neurofibromatosis type 2 (NF2) (Shollar et al. 2004), which might
contribute to its tumor induction potential.
At present, there are no reported in vitro transformation systems for KIV, WUV,
or MCV.
MPyV Middle T Antigen

MT is uniquely encoded only by MPyV and hamster polyomavirus. MT is required
for an efficient MPyV life cycle, which reflects contributions at several levels
(reviewed in Fluck and Schaffhausen 2009). It is required for VP1 phosphorylation
and capsid assembly, but also plays a key role in regulation of viral transcription and
replication. The latter effects are mediated by MT activation of AP1 and PEA3
(ETS family) binding sites within the MPyV enhancer (Chen et al. 1995). MPyV
differs from SV40 in its absolute dependence on the enhancer for viral replication
(de Villiers et al. 1984), whereas for SV40 the core origin suffices. Presumably, the
MPyV enhancer allows for the broad tissue tropism of this virus.
MT is always required for MPyV transformation and often sufficient (reviewed
in Fluck and Schaffhausen 2009; Schaffhausen and Roberts 2009; Ichaso and
Dilworth 2001). Its transformation mechanism is fundamentally different from that
of other T antigens. Notably, MT is a potent transforming protein in established cell
lines. However, transformation of primary cells requires cooperation with LT or ST
(Rassoulzadegan et al. 1982, 1983; Asselin et al. 1983, 1984; Lomax and Fried
2001). Efficient transformation of human cells in cooperation with a dominant
negative p53 has also been demonstrated (Utermark et al. 2007).
Due to a hydrophobic sequence at its C-terminus, MT is membrane-localized
(Ito 1979). This includes localization both at the plasma membrane as well as internal
membranes and cytoskeletal elements. Membrane localization is essential for its
transforming activity (Carmichael et al. 1982). MT has often been envisioned to
mimic a constitutively active growth factor receptor (like Her2/Neu) because it acts
by assembling signaling molecules that activate downstream cytoplasmic signaling
cascades. A schematic drawing of MT is outlined in Fig. 14.5.
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Fig. 14.5 Schematic drawing of MPyV MT. The N-terminus contains a DnaJ domain wherein lies
determinants also for binding of TAZ. MT may be viewed as a mimic of a constitutively active
growth factor receptor, membrane-localized through its C-terminal hydrophobic anchor sequence.
MT binds to PP2A and uses it to build a scaffold leading to recruitment and activation of PTKs like
Src, Fyn, and Yes that in turn phosphorylate MT on the major sites Y250, Y315, and Y322. Each
of these phosphorylation site acts as a docking platform for a major signal generator. Y250 signals
through binding of the Shc adaptor protein to mediate activation of the Ras/MAPK pathway. Y315
recruits the p85 subunit of PI3K to activate downstream signaling through Akt and Rac. Y322 is
important for recruitment of PLCg1, which generates diacylglycerol (DAG) and inositol trisphosphate (IP3) that in turn can release Ca2+ and activate protein kinase C (PKC). Shown are also
the S257 phosphorylation site involved in 14-3-3 recruitment and the proline-rich region (PPP)
implicated in transformation via an unknown mechanism
MT contains a DnaJ domain at its N-terminus whose function per se has not been
linked with transformation (Campbell et al. 1995); however, it is required for overall folding of the protein and binding of more C-terminal interactors. The extreme
N-terminus (amino acids 24) has been found to bind a protein called TAZ, which
is playing a role in transcriptional regulation and regulated protein degradation
through the b-TrCP E3 ubiquitin ligase complex (Tian et al. 2004). Binding of
TAZ appears to be important for transformation, but since the mutation affects all
three T antigens, it remains uncertain wherein the defect lies. Similarly to ST, MT
also binds to PP2A (Pallas et al. 1990), but the signaling outcome is different
because of MT localization at the membrane (Mullane et al. 1998). Binding of MT
to PP2A is absolutely required to build a scaffold for recruitment of protein tyrosine
kinases (PTKs) like c-Src, c-Fyn, and c-Yes (Courtneidge and Smith 1983), reviewed
in (Fluck and Schaffhausen 2009). C-Src is the cellular counterpart of the retroviral
oncogene v-Src. Importantly, tyrosine phosphorylation was first discovered as a
major signaling mechanism by studying MT immunoprecipitates (Eckhart et al.
1979). How PP2A mechanistically contributes to PTK recruitment is not clear and
further confounded by the observation that catalytic activity of PP2A is not required
(Ogris et al. 1999). Moreover, it remains unclear if PP2A and PTKs act on targets
outside the MT complex.
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O. Gjoerup
The recruitment of PTKs is critical to MT transformation. MT binding leads to

the activation of PTK activity, and in turn PTKs phosphorylate the major sites Y250,
Y315, and Y322 on MT. Each of these tyrosine phosphorylation site constitutes a
docking site for a major signaling molecule. Y250 is part of the sequence NPTY,
which is required for binding to the PTB domain of the adaptor molecule Shc
(Dilworth et al. 1994; Campbell et al. 1994). Shc can signal to another adaptor
molecule, Grb2, which in turn can associate with and activate the Ras guanine
nucleotide exchange factor Sos. Sos activation leads to Ras activation and activation of the downstream MAPK signaling cascade. Grb2 can also, alternatively,
signal to the Gab1 docking molecule, which is linked to PI3K signaling. The Y250
site can have a dramatic effect on MT transformation in murine cells, but in other
contexts like human cell transformation assays, there is no effect (Utermark et al.
2007). This reflects the importance of different signaling circuits required for transformation in different cell systems.
The Y315 site connects MT with the important signaling molecule, PI3K
(Whitman et al. 1985; Kaplan et al. 1986). In fact, PI3K was first discovered in
studies of MT immunoprecipitates (Whitman et al. 1985). PI3K consists of a regulatory p85 subunit and a catalytic p110 subunit. Y315 is part of a YMPM motif,
which binds to the SH2 domains of p85. Thus, MT recruits PI3K to the membrane,
which stimulates its activity. PI3K causes production of PIP3 and the membrane
recruitment of proteins with specific pleckstrin homology domains. Downstream
targets of PI3K include PDK1, Akt, and Rac. Akt is a kinase that protects against
cell death and contributes to transformation. Rac is a small GTP-binding protein
involved in cytoskeletal organization, which also contributes to MT transformation
(Urich et al. 1997).
The Y315F mutant exhibits a striking defect in many, but not all, cell transformation and tumor induction systems. In human cells, MT transformation is abrogated
(Utermark et al. 2007). Also, it was demonstrated that knockout of p110a abrogates
MT transformation of mouse embryo fibroblasts, proving that it is essential for MT
oncogenesis (Utermark et al. 2007). The discovery of PI3K through its association
with MT is especially significant given the recent findings of frequent activating
p110a mutations in human cancers of breast, colon, prostate, and liver (Samuels
et al. 2004). PI3K inhibitors are in clinical trials and may provide an efficient therapeutic for certain cancers (Wong et al. 2010; Liu et al. 2009).
The Y322 site, part of the sequence YLDI, is required for binding of PLCg1 (Su
et al. 1995). The effects of signaling through PLCg1 have been more difficult to
discern than through Shc and PI3K. A Y322F mutant is not defective under normal
growth conditions, but does exhibit a defect in transformation, when the assay is
conducted under reduced serum conditions (Su et al. 1995). Consistent with binding
of PLCg1, an increase in inositol trisphosphate can be observed. This can potentially lead to protein kinase C (PKC) activation and release of Ca2+.
Two other regions of MT are of interest. The S257 phosphorylation site is implicated
in recruitment of 14-3-3 proteins (Cullere et al. 1998). While the S257 mutant does
not show a phenotype in cell culture assays for transformation, there is a striking
deficiency of this mutant virus to specifically induce salivary gland tumors (Cullere
et al. 1998). The mechanism involved is not known. Furthermore, a proline-rich
355
region between amino acids 332347 plays an unknown role in transformation

(reviewed in Fluck and Schaffhausen 2009; Schaffhausen and Roberts 2009). It
appears that all the known binding proteins are recruited normally, yet mutants in
this region fail to transform. Finally, it should be emphasized that MT transgenic
models, especially the MMTV-MT mammary carcinoma model (Guy et al. 1992),
continues to inform us about metastasis, the role of stromalepithelial interactions
for tumor progression, and the contribution of different genetic backgrounds for
carcinogenesis.
Small T Antigen
ST is not strictly required for the viral life cycle but can stimulate viral replication
to various extents depending on the polyomavirus (Sleigh et al. 1978; Cicala et al.
1994; Berger and Wintersberger 1986; Kwun et al. 2009). While ST is not sufficient
to transform cells, it can cooperate with LT for transformation of certain cell types,
where LT is not sufficient, notably human cells (de Ronde et al. 1989; Chang et al.
1985; Hahn et al. 1999; Porras et al. 1996; Yu et al. 2001). ST expression by itself
does allow cells to grow to a higher saturation density (Cherington et al. 1986; Noda
et al. 1986). ST can also cooperate with LT to allow rodent cell lines to grow
independent of anchorage (Bouck et al. 1978; Sleigh et al. 1978; Jog et al. 1990;
Mungre et al. 1994; Bikel et al. 1986). Much emphasis has centered on the remarkable demonstration that human cells can be fully transformed by defined genetic
elements, such as combinations of LT, ST, hTert, and oncogenic H-Ras (Hahn et al.
1999). These observations have definitively highlighted a critical function of ST in
oncogenesis. The function of the ST DnaJ domain has not been elucidated (Boyapati
et al. 2003). ST is localized in both the cytoplasm and the nucleus. The unique
region contains two CxCxxC clusters that coordinate Zn2+ and confer conformational
stability (Turk et al. 1993). These are highly conserved in most of the polyomaviruses, with the exception of the avian (Pipas 1992).
Virtually, all of the known functions of ST, including its contributions to transformation, can be attributed to its binding of the important cellular serinethreonine
phosphatase PP2A (Rundell and Parakati 2001; Sablina and Hahn 2008; Skoczylas
et al. 2004). PP2A is a heterotrimeric enzyme composed of a scaffold subunit (A),
a regulatory subunit (B), and a catalytic subunit (C). It is a diverse class of enzymes,
since there are 2 different A subunits, 2 C subunits, and 17 B subunits (reviewed
in Sablina and Hahn 2008). While it is generally believed that ST binds the AC
holoenzyme and displaces or prevents the binding of B subunits, thus causing overall
inactivation of PP2A activity (Pallas et al. 1990; Yang et al. 1991), there are also
some examples of substrates, where ST promotes their dephosphorylation (Yang
et al. 1991, 2007; Yu et al. 2005). Interestingly, in human transformation assays, the
effect of ST can be partially mimicked by specific knockdown of the B56g subunit,
highlighting its importance (Chen et al. 2004). Significantly, mutations in PP2A subunits
have been discovered in certain human cancers, consistent with a tumor-suppressor
function (Sablina and Hahn 2008; Arroyo and Hahn 2005).
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O. Gjoerup
The binding of ST to PP2A leads to a panoply of outcomes associated with

increased proliferation and disruption of the actin cytoskeleton (reviewed in Rundell
and Parakati 2001; Sablina and Hahn 2008; Skoczylas et al. 2004). Some of the
important downstream targets are kinases like Akt, MAPK, and PKCz (Yuan et al.
2002; Rodriguez-Viciana et al. 2006; Sontag et al. 1993, 1997), as well as stabilization
of the proto-oncogene c-myc (Yeh et al. 2004). There is also clear promotion of the
G1-S cell cycle transition likely mediated through activation of the cyclin A and
cyclin D1 promoters combined with the degradation of the cdk inhibitor p27kip1
(Sontag et al. 1993; Howe et al. 1998; Porras et al. 1999; Skoczylas et al. 2005;
Watanabe et al. 1996). ST is a somewhat promiscuous transcriptional activator
(Loeken et al. 1988). Microarray experiments with SV40 ST indicate that it activates
some genes in a non-PP2A-dependent manner, but the exact nature of other functions
is not known (Moreno et al. 2004).
Although polyomaviral ST proteins other than SV40 are not as well-characterized,
the general theme of PP2A binding appears to hold true. MPyV ST also induces
proliferation alone (Mullane et al. 1998) or together with LT (Ogris et al. 1992),
and it can cooperate with MT for transformation or tumor induction (Asselin et al.
1983, 1986). The complementation of MT for transformation of REF52 or primary
cells by ST has been reported to reflect a disconnection of p19ARF, which MT induces,
from p53 induction (Lomax and Fried 2001). The exact mechanism involved is
unclear, but ST must bind PP2A to overcome p19ARF signaling to p53 (Moule et al.
2004). Whether this mechanism operates with other ST proteins remains to be
shown. MPyV ST can also induce cell death in growing cells but protect against
apoptosis when cells are starved, in both cases dependent on PP2A binding (Andrabi
et al. 2007). Toggling between these two opposing outcomes likely has to do with
Akt phosphorylation at its two activating sites T308 and S473, which is in turn regulating downstream substrate selection.
Both JCV and MCV ST also bind PP2A and stimulate viral DNA replication
(Bollag et al. 2010; Kwun et al. 2009). Surprisingly, it was recently shown that JCV
ST has 2 LxCxE motifs and is capable of binding members of the pRB family
(Bollag et al. 2010). Little is known about BKV ST, except that it appears to bind
PP2A (Rundell et al. 1981).
Polyomavirus-Induced Tumors in Animals

Polyomavirus tumor induction in animals can be determined in either of the three
different ways: inoculation of virus into sites of a susceptible animal, transgenic
expression of LT, or the whole early region, with a tissue-specific promoter or
injection of cells expressing the T antigens into nude mice to produce xenografts.
Early studies clearly demonstrated the ability of SV40 to induce tumors in newborn
hamsters. Different tumors are produced dependent on the route of inoculation.
Intracranial injection of SV40 resulted in ependymomas (Kirschstein and Gerber
1962), whereas intravenous injection of hamsters yielded leukemias, lymphomas,
357
and sarcomas (Diamandopoulos 1972). Intrapleural injection produced mesothelial

tumors in weanling hamsters (Cicala et al. 1993).
Transgenic expression of SV40 in mice using its natural promoter potently
induces choroid plexus tumors of the brain epithelium (Brinster et al. 1984). Interestingly,
transgenic expression of LT can induce a spectrum of different responses ranging
from no stimulation of proliferation, hyperplasia, dysplasia, and carcinoma to fullblown invasive or metastatic phenotypes. A wide variety of other tumors have been
induced by transgenic expression of SV40 LT alone, or in combination with ST,
when driven by various tissue-specific promoters (reviewed in Ahuja et al. 2005;
Saenz Robles and Pipas 2009). Transgenic model systems have been developed for
intestine, eye, pancreas, prostate, salivary gland, stomach, lung, and liver in addition
to brain to study SV40-induced tumorigenesis. Tumorigenesis invariably depends
on binding of pRB family members, whereas the requirement for p53 binding is
highly variable. Frequently, expression of a truncated N-terminal fragment of LT
(1136 or 1121), which carries the DnaJ and pRB binding sequences, is sufficient
to trigger tumorigenesis (Chen et al. 1992; Symonds et al. 1994; Bennoun et al.
1998; Rathi et al. 2007). Collectively, these model systems have provided extraordinary insight to the development of cell type-specific tumors, and together these
studies have underscored the importance of cell context for tumor progression.
BKV and, especially, JCV have also been shown to be highly oncogenic in animals
(reviewed in White et al. 2005). JCV induces brain tumors in Golden Syrian hamsters,
but astrocytomas, glioblastomas, and neuroblastomas in owl and squirrel monkeys.
Monkeys are nonpermissive for JCV, and the virus is integrated in the tumors. JCV
has also been reported to induce neuroectodermal tumors in hamsters and rats.
Conversely, transgenic expression of JCV early region results in adrenal neuroblastomas, neuroectodermal tumors, as well as tumors originating from the cerebellum
and pituitary neoplasia. Interestingly, dysmyelination of the central nervous system
was also observed, reminiscent of PML. JCV is suspected of playing a role in human
medulloblastomas, glioblastomas, and astrocytomas. Linking BKV and JCV to human
cancer is difficult since they are ubiquitous in the human population and most of the
proposed neoplasias, where a causal link might exist, are not characterized by integration of the viral genome (Abend et al. 2009a; Maginnis and Atwood 2009).
BKV is oncogenic in young or newborn rodents, but the efficiency of tumorigenesis depends on inoculation route (White et al. 2005). Transgenic expression of
the BKV early region also results in tumors, but the spectrum is different from JCV.
Mainly hepatocellular carcinomas, renal adenocarcinomas, thymomas, or lymphomas are observed with BKV.
Genome Destabilization
Much accumulated evidence indicates that LT can induce genomic instability manifested in both structural and numerical chromosome aberrations (Ray et al. 1990, 1992,
1998; Ray and Kraemer 1993; Woods et al. 1994; Stewart and Bacchetti 1991;
358
O. Gjoerup
Friedrich et al. 1992; Chang et al. 1997; Schaefer et al. 1993; Levine et al. 1991;
Sargent et al. 1997; Ewald et al. 1996). This has been demonstrated to occur rapidly
after LT expression, thus preceding the transformed phenotype and excluding it as
a secondary effect of transformation (Chang et al. 1997; Ray et al. 1990). Most of
these experiments have been performed with SV40 LT, but limited studies of BKV
and JCV LT suggest that they exhibit similar characteristics with regard to induction
of chromosome instability (Neel et al. 1996; Darbinyan et al. 2007; Trabanelli et al.
1998; Theile and Grabowski 1990; Ricciardiello et al. 2003). Cytogenetic analysis
indicates that LT-expressing rodent or human cells often are tetraploid or aneuploid
and harbor structural alterations, such as chromosome breaks, fragments, rings,
double minutes, translocations, as well as frequent dicentric chromosomes, perhaps
arising from telomeric fusion (Chang et al. 1997; Woods et al. 1994; Stewart and
Bacchetti 1991; Ray et al. 1992). Furthermore, LT is mutagenic (Theile and
Grabowski 1990), recombinogenic (St-Onge et al. 1990; Xia et al. 1997), and causes
sister chromatid exchanges (Nichols et al. 1978), along with frequent micronuclei
formation (Hein et al. 2009).
Interestingly, expression of the first 147 amino acids of LT appears to induce
genome destabilization with wild-type efficiency (Woods et al. 1994). Moreover,
binding to pocket proteins was found not to be essential for the phenotype. It was
reported that interference with mitotic checkpoints might contribute toward the
induced genomic instability (Chang et al. 1997). While the exact molecular mechanism underlying genome destabilization remains to be elucidated, the binding of
LT to the mitotic kinase Bub1 is a good candidate (Cotsiki et al. 2004). Bub1 is
primarily involved with the spindle checkpoint, which is a cellular quality control
mechanism that monitors bivalent attachment of microtubules to the kinetochores
at the metaphase-to-anaphase transition of mitosis (Meraldi and Sorger 2005;
Perera et al. 2007; Williams et al. 2007). Failure of Bub1 to act can result in
chromosomal instability, aneuploidy, chromosome missegregation, and enhanced
tumorigenesis (Cahill et al. 1998; Jeganathan et al. 2007; Kawashima et al. 2010;
Meraldi and Sorger 2005; Perera et al. 2007; Schliekelman et al. 2009; Tang et al.
2004; Baker et al. 2009). LT binding to Bub1 results in attenuation, but not
complete inactivation, of the spindle checkpoint (Cotsiki et al. 2004). Strikingly,
while wild-type LT induces tetraploidy in ~20% of normal human fibroblasts, the
Bub1 binding mutant of LT is completely defective in this regard (Hein et al. 2009).
Of further interest, LT point mutant analysis has correlated Bub1 binding with
transformation of Rat-1 fibroblasts (Cotsiki et al. 2004). Notably, tetraploidy has
recently been demonstrated in a p53-deficient background to promote tumor development (Fujiwara et al. 2005).
A key unanswered question is whether LT induction of genomic instability
contributes to, or even drives, long-term tumorigenesis. In the case of human papillomavirus (HPV), expression of the viral oncoproteins E6 and E7 is not sufficient to
drive tumorigenesis; genomic instability is required as well (Hurlin et al. 1991).
This likely explains a long latency period between the initial infection and the occurrence of cervical cancer. For polyomaviruses, the evidence is more tenuous, although
a number of observations suggest a role.
359
SV40 LT is a highly versatile oncoprotein but not particularly potent when

compared with MT or oncogenic H-Ras. Even when LT is delivered into every
single cell using a retroviral vector, only few transformed foci grow out, suggesting
that additional events may be required (Jat et al. 1986). When LT is expressed transgenically in the mouse salivary gland using a tetracycline-regulatable promoter,
hyperplasia and polyploidy can be reversed after 4 months of age, but not after 7
months (Ewald et al. 1996). Moreover, while most cell clones expressing temperaturesensitive mutants of LT can be reversed to a normal phenotype at the nonpermissive
temperature, the A-type transformants remain neoplastic following LT inactivation
after temperature shift (Rassoulzadegan et al. 1978; Seif and Martin 1979). Finally,
although stable expression of an origin-defective SV40 genome in keratinocytes
failed to elicit a tumorigenic response in nude mice, 46 passages of these cells
in vitro allowed the induction of invasive squamous cell carcinomas (Brown and
Gallimore 1987). Taken together, these observations suggest that LT expression
might induce chromosomal aberrations that contribute toward tumorigenesis, for
example through loss of tumor-suppressor genes or oncogene copy number gains.
Induction of DNA Damage Responses

Recent evidence illustrates that viruses like polyoma not only induce S phase in
quiescent cells, but that they have also targeted the DNA damage response (DDR)
to regulate their own replication (Dahl et al. 2005; Hein et al. 2009; Shi et al. 2005;
Zhao et al. 2008; Boichuk et al. 2010). It is a relatively recent discovery that most
viruses interact with components of the DDR to stimulate or inhibit it (reviewed in
Chaurushiya and Weitzman 2009; Lilley et al. 2007). Adenoviruses appear to mainly
inactivate the DDR by targeting the Mre11-Rad50-Nbs1 (MRN) DDR sensor complex
(Stracker et al. 2002). In contrast, the polyomaviruses activate the DDR and exploit
it (Dahl et al. 2005; Shi et al. 2005; Zhao et al. 2008; Boichuk et al. 2010). The DDR
can be divided into two major branches. The ATM kinase responds to DNA doublestrand breaks, whereas ATR kinase responds to replication-associated single-stranded
DNA lesions. MPyV was demonstrated to activate the ATM pathway upon infection,
and loss of ATM led to reduced replication and virus yield (Dahl et al. 2005). It was
proposed that maintaining an S/G2-phase environment via ATM activation might
cause the replication enhancement (Dahl et al. 2005). JCV was shown to induce
both the ATM and ATR pathways, thus eliciting a G2 checkpoint-mediated arrest
and increased genome replication (Orba et al. 2010).
SV40 infection of monkey kidney cells also results in ATM/ATR and Chk1/2
activation (Zhao et al. 2008; Shi et al. 2005; Okubo et al. 2003; Boichuk et al. 2010).
ATM signaling was shown to promote viral replication in part via phosphorylation
of LT on Ser120, which plays a regulatory role in replication (Shi et al. 2005). After
SV40 infection of human or monkey cells, LT colocalizes with activated ATM, the
phosphorylated form of histone H2AX (g-H2AX) and the MRN complex in nuclear
360
O. Gjoerup
foci corresponding to replication centers (Zhao et al. 2008; Shi et al. 2005; Boichuk
et al. 2010). ATM signaling was shown to be important for establishment of replication
centers and for proteasomal degradation of the MRN complex via the Cul7 ubiquitin
ligase late in infection (Zhao et al. 2008).
Interestingly, not just SV40 virus, but even LT expression in the absence of a
functional origin, induces multiple DDR signaling responses in normal human
fibroblasts via different LT domains (Boichuk et al. 2010; Hein et al. 2009). LT
induces large nuclear foci of g-H2AX/53BP1, a hallmark of the DDR, via Bub1
binding (Hein et al. 2009). In addition, other downstream ATM/ATR targets are
activated, including Chk1, Chk2, and p53, in the latter case leading to its stabilization.
LT induces overt DNA damage, in part double-strand breaks, as demonstrated in
comet assays (Boichuk et al. 2010). Besides g-H2AX/ 53BP1 DDR foci, LT also,
via distinct domains, induces foci of FancD2 and Rad51, consistent with their
activation on chromatin. FancD2 is a key member of the Fanconi anemia family,
which is characterized by enhanced susceptibility to interstrand cross-links probably
due to a role in repair of stalled or collapsed replication forks (Moldovan and DAndrea
2009; Howlett et al. 2005). Fanconi anemia patients suffer from genomic instability
and are predisposed to cancer. Rad51 is essential for homologous recombinationmediated DNA repair and acts to enhance SV40 replication (Boichuk et al. 2010).
Exactly what LT domains or functions trigger the distinct responses involving
FancD2 and Rad51 remains to be shown. It seems reasonable to hypothesize that LT
induces a form of replication stress by deregulating the firing and dynamics of
cellular replication origins.
These extensive perturbations of DDR signaling are likely to influence oncogenesis perhaps in part through effects on genomic stability and generation of
aneuploidy. Traditionally, the DDR is viewed as an early barrier to tumorigenesis
(Bartkova et al. 2005). Loss of several of the genes involved in the DDR is associated with genetic predisposition to human malignancy, for example ATM, Nbs1,
BRCA1, and FancD2. This is consistent with fibroblasts from Fanconi anemia
patients being hypersensitive to SV40-mediated transformation (Todaro et al. 1966;
Liu et al. 1996). However, various reports indicate that the stabilized form of p53,
presumably a by-product of DDR activation (Hein et al. 2009), can acquire a gain
of function in transformation (Deppert et al. 1989; Hermannstadter et al. 2009;
Bocchetta et al. 2008). The 17k gene product, when rendered pRB binding
deficient, can induce both premature cellular senescence and DDR activation
(Gjoerup et al. 2007; Hein et al. 2009), two cellular responses that have previously
been linked together in the context of oncogene-induced senescence (Bartkova et al.
2006; Di Micco et al. 2006). Interestingly, senescence induction can cause the
acquisition of the senescent-associated secretory phenotype, which entails secretion
of proinflammatory cytokines that might promote tumor progression (Rodier et al.
2009). Hence, senescence is a double-edged sword with regard to malignancy
(Davalos et al. 2010). The exact contribution of DDR signaling to polyomavirusinduced transformation and tumorigenesis has not been established but is likely to
depend on the viral oncoprotein and cellular context.
361
Concluding Remarks
In the last 3 years, we have witnessed a revitalization of the polyomavirus field with
the discovery of six new human polyomaviruses, including MCV, which appears to
be directly linked to human cancer causality (Feng et al. 2008). Technological
advances have allowed us to make these new virus discoveries. They provide us
with a framework of expectations for what the next several years might bring:
Are there yet more undiscovered human polyomaviruses out there? And more
importantly, how do we decisively determine if they are associated with human
disease, if their prevalence is widespread? For viruses that might act as cofactors
in carcinogenesis, causality is ever more challenging to prove (Harris et al. 1998a).
Indeed, here, the example of MCV may guide future explorations. First, a cancer
was chosen with a likely infectious etiology because of an increased incidence in
immunodeficient patients. Second, an infectious agent was identified to be MCV
based on DTS technology (Feng et al. 2008). Third, it was demonstrated that the
MCV genome is monoclonally integrated in most of the MCC samples, indicating
that integration preceded tumor formation. Fourth, the integration event invariably
was accompanied with point mutations in the LT-coding sequence leading to loss of
the helicase domain but retention of DnaJ, pRB binding domains, and ST coding
(Shuda et al. 2008). This is consistent with observations from other polyomaviruses,
where it was also found that viral replication functions like the helicase activity are
deleterious in the context of tumor formation, perhaps due to localized collision of
replication forks and DNA damage responses at the integration site (Israel et al.
1980; Lania et al. 1981). This observation establishes that MCV is not just a passenger
virus replicating well in tumors. Fifth, in the infected tumors, T antigen is expressed
only in the tumor cells (Shuda et al. 2009). Sixth, it was demonstrated that knockdown
of the truncated MCV LT and ST with short hairpin RNA (shRNA) leads to growth
arrest, or cell death, but only in MCV-positive MCC samples (Houben et al. 2010).
Collectively, this is providing strong evidence for a continuous requirement for
MCV T antigens in maintaining the tumor.
Despite the recent progress on MCV, there is still much that we do not understand.
MCV, as well as cutaneous polyomaviruses HPyV6 and HPyV7, was detected in
skin samples along with many cutaneous HPVs, perhaps illustrating a closer kinship
with HPVs than previously anticipated (Schowalter et al. 2010). Virions are shed
from the skin. Is the life cycle, like HPV, also tied to epidermal differentiation?
Indeed, it has not been possible to establish a cell culture system yet where KIV,
WUV, and MCV can be grown likely because we are missing the right cell type or
culturing conditions. Can recombination events occur, either between different
polyomaviruses coinhabiting the same cell or even with HPVs? Hybrid genomes
between the polyomaviruses and papillomaviruses can apparently be created occasionally by recombination. Woolford et al. discovered a new virus in papillomas and
carcinomas of bandicoots, whose early region encodes T antigens resembling those
from polyomaviruses, whereas the late region encodes capsid proteins similar to
those of HPV (Woolford et al. 2007).
362
O. Gjoerup
Furthermore, it becomes important to know if there are different serotypes of

MCV that can be categorized as high risk versus low risk for MCC, similarly to
HPV and cervical cancer. Moreover, have the tumor mutants of MCV acquired any
gains in transformation properties relative to the wild-type T antigens? MCV is
present in many tissues; however, it is only associated with MCC. Why are some
tissues apparently more susceptible to tumorigenesis than others? The observations
for SV40, BKV, and JCV also suggest extensive perturbations of DNA damage
responses and chromosome stability. Is this a common phenomenon of the polyomaviruses, and more importantly, does it contribute to long-term tumorigenesis?
The recent dramatic increase in discovery of new polyomaviruses, and the first solid
link to human cancer etiology, reinforces our beliefs that these viruses continue to
serve as important model systems for the development and progression of cancer.
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Chapter 15
Polyomavirus SV40: Model Infectious

Agent of Cancer
Janet S. Butel
Introduction
Polyomavirus SV40 is a small DNA-containing tumor virus that was identified 50 years
ago. It gained immediate scientific attention because of its unexpected presence in poliovaccines of the time. SV40 has been a valuable tool for discovery of fundamental
processes in cell biology as its limited genetic content makes it dependent on cellular
machinery for virus replication. These discoveries have provided insights into mechanisms that are at the root of cell transformation and neoplasia. SV40 earned its status as
a model infectious agent of cancer because of its ease of culture and quantitation in vitro,
its capacity to transform cells in culture, and the susceptibility of small animal models
to infection and tumor induction. It is also the most tractable polyomavirus and often
serves as the representative in studies of fundamental properties of the family.
This review will focus on selected features of the SV40 system that have contributed
to our understanding of cancer, including elements of viral biology that make it a
tumor virus, factors that affect its oncogenic potential, mechanistic insights gained
from animal models, and its status as a potential human pathogen.
The SV40 Viral Genome and Virus-Encoded Proteins

SV40 is the type species member of the family Polyomaviridae. Polyomaviruses are
small, nonenveloped, icosahedral DNA viruses (Imperiale and Major 2007). Their
genomes consist of a single copy of double-stranded, circular, supercoiled DNA
J.S. Butel (*)

Department of Molecular Virology and Microbiology, Baylor College of Medicine,
Houston, TX 77030, USA
Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA
e-mail: jbutel@bcm.edu
377
378
J.S. Butel
Regulatory region
5192
31 40
103 107
250
300
21-bp
repeats
Ori
72 bp
Early promoter
SES
72 bp
Enhancer
5163
5163
Ori
5243 0
.7
.6
.8
.5
SV40
.4
.9
5243 bp
1
.3
0
.2
.1
2693
Fig. 15.1 Genome organization of polyomavirus simian virus 40, strain 776. The circle represents
the circular SV40 DNA genome. Nucleotide numbers begin and end at the origin (Ori) of viral
DNA replication (0/5,243). Unique Bgl-1 and Sfi-1 sites flank the Ori. A unique EcoRI site is
shown at map unit 0/1. Boxed arrows indicate the open reading frames that encode the viral
proteins. Arrowheads point in the direction of transcription; the beginning and end of each open
reading frame is indicated by nucleotide numbers. The regulatory region is shown at the top; Ori,
origin of DNA replication; 21-bp repeats, GC-rich SP1 binding sites and location of the SV40
packaging signal (SES); 72-bp, tandemly repeated 72-bp sequences within the enhancer. Numbers
above the diagram identify nucleotide positions that border specific regulatory region segments.
From Lednicky and Butel (2010)
about 5 kb in length (Fig. 15.1). Although there appears to be only one serotype of
SV40, genetic strains exist that can be distinguished by nucleotide differences in the
early region. Isolates also display differences in the regulatory region of the viral
genome (Stewart et al. 1998; Lednicky and Butel 2001; Forsman et al. 2004).
15
SV40 and Cancer
379
Fig. 15.2 Regulatory region DNA sequence profiles of SV40 viral ioslates. Shown are examples
of simple and complex regulatory regions. Landmarks are as identified in the legend to Fig. 15.1.
The rearranged enhancer region of SVPML-1 is represented with boxes labeled 38, 23, 20 and a
shaded box referring to nucleotides 252263. Nucleotide numbers are based on that of SV40-776.
Origins of strains: SVCPC, human tumor; SV40-776, adenovirus vaccine; Baylor, oral poliovaccine; VA45-54, monkey kidney cells; SVPML-1, human brain (Forsman et al. 2004). Modified
from Stewart et al. (1998)
Viral Regulatory Region

The nontranslated regulatory region contains a single origin (Ori) of DNA replication
and elements controlling transcription and replication. The regulatory region can be
divided into the Ori, the GC-rich 21-bp repeat region containing Sp1 binding sites
(part of the early promoter), the enhancer containing the 72-bp element, and a region
containing the late promoter/initiator (Fig. 15.2) (Lednicky and Butel 2001; Yaniv
2009). Different strains of SV40 possess variations in the structure of the regulatory
region. Among most laboratory strains there are duplications and rearrangements
involving the 72-bp element (complex, 2E), whereas viruses detected in natural
infections in monkeys or humans often contain no duplications in the enhancer
(simple, archetypal, 1E) (Ilyinskii et al. 1992; Lednicky et al. 1998; Newman et al.
1998; Stewart et al. 1998). A 1E SV40 sequence also was found within a natural
SV40-adenovirus 7 hybrid virus (Lanford et al. 1986).
380
J.S. Butel
Regulatory region rearrangements can arise within a given host; in rhesus

monkeys immunosuppressed due to simian immunodeficiency virus infection, SV40
versions with simple or complex regulatory regions but a common T-antigen (T-ag)
variable domain were detected (Lednicky et al. 1998). Although viral regulatory
region rearrangements are commonly observed in SV40-infected immunocompromised monkeys, those changes do not appear to be required for induction of
progressive multifocal leukoencephalopathy disease in those animals (Dang et al. 2005).
Numerous examples of complex rearrangements in the viral regulatory region from
various sources have been described (Lednicky et al. 1998; Butel et al. 1999;
Lednicky and Butel 2001; Sroller et al. 2008). As the regulatory region is not invariant,
it cannot be used for strain identification purposes. In addition to these large structural changes, several single-nucleotide polymorphisms have been detected in the
regulatory region that are useful in distinguishing different isolates (Butel et al. 1999;
Lednicky and Butel 2001).
Viruses with complex regulatory regions tend to replicate better in tissue culture
than those with simple regulatory regions (Lednicky et al. 1995b). In low-passage,
uncloned virus stocks of two laboratory strains (Baylor, VA45-54), both simple and
complex versions of the regulatory region could be detected whereas higher passage
stocks of both strains contained only a complex version (Lednicky and Butel 1997).
This indicates that 2E viruses were selected during passage in tissue culture cells,
presumably due to superior growth, and explains why other laboratory strains also
have complex regulatory region structures. It has been suggested from studies with
mouse polyomavirus that 2E viruses may induce more acute disease whereas 1E
viruses produce low-grade, benign infections (Shadan and Villarreal 1993). It can
be envisioned that the host immune response might more readily eliminate the
better-replicating 2E virus, while allowing the survival of the 1E strains found in
most natural infections.
Viral Late Proteins

SV40 encodes three late structural viral proteins (VP13) (Fig. 15.1). The major
capsid protein, VP1, contains 362 amino acids and forms the pentameric capsomeres
that make up the surface of the virus particle. Viruses initiate infection by VP1 binding
to specific receptors (GM1 gangliosides and major histocompatibility class I molecules) on the surface of host cells (Breau et al. 1992; Tsai et al. 2003); many cell
types express these receptors, allowing for a broad cellular tropism for the virus. The
VP1 gene of SV40 is very highly conserved and cannot be used for genotyping. Most
SV40 strains share an identical VP1 amino acid sequence, although SV40-776
contains a nucleotide polymorphism that changes VP1 amino acid 83 from aspartic
acid (Asp) to glutamic acid (Glu), a conservative change. VP1 residue 83 does not
interact with the ganglioside receptor of SV40 (Neu et al. 2008) and has no effect on
induction or detection of neutralizing antibody. Perhaps antigenic variation is limited
SV40 and Cancer
P P P PP
200
100
126
DnaJ domain
HPDK
Hsc70 binding
Small t-ag &

1 17KT common
300
302 320
Zn
finger
132
NLS
70
131 Ori DNA259

binding
82
102
Cul7
69 binding 83
Bub1
pRb/ p107/
p130 binding
LXCXE
89 binding 97
147
271
708
682
Helicase
517
418
259
351
p53
binding
622 Variable 708

domain
ATP binding/ ATPase

450
708
Host
range
627
Pol
binding
115
Nbs1
binding
PP
600
500
400
S677
S679
T701
S639
381
S106
S112
S120
S123
T124
15
533
627
682
p53
626
binding
708
Fbw7
binding
Fig. 15.3 Functional domains of SV40 large T-ag. Specific regions assigned to functional activities
or as binding sites for target proteins are indicated below the horizontal bar representing the T-ag
molecule. Numbers given are the amino acid residues using the numbering system for SV40-776.
Regions are indicated as follows. Small t-ag and 17KT common: region of large T-ag encoded in
the first exon; the amino acid sequence in this region is common to large T-ag, small t-ag, and
17KT. DnaJ domain: region required for binding the chaperone heat shock protein hsc70; the
HPDK motif is required for binding hsc70. pRb/p107/p130 binding: region required for binding of
the Rb tumor suppressor protein, and the Rb-related proteins p107 and p130; the LXCXE motif is
required for binding these proteins. NLS: contains the nuclear localization signal (PKKKRKV).
Ori DNA binding: minimal region required for binding to SV40 Ori DNA to initiate viral DNA
replication. Helicase: region required for full helicase activity. Pola binding: region required for
binding to DNA polymerase a-primase; necessary for viral DNA replication. Zn finger: region
which binds zinc ions. p53 binding: regions required for binding the p53 tumor suppressor protein.
ATP binding/ATPase: region containing the ATP binding site and ATPase catalytic activity. Variable
domain: region containing amino acid differences among viral strains and used for genotyping.
Host range: region defined as containing the host range and Ad helper functions. Cul7 binding,
Bub1 binding, Nbs1 binding, and Fbw7 binding: regions required for binding of Cul7, Bub1,
Nbs1, and Fbw7 proteins, respectively. The circles containing a P above the bar indicate sites of
phosphorylation found on large T-ag expressed in mammalian cells. S indicates a serine and
T indicates a threonine residue. Modified from McNees and Butel (2008)
due to restrictions imposed by capsid symmetry, allowing negligible deviation in the

sequence of VP1 and no change in serotype. There are also two late nonstructural
proteins that are not present in mature virions. The agnoprotein has a maturation
function that facilitates virus assembly; VP4 induces cell lysis late in infection.
The Large T-Antigen Early Protein

SV40 encodes two major early nonstructural proteins, which share 82 N-terminal
amino acids as a result of alternative splicing of viral transcripts. The large T-ag of
SV40 contains 708 amino acids and is a highly multifunctional protein that
comprises a series of functional domains (Fig. 15.3). It is an essential replication
protein for the virus. T-ag activities and functions expressed during the viral life
cycle include initiation of viral DNA replication, ATPase and helicase activities,
382
J.S. Butel
autoregulation of early transcription, and induction of late transcription. T-ag

undergoes post-translational modifications, including phosphorylation, N-terminal
acetylation, O-glycosylation, poly-ADP-ribosylation, palmitylation, and adenylation.
The sites of phosphorylation are clustered near the ends of the molecule, with the
majority of residues being serines (Fig. 15.3). Unlike many oncoproteins, T-ag is
not phosphorylated at tyrosine residues.
Dysregulation of Cell Cycle
T-ag functions to prepare a cellular environment conducive to viral replication. This is
necessary as the polyomavirus genome is too small to encode homologs for components
of the cellular replication machinery. Rather, the viral early proteins target cellular
proteins to exert control over essential cellular processes, with the end result of forcing
quiescent cells to enter into S phase and thus providing factors needed for cellular
DNA replication. Viral DNA replication can then proceed. Binding of T-ag to cellular
tumor suppressor proteins is key to expression of viral effects on host cells, both for
producing a permissive environment for viral replication and for inducing cell transformation (Butel and Lednicky 1999; Butel 2000; Arrington and Butel 2001; SenzRobles et al. 2001; Sullivan and Pipas 2002; Ahuja et al. 2005; Levine 2009; Pipas 2009;
Gjoerup and Chang 2010). Particular T-agcell protein interactions are described in a
following section Cellular Proteins Targeted by SV40 T-Antigen.
Maintenance of Transformation
The essential role of T-ag in maintenance of cellular transformation in vitro was
established using temperature-sensitive (ts) mutants of the A gene protein (T-ag)
(Brugge and Butel 1975; Martin and Chou 1975; Tegtmeyer 1975; Noonan et al.
1976). Mouse, hamster, and human cells were transformed by early region mutants
(tsA) of SV40. When cultured at the permissive temperature, the cells exhibited the
phenotype of transformed cells, including elevated saturation density, colony formation on plastic and in soft agar, and increased uptake of hexose. However, when
grown at the high, restrictive temperature, the cells reverted to the phenotype of
normal, untransformed cells. When the mutant-transformed cells were shifted down
from the high temperature to the permissive temperature, they formed transformed
foci, indicating that the cells were alive and that the phenotype was reversible.
Wild-type (WT) virus-transformed cells exhibited transformed characteristics under
both conditions. This showed that the continual expression of the viral T-ag protein
was required to maintain the transformed phenotype (Brugge and Butel 1975).
Cytoplasmic T-Antigen
An odd adenovirus-SV40 hybrid carrying a defective SV40 genome was discovered in
1964 in a strain of adenovirus 7 that had been adapted to grow in monkey kidney cells;
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SV40 and Cancer
383
Fig. 15.4 Localization of SV40 T-ag in monkey kidney cells as revealed by indirect immunofluorescence. (a) Typical nuclear localization of T-ag in an SV40 hamster tumor cell line. (b) Green
monkey kidney cells infected with hybrid virus PARA(2cT)-adenovirus 7, showing the retention
of T-ag in the cytoplasm. This mutant resulted in the identification of the SV40 NLS and shows that
nuclear translocation of T-ag is dependent on the nuclear localization signal. (c) Nuclear T-ag
expression in an SV40-immortalized green monkey kidney cell line. (d) Same cells as in panel c
showing cytoplasmic localization of T-ag 24 h after infection with the cytoplasmic mutant hybrid
virus, showing that the cT-ag was dominant-acting and prevented the movement of WT T-ag into
the nucleus
the adenovirus stock had been contaminated with SV40 that was removed by passage
in the presence of SV40 antiserum. The hybrid virus was recognized by the induction
of synthesis of SV40 T-ag that could be prevented by neutralization with adenovirus
antiserum but not by SV40 antiserum (Huebner et al. 1964; Rapp et al. 1964; Rowe and
Baum 1964). Three of 112 doubly plaque-purified clones from the parental hybrid
virus were found to induce the synthesis of SV40 T-ag that was retained in the cytoplasm (Butel et al. 1969), in striking contrast to the typical nuclear localization of T-ag
(Fig. 15.4a, b). It was later shown that an SV40 DNA insert containing the entire SV40
early region had replaced the adenovirus 7 fiber gene in the hybrid virus (Lanford et al.
1986). An SV40 mutant virus [SV40(cT)] was constructed from the cytoplasmic hybrid
virus. This construct was defective for replication, but could be propagated in COS-1
cells. Sequence analysis revealed a single point mutation at nucleotide 4434 which
would convert a positively charged lysine at residue 128 of T-ag to a neutral asparagine,
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J.S. Butel
Fig. 15.5 Effect of T-ag on localization of p53 in transformed mouse cells. (Panels a and b)
BALB-3T3 mouse cells transformed by WT SV40. (Panels c and d) BALB-3T3 cells transformed
by the SV40(cT) cytoplasmic construct. Panels a and c were stained for SV40 T-ag and panels b
and d were stained for p53 by indirect immunofluorescence. (a) WT T-ag localized in the nucleus.
(b) Nuclear p53 in WT transformed cells. (c) T-ag induced by the cytoplasmic mutant present in
the cytoplasm. (d) p53 retained in the cytoplasm in SV40(cT) mutant-transformed cells. Note that
the subcellular localization of p53 matched that of T-ag. From Lanford et al. (1985a)
a finding that resulted in the identification of the SV40 nuclear localization signal
(NLS; Fig. 15.3) (Lanford and Butel 1984). A mutagenesis approach independently
identified the SV40 NLS (Kalderon et al. 1984). The discovery of the SV40 NLS was
of broad interest to the fields of virology and cell biology.
The cytoplasmic mutant of SV40 was competent to transform established mouse
3T3 cells at an efficiency comparable to that of WT SV40, whereas its transforming
ability was substantially reduced in primary mouse embryo fibroblasts under stringent
growth conditions (Lanford et al. 1985b). The cT-ag transformants displayed similar
growth properties to those of WT transformants, showing that it is possible for T-ag to
mediate and maintain transformation without translocating to the nucleus (Fig. 15.5a, c).
This property may be related to the observation that p53 was also retained in the cytoplasm of SV40(cT)-transformed cells, suggesting that the cT-ag was sequestering p53
and preventing its function (Fig. 15.5b, d). The transport-defective form of T-ag was
dominant acting and blocked the translocation of WT T-ag to the nucleus in infected
and transformed cells (Lanford and Butel 1980, 1984) (Fig. 15.4c, d).
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385
The SV40(cT) mutant was able to induce tumors in newborn hamsters, but at a
much reduced frequency compared to WT virus and after extended latent periods.
Cells in the tumors that arose expressed T-ag in the cytoplasm (Lanford et al. 1985b).
This same cT-ag mutant was able to produce rapidly appearing brain tumors in
transgenic mice (Section Animal Models and Insights into SV40 Biology).
The functions necessary for tumor formation in inoculated newborn hamsters that
were dispensable in transgenic mice are unclear.
T-Antigen Variable Domain

There is a variable domain at the C-terminus of T-ag (T-agC) encompassing residues 622708 (Fig. 15.3). SV40 strains can be distinguished on the basis of nucleotide differences in this region, including polynucleotide insertions and deletions as
well as single nucleotide changes, which often would change predicted amino acids
(Stewart et al. 1996). In contrast, the first 622 residues of T-ag are completely conserved (Fig. 15.6a). Phylogenetic analysis established that SV40 strains can be
grouped into clades (genogroups) based on the sequence of the T-ag variable domain
(Forsman et al. 2004) (Fig. 15.6b). Analysis based on the T-agC was highly congruent with whole-genome analysis. (A segment of the regulatory region (nucleotides
29246) was excluded from the whole-genome analysis because genetic changes can
occur within that region during viral growth in vivo.) Several strains were outliers
and did not map to one of the clades, suggesting that other viral genogroups may
occur. The existence of different SV40 genogroups raised the question of whether
viral strains may differ in biological properties. This intriguing possibility was
answered affirmatively in the hamster animal model (Section Animal Models and
Insights into SV40 Biology). The viral encoded microRNAs (miRNAs) target early
viral transcripts within this region (Sullivan et al. 2005) (Fig. 15.1). The SV40 miRNAs accumulate at late times during infection in cultured cells and mediate cleavage
of early viral mRNA, thereby reducing expression of viral tumor antigens.
At the extreme C-terminus of large T-ag, embedded within the variable domain,
is the host range/adenovirus (Ad) helper function (hr/hf) domain (amino acids 682709)
(Fig. 15.3). A C-terminal fragment of T-ag can relieve the Ad replication block in
monkey cells (Cole et al. 1979) by an unknown mechanism. The hr function was
identified because T-ag C-terminal deletion mutants grew very poorly in the CV-1
monkey kidney cell line, but grew well in the BSC-1 and Vero monkey kidney cell
lines (Pipas 1985; Tornow et al. 1985). Although viral DNA was replicated to near
WT levels in all three cell types (Pipas 1985; Stacy et al. 1989), virions produced by
the hr/hf mutants did not assemble properly, seemingly due to an inability to add
VP1 to the 75S assembly intermediates (Spence and Pipas 1994). Interestingly, not
all primate polyomavirus large T-ags contain an hr domain. In addition to SV40,
those with an hr domain include JCV, BKV, and SA12, whereas LPV, KIV, WUV,
and MCV apparently lack such a domain. The precise function of the hr domain and
the explanation for this difference among viral species are not known.
SV40-776
SVCPC
No amino acid changes
SV40-Baylor
VA45-54
SVPML-1
b
Clade C
H388(98)
OST9 (97)
I508(98)
EP14 (95)
Clade B
Baylor(56)
K661(98)
Rh911(62)
GM00637H (99)
CPP15 (95)
SVCPC (95)(SVMEN (84), NHL-8 (02), Men-99 (03))
60
74
777(62)
PML-1
(72)
62
777*(79)
N128(65)
62
69
T302(98)
MC028846B(55)
(NHL-28 (02), NHL-170 (02), NHL-416(02))
62
84
PA-57(61)
OST3 (97)
100
OST2
776(60)
(97)
6593(98)
GN13 (95) VA45-54(60)

776*(60)
H328(98)
Clade A
Fig. 15.6 SV40 large T-antigen variable domain. (a) Examples of diversity among variable
domains at the C-terminus of T-ag (T-agC). The rectangles represent the amino acid sequence of
the protein. Viral strains are identified on the left. Sequence differences in the T-agC compared
to SV40-776 are indicated above the rectangle on the right using the SV40-776 numbering system.
Numbers below the rectangle are actual amino acid numbers for that particular protein. Hatch marks
indicate regions of complete identity with SV40-776. The open portion indicates the sequence
variability in the variable domain among strains. From Stewart et al. (1996). (b) Phylogenetic tree
for SV40 based on T-agC sequences. Unrooted consensus tree of 1,000 bootstrap replicates of
available T-agC sequences, generated by maximum-parsimony analysis. Conventions for
labels are as follows: monkey isolates = roman type; vaccine isolates = bold-face type; human
isolates = underscored italic type. Sequences in parentheses are from independent sources, but are
the same as the sequence preceding the parentheses. Year of sample origin/isolation/detection is
indicated in superscript in parentheses. From Forsman et al. (2004)
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SV40 and Cancer
387
The Small t-Antigen Early Protein

The second major early nonstructural protein, small t-antigen (t-ag), contains 174
residues, the first 82 of which are in common with large T-ag (Fig. 15.1). Through its
unique region, small t-ag complexes with cellular protein phosphatase-2A (PP2A), a
serine/threonine phosphatase that regulates many biological processes in mammalian
cells. PP2A contains three subunits: a scaffold A subunit, a catalytic C subunit, and
a regulatory B subunit, the latter of which is displaced by t-ag. This has the effect of
modifying the substrate specificity of PP2A activity or inhibiting its enzyme activity.
Small t-ag is able to induce proliferation of cells. It enhances transformation by large
T-ag, although its precise contribution is unclear (Khalili et al. 2008; Gjoerup and
Chang 2010). Tumors induced in hamsters by a t-ag deletion mutant of SV40 often
arose in lymphoid tissue and not in all tissues affected by WT virus, suggesting that
small t-ag may be required to stimulate proliferation of quiescent cells in vivo
(Carbone et al. 1998). Small t-ag is essential for SV40 transformation of normal
human cells (Hahn et al. 2002). Complete transformation of several types of human
cells (fibroblasts, mammary epithelial cells and kidney epithelial cells) required the
combination of SV40 large T-ag (disruption of pRb and p53 pathways), small t-ag
(alteration of PP2A functions), telomerase (hTERT), and oncogenic H-ras (stimulation of growth signals). Thus, five regulatory pathways must be altered for transformation of those normal human cells, and SV40 is able to affect three of them,
indicating the genetic underpinnings that make SV40 a strongly oncogenic virus.
Cellular Proteins Targeted by SV40 T-Antigen

T-ag forms complexes with several cellular proteins in order to carry out its multiple
functions (Fig. 15.3). Some of the molecules targeted as part of the viral replication
program can also contribute to cell transformation, making T-ag an oncoprotein and
endowing SV40 with cancer-causing potential. Recent reviews have described these
protein interactions in detail (Sullivan and Pipas 2002; Ahuja et al. 2005; DeCaprio
2009; Fanning and Zhao 2009; Levine 2009; Pipas 2009; Gjoerup and Chang 2010;
Rathi and Pipas 2010).
DnaJ Domain and hsc70

The T-ag amino terminus contains a DnaJ (or J) domain (Srinivasan et al. 1997;
Sullivan et al. 2000; Sullivan and Pipas 2002; Ahuja et al. 2005; Gjoerup and
Chang 2010). DnaJ proteins are molecular chaperones that, together with members
of the DnaK class of chaperone proteins, bind substrate proteins and modify them
in some way, such as refolding or disrupting protein complexes. The J domain of
T-ag (amino acids 170) binds cellular heat-shock protein hsc70 and stimulates its
388
J.S. Butel
ATPase activity. It is thought that the T-ag J domain functions in the disruption of
Rb-E2F complexes, using energy provided by the hsc70 ATPase activity. A functional
J domain is necessary for SV40 viral replication and for many instances of cell
transformation.
Rb Family of Tumor Suppressor Proteins

A separate domain of T-ag (amino acids 102115) binds to retinoblastoma (Rb)related tumor suppressor proteins (pRb, p107, p130/pRb2) through its LXCXE
motif. The first recognition of an interaction between an oncogene and an antioncogene involved adenovirus E1A and pRb (Whyte et al. 1988), followed quickly
by the finding that SV40 T-ag also bound pRb (DeCaprio et al. 1988). The Rb protein functions as a transcriptional repressor and is regulated by phosphorylation
throughout the cell cycle. T-ag selectively complexes with the underphosphorylated
form of pRb found in the G0/G1 phase of the cell cycle and not the more highly
phosphorylated forms present in the later stages of the cell cycle; in so doing, T-ag
abolishes G1/S checkpoint control. This causes unscheduled dissociation of pRb:E2F
complexes and release of E2F to activate expression of growth-stimulatory genes
(Senz-Robles et al. 2001; Sullivan and Pipas 2002; Ahuja et al. 2005; Javier and
Butel 2008; DeCaprio 2009; Pipas 2009; Gjoerup and Chang 2010). T-agpRb
binding is required for SV40 transformation to occur. There are two other Rb-related
proteins, p107 and p130, that also bind T-ag at the same LXCXE site. Presumably,
inactivation of p107 and p130 are also important in SV40 transformation. There are
multiple E2F proteins and it appears that different ones are preferentially bound by
the individual Rb family members. pRb binds activating molecules E2F13, whereas
p107 and p130 bind to the repressors E2F45. T-ag disrupts this pathway by binding
the Rb proteins and blocking their ability to regulate E2Fs (Ahuja et al. 2005;
DeCaprio 2009; Gjoerup and Chang 2010). Many human tumors contain mutations
in the Rb pathway.
p53 Tumor Suppressor Protein

A region toward the C-terminus of T-ag contains the bipartite binding site (amino
acids 350450 and 533626) for the p53 tumor suppressor protein. The p53 protein
was discovered as a cellular protein in complex with SV40 T-ag (Lane and Crawford
1979; Linzer and Levine 1979; Levine 2009). However, a decade of research was
required to establish p53 as a tumor suppressor and not an oncogene, culminating in
the generation of p53 knock-out mice that were highly prone to spontaneous tumors
(Donehower et al. 1992). The early studies were confounded because some p53
clones contained gain-of-function mutations and behaved differently, and it was not
clear which was the WT version. The p53 protein acts to detect stress signals that
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SV40 and Cancer
389
would affect the fidelity of DNA replication, such as DNA damage, hypoxia, heat
shock, spindle damage, virus infection, and oncogene activation (Levine 2009).
It then either pauses the cell in late G1 (cell cycle arrest) to permit DNA repair,
directs the cell to terminate cell division (senescence), or initiates programmed cell
death via the apoptotic pathway. SV40 T-ag binding sequesters p53, abolishing its
function and allowing infected cells to survive to complete virus replication. That
may also allow cells with genetic damage to survive and proliferate, resulting in
T-ag-expressing cells that accumulate genomic mutations (Senz-Robles et al. 2001;
Ahuja et al. 2005; Javier and Butel 2008; Levine 2009; Pipas 2009; Gjoerup and
Chang 2010). T-ag binding stabilizes p53 and extends its half-life so that there are
large amounts of p53 in T-ag-expressing cells. In contrast, WT p53 is present in
normal cells at very low levels with a short half-life of less than 30 min. It has been
envisioned that T-ag blocks p53 sequence-specific DNA binding and transcriptional
activation. However, it is still not clear if T-ag binding inactivates all p53 activities,
equivalent to a p53-null environment, or if some p53 activity might be retained or if
a gain-of-function might be generated. The T-agp53 interaction is clearly important
for virus replication and cell transformation, with T-ag mutants unable to bind p53
being defective for cell transformation (Kierstead and Tevethia 1993). However,
studies from transgenic mouse systems have revealed that in certain specific cell
types p53 inactivation is not a requirement for SV40 transformation (Section
Animal Models and Insights into SV40 Biology). The power of the p53 tumor
suppressor system is illustrated by the fact that at least half of human cancers contain
alterations in the p53 protein or the p53 pathway.
Other Targets
Several other cellular proteins have been identified as potential targets for SV40
T-ag. However, additional characterizations are necessary to understand the role of
these interactions in viral replication and/or transformation.
There are adjacent binding sites in the amino terminus of T-ag for Cul7 (residues 6983) and Bub1 (residues 8997). Cul7 is part of an E3 ubiquitin ligase that
is involved in proteasomal degradation of proteins (Ali et al. 2004). Deletion mutants
suggest that Cul7 binding may be involved in SV40 transformation. Bub1 is a mitotic
spindle checkpoint protein. The T-agBub1 interaction compromises the spindle
checkpoint and may contribute to chromosomal instability in SV40-transformed
cells (Cotsiki et al. 2004). If the T-agBub1 interaction is abolished, SV40-induced
focus formation in Rat-1 cells appears inhibited. However, the role Bub1 may play
in SV40 transformation is unclear.
T-ag reportedly binds Nbs1, the Nijmegen breakage syndrome protein 1,
a component of the MRN (Mrell/Rad50/Nbs1) complex, through the origin-binding
domain (residues 147259) (Digweed et al. 2002; Wu et al. 2004). The MRN
complex functions in DNA repair. It was reported that the number of nuclear repair
foci following irradiation of cells was reduced in the presence of T-ag. Fbw7 is
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J.S. Butel
another cellular protein reported to interact with T-ag (Welcker and Clurman 2005).
Fbw7 is part of the cellular ubiquitination machinery that binds to a motif (phosphodegron) that is present at the C-terminus of T-ag (residues 682708). Whether these
interactions with Nbs1 or Fbw7 contribute to genomic instability or transformation
by SV40 is unknown.
T-ag associates with p300/CBP, also called E1A binding protein p300/CREB
binding protein, two closely related transcriptional coactivators that interact with
multiple transcription factors and increase target gene expression. Thus, they are
involved in many cellular processes. The association of p300/CBP with T-ag is
indirect, with p53 serving as a bridge (Poulin et al. 2004). p300/CBP possess intrinsic
histone acetyltransferase activity and acetylate T-ag on K697 in a p53-dependent
process. The biological effects of T-ag interactions with p300/CBP on virus replication
or cell transformation have not been established.
Animal Models and Insights into SV40 Biology

Small animal models are invaluable for studies of the biology of infectious agents.
Steps in the viral life cycle and consequences of infection, including development
of neoplasia, can be evaluated in the context of differentiated cell types, complex
tissues, and host responses. Animal models for SV40 include Syrian golden
hamsters and mice.
Hamster Model of SV40 Infection and Oncogenesis

Syrian golden hamsters (Mesocricetus auratus) have long been recognized as the
tumor model for SV40 (Butel et al. 1972; Butel and Lednicky 1999; Butel 2000).
The oncogenic potential of SV40 was demonstrated soon after its discovery by
inoculation of newborn hamsters (Eddy et al. 1962; Girardi et al. 1962). Eddy had
previously shown that rhesus monkey kidney cells contained an oncogenic substance
able to induce tumors in newborn hamsters (Eddy et al. 1961) and subsequently
identified the factor as SV40. Several host and viral factors influence the development
of SV40-mediated neoplasia, including age at the time of infection, route of inoculation,
dose of inoculum, and viral genetic variation. The frequency of tumor development
is high in animals infected as newborns, but is reduced in older animals. This suggests
that the host immune response is capable of controlling tumor development, but can
be overwhelmed. Environmental carcinogens can be evaluated in this system;
asbestos and SV40 infection cooperate as co-carcinogens in causation of malignant
mesotheliomas in hamsters (Kroczynska et al. 2006).
The types of tumors that develop in SV40-infected hamsters vary, depending on
the route of inoculation (which determines tissue exposures). Subcutaneous inoculation
usually results in sarcomas at the site of injection; intrapleural and intraperitoneal
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SV40 and Cancer
391
inoculation leads to a high proportion of mesotheliomas (Cicala et al. 1993; Vilchez

et al. 2004; Sroller et al. 2008); intracerebral inoculation produces ependymomas
(Kirschstein and Gerber 1962); and intravenous inoculation induces a spectrum of
tumors, especially lymphomas, mesotheliomas, and osteosarcomas (Diamandopoulos
1973; Carbone et al. 1989; McNees et al. 2009). These studies showed that SV40
has a broad tissue tropism with the capacity to transform a variety of target cell
types. In addition, tumors induced in hamsters turned out to predict the types of
human tumors later found to be associated with SV40 (Section Human Infections
and Cancer Associations).
Influence of Viral Regulatory Region on Tumor Induction

and Vertical Transmission
Recent studies have established that the viral regulatory region is a genetic determinant
of SV40 oncogenic potential whereas the T-ag variable domain exerts negligible
influence (Sroller et al. 2008). These two genomic regions were described above as
being sites of variation among SV40 isolates. A panel of recombinants was constructed
that mixed different regulatory regions and T-agC variable domains. Interestingly,
the parental and recombinant viruses displayed similar transforming frequencies
in vitro in mouse cells (Sroller et al. 2008). However, when 21-day-old weanling
hamsters were inoculated intraperitoneally, the oncogenic potential in vivo of the
viruses ranged from 83% to 0% (Fig. 15.7). Significantly more animals developed
tumors after exposure to viruses with simple (1E) regulatory regions than to those
with complex (2E) regulatory regions. In contrast, the T-agC variable domain
exhibited no particular patterns related to oncogenicity.
As the SV40 variants showed no differences in transforming activities in vitro, the
in vivo results must have reflected the consequences of variable virushost interactions.
One possible explanation is that the viruses with complex regulatory regions replicate
more abundantly and are recognized and cleared more efficiently by the hosts immune
response than are the slower-replicating viruses (those with simple regulatory regions).
This concept is supported by observations with mouse polyoma virus and lymphocytic
choriomeningitis virus, which showed that slower-growing viruses elicited milder host
responses and were more apt to persist long-term (Rochford and Villarreal 1991;
Bocharov et al. 2004). More residual persistently infected cells by the SV40 1E variants
could account for their higher level of subsequent tumor development.
An experiment designed to examine vertical transmission of SV40 in hamsters,
rather than tumor formation, supported the interpretation that relative levels of viral
replication affect biological outcomes (Patel et al. 2009). Pregnant hamsters inoculated
intraperitoneally with SV40 strains containing complex regulatory regions transmitted
virus to their offspring more frequently than those infected with simple enhancer
viruses (p < 0.001). Virus was detected in maternal kidney and spleen tissues in some
animals for at least 24 days after infection. These data suggested that SV40 strains
with complex regulatory regions replicated to higher levels in the pregnant hamsters
than 1E viruses, increasing the likelihood of transmission to their litters.
392
J.S. Butel
100
Tumor development (%)
90
Regulatory region
1E
80
70
2E
60
50
40
30
20
10
Ba
y(
1
PC E)
-P
SV ML
C CPC
PC
C -Ba
PC y
77 776
6(
C 1E)
PC
77 -VA
6VA Bay
45
77 -54
677 VA
6
77 (2E
6- )
C
Ba PC
y
77 (2E
6- )
P
SV ML
P
co ML
nt
ro
ls
SV40 parental and recombinant viruses
Fig. 15.7 Influence of the SV40 regulatory region on tumor induction in hamsters. Weanling
hamsters were inoculated intraperitoneally with parental and recombinant viruses and observed for
1 year for tumor development. Each bar shows the tumor incidence in the group inoculated with
the virus identified below the bar. Black bars indicate viruses with simple regulatory regions (1E);
hatched bars indicate viruses with complex regulatory regions (2E). Viruses with simple regulatory
regions were more oncogenic than those with complex regulatory regions. The difference between
the two groups (1E vs. 2E) was statistically significant (66/152, 43% vs. 18/155, 12%; p = 0.0001).
From Sroller et al. (2008)
Other lines of evidence also indicate that SV40 can infect and replicate in hamsters.
First, many virus-inoculated but tumor-free animals develop antibodies to SV40
T-ag, with more responders among animals inoculated with 2E viruses as compared
to 1E viruses (Sroller et al. 2008; McNees et al. 2009). SV40 T-ag is not a component
of the virus particle, but rather is synthesized in infected cells. Second, infectious
SV40 was recovered from many (39%) tumor cell lines established from primary
tumors that arose months after virus infection of the hamsters (Sroller et al. 2008).
This is in keeping with earlier studies that had reported recovery of infectious virus
from SV40-induced hamster tumor cells (Sabin and Koch 1963; Black and Rowe
1964; Gerber 1964; Boyd and Butel 1972; Lednicky and Butel 1999). Third, SV40
regulatory region rearrangements were detected in several SV40 hamster tumors
induced by 1E viruses (Sroller et al. 2008). The mechanism of rearrangement is
unproven, but it has been suggested that it reflects a recombination event during
viral DNA replication (Yogo and Sugimoto 2001).
Model of SV40 Pathogenesis of Infection and Disease

These hamster data showed that SV40 strains differ in biological properties and
suggested that the risk of disease may depend on the viral isolate causing an infection.
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393
Exposure
No infection
Infection Mucosal surface

(respiratory, gastrointestinal) (?)
Initial replication
(lymphoid tissue, GI tract) (?)
SV40 genetic
variation?
Dissemination
(blood)
Eradication
Viral load?
SV40 microRNA?
Secondary organs
(kidney, liver, lung, spleen, tonsil, brain)
Persistence
Viral load?
Excretion
(urine, feces)
Disease
(cancer)
Fig. 15.8 Model of SV40 pathogenesis of infection and disease. This model proposes that attributes
of persistent infections established by SV40 during early stages of infection are key factors in
determining the relative risk of subsequent development of disease. As viral infection occurs and
spreads to secondary organs, the host response attempts to eradicate the infection. Factors influencing this process are proposed to be SV40 genetic variation, SV40 microRNA, and viral load.
Viral load refers to the number of persistently infected cells in the host; the more that survive
elimination are predicted to produce an increased chance of subsequent tumor development.
Proposed steps in the pathogenesis of infection are based on observations from hamsters and
humans. Question marks indicate details of the model that have not been experimentally proven
Those insights inspired the development of a model of SV40 pathogenesis of infection

and disease (Fig. 15.8). The model envisions that the risk of virus-induced disease
is dependent on the particular characteristics of a persistent infection, with those
characteristics determined by the virushost interaction during the acute phase of
infection in each given host. A significant feature is predicted to be the viral load,
which would likely reflect the absolute number of persistently infected cells in the
host. This hypothesis can be tested experimentally in Syrian golden hamsters as
they are both an infection and a tumor model for SV40. Studies could be designed
to identify cell types that support virus replication, as well as sites of long-term
persistence. Genetic factors that do not affect virus replication in vitro, such as the
viral miRNA, could be examined for effects in vivo. General principles that emerge
would in all likelihood be applicable to other polyomavirus systems. Cancer viruses
establish persistent infections in susceptible hosts and a better understanding of the
origin and evolution of persistently infected cells prior to the initiation of transformation
would help clarify the risk of development of virus-associated cancer.
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J.S. Butel
Mouse Model of Cellular Immune Responses to SV40 T-Antigen

Tumor-bearing hamsters usually develop antibodies to SV40 T-ag, as do many
virus-infected animals without tumors (Sroller et al. 2008). Virus-inoculated rabbits
and monkeys also produced T-ag antibody responses (Rapp et al. 1967; Vonka
et al. 1967; Tevethia 1970). Such observations showed that SV40 T-ag is a strongly
immunogenic protein and that development of antibodies to T-ag is a sign of SV40
infection and not necessarily a sign of neoplasia (Tevethia 1990; Tevethia and Schell
2001). Immunological studies of cell-mediated immunity in hamsters are difficult
because of the limited knowledge of hamster immunology and the lack of genetically
inbred lines. Although mice fail to develop tumors after SV40 inoculation and do
not support SV40 replication, they have been valuable models for defining basic
mechanisms of cytotoxic T lymphocyte (CTL) responses to SV40 proteins.
CTL Epitopes on T-Antigen

Tevethia and colleagues have identified four distinct CTL epitopes for T-ag in mice.
These epitopes were mapped using deletion mutants and synthetic peptides to the
following T-ag residues: epitope I, residues 206215; epitope II/III, 223231; epitope
IV, 404411; and epitope V, 489497. Three of the epitopes are H-2Dd restricted,
whereas one (IV) is H-2Kb restricted. Epitope V is immunorecessive (Tevethia 1990;
Deckhut et al. 1992; Lippolis et al. 1995; Mylin et al. 1995; Schell and Tevethia 2001;
Tevethia and Schell 2001) (Fig. 15.9). Four additional epitopes have been identified:
residues 362384 (Frster et al. 1995), 499507 (Newmaster et al. 1998), 529543
(Schell TD 2010, personal communication), and 560568 (Zheng et al. 2002). These
are H-2Ak, H-2Kd, H-2Ab, and H-2Kk restricted, respectively.
These multiple CTL determinants provide effective immunosurveillance against
the outgrowth of SV40 tumor cells in mice. In fact, CD8+ CTLs against a single T-ag
epitope (404411) were able to control progression or cause regression of T-aginduced pancreatic tumors and brain tumors in vivo in two different transgenic systems
(Otahal et al. 2006; Tatum et al. 2008; Yorty et al. 2008). However, tumor cells that
escape CTL immunosurveillance are known to emerge. In vitro selections of SV40transformed mouse cells using individual epitope-specific CTL clones resulted in
CTL-resistant cell populations. Genetic analyses revealed that the resistant cells
expressed T-ag with point mutations or deletions that affected the respective CTLrecognition epitope. A multiplicity of escape mutations were identified, with residue
substitutions more common than sequence deletions. Thus, single residue changes
within CTL recognition sites are sufficient to abrogate transformed cell destruction
by particular CTL clones (Lill et al. 1992; Mylin et al. 2007). These studies revealed
a mechanism for tumor cell escape from immunosurveillance while allowing for
maintenance of cell transformation. They also suggested potential complications of
certain immunotherapy strategies.
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SV40 and Cancer
395
H-2b-restricted epitopes
1
82 83
Residues:
Sequence:
206-215
II/
III
223-231
708
IV
404-411
489-497
529-543
SAINNYAQKL CKGVNKEYL VVYDFLKC QGINNLDNL NEYSVPKTLQARFVK
MHC restriction:
H-2Db
H-2Db
H-2Kb
H-2Db
H-2Ab
HLA-restricted epitopes
1
82 83
708
Residues:
138-146
281-289
Sequence:
FPSELLSFL
KCDDVLLLL
MHC restriction:
HLA-B7
HLA-A2.1
285-293
VLLLLGMYL
HLA-A2.1
577-585
LMLIWYRPV
HLA-A2.1
Fig. 15.9 Relative location of murine H-2b-(top) and human HLA-(bottom) restricted epitopes in
SV40 T-ag recognized by CD8+ T cells. Roman numerals in top diagram indicate the designations
for the corresponding MHC class I-restricted epitopes. Arabic numbers indicate the amino acid
positions within the T-ag sequence. Only the epitopes found in C57BL/6 mice are shown in the top
panel. See text for details. Courtesy of T.D. Schell and S.S. Tevethia
As the T-antigens of polyomaviruses SV40, JCV and BKV are related, the possibility
of CTL epitope cross-reactivity was examined. CTL clones specific for epitope II/III
reacted with both JCV- and BKV-transformed cells, identifying a particular epitope
that is found on the T-antigens of all three polyomaviruses. In contrast, epitope V was
specific for SV40. Particular CTL clones against epitope I and epitope IV of SV40
recognized JCV T-ag as well, but not BKV T-ag (Tevethia et al. 1998). Thus, CTL
reagents exist that can discriminate among the T-antigens of SV40, JCV and BKV,
whereas others show that cross-reactivity exists for certain epitopes in mice.
Four epitopes have been identified on T-ag that are recognized in humans: residues
138146 (Coleman et al. 2008), residues 281289 (Schell et al. 2001), residues 285
293 (Bright et al. 2002), and residues 577585 (Velders et al. 2001) (Fig. 15.9). The
first is HLA-B7 restricted and the others are HLA-A2.1 restricted. The T cell responses
against epitopes 138146 and 285293 were found in patients with malignant pleural
mesothelioma and were capable of recognizing and lysing T-ag-expressing target cells
in vitro (Bright et al. 2002; Coleman et al. 2008). It is apparent that the human T cell
responses discovered to date are against epitopes different from those identified in
mice. More work is needed to identify epitopes on T-ag that can be recognized by
other HLA types in humans. Both epitopes 138146 and 281289 were shown to be
specific for SV40 and not shared with JCV or BKV T-ag (Schell et al. 2001; Coleman
et al. 2008). Whether other T-ag epitopes recognized in humans might cross-react with
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J.S. Butel
those of JCV or BKV and what effect such immunity might have on SV40 infections
in humans are unknown. It is possible, for example, that pre-existing cross-reactive
immunity might dampen specific antibody responses to SV40.
SV40 MicroRNA and CTL Evasion

To experimentally test if specific CD8+ T cell clones could recognize and attack
SV40-infected cells, TC-7 monkey kidney cells were transfected with murine H2-Kb
and H2-Db class I antigens, allowing murine CTL clones against SV40 T-ag epitopes
to be utilized. Following infection of the modified TC-7 cells with SV40, exposure
to T-ag-specific CTL clones significantly reduced viral yields (Bates et al. 1988).
This showed that CTLs that target SV40 T-ag epitopes can mediate the elimination
of virus-infected cells. Subsequently, this system was applied to address the possibility
that SV40 miRNAs function to facilitate CTL evasion by infected cells. It was found
that cells infected with a virus unable to express viral miRNAs were more susceptible
to CTL-mediated lysis and produced more interferon gamma than companion
cultures infected with WT SV40 (Sullivan et al. 2005). Perhaps this mechanism to
delay or reduce elimination of infected cells by the host facilitates virus production
or establishment of persistently infected cells; it would seem it might also help the
survival of transformed cells if they were to arise in an infected host and express any
viral miRNA.
Transgenic Mouse Models of SV40 T-Antigen Effects

The development of transgenic mouse technology added a new dimension to studies
of viral oncogenes (Hanahan 1989; Adams and Cory 1991; Van Dyke 1994). It became
possible to analyze tumor virus proteins in host animals to complement studies with
cultured cells in vitro. Tissue-specific promoters could be used to examine viral
gene effects on different cell types, mutated genes could be tested to dissect viral
transforming functions, and different host genetic backgrounds could be utilized to
expand insights into host responses in vivo. SV40 T-ag was the first viral oncoprotein
expressed in transgenic mice; under the control of its natural regulatory region the
virus induced brain tumors in the choroid plexus of the animals (Brinster et al. 1984).
Since that beginning, SV40 T-ag expression has been directed to many different
tissues and cell types by the use of tissue-specific promoters.
Transgenic models have contributed to fundamental principles related to SV40
T-ag transformation and tumor development (Lednicky and Butel 1999; Butel 2000;
Ahuja et al. 2005; Pipas 2009; Senz Robles and Pipas 2009; Gjoerup and Chang 2010).
These general principles include the following: T-ag is a potent oncoprotein able to
induce neoplasms in a variety of tissues, T-ag can stimulate resting cells to enter the
cell cycle and proliferate, T-ag inhibition of the Rb and p53 pathways is central to
the SV40 transformation process, and additional genetic changes beyond viral
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SV40 and Cancer
397
oncogene expression are necessary for tumor formation. Transgenic studies also
have shown that there are exceptions to those general rules and that T-ag expression
may exhibit tissue- and cell-type-specific effects (Ahuja et al. 2005; Pipas 2009;
Senz Robles and Pipas 2009; Gjoerup and Chang 2010). Thus, another basic principle
is that cell context is important for expression of T-ag-mediated transforming events.
Several examples are described to illustrate the significance of transgenic systems
in broadening an understanding of viral carcinogenesis.
Rb and p53 Pathways in SV40 Transformation

The effects of the Rb and p53 pathways on T-ag tumor induction was first analyzed
in the choroid plexus model. The choroid plexus epithelium lines the ventricles of
the brain and maintains a bloodcerebrospinal fluid barrier. A truncated protein
containing only the first 121 amino acids of T-ag and unable to bind p53 (T121)
induced choroid plexus tumors, but at a lower rate than WT T-ag, and the tumors
that appeared grew more slowly and displayed many apoptotic cells compared to
tumors expressing full-length T-ag. If the Rb-binding sequence were mutated, the
T121 T-ag fragment failed to induce tumors, showing the absolute requirement for
T-ag inhibition of the Rb pathway in this system (Chen and Van Dyke 1991; Chen
et al. 1992; Symonds et al. 1993; Senz-Robles et al. 1994). The observed apoptosis
was p53-dependent because when the amino terminal truncated mutant was
expressed on a p53-deficient background, the tumors that arose showed negligible
apoptosis and grew rapidly (Symonds et al. 1994). The interpretation was that T-ag
binds and inactivates pRb proteins, inducing cell proliferation and unscheduled
DNA synthesis. This triggers a p53 response poised to eliminate the damaged cells
via apoptosis, which T-ag blocks by binding and inactivating p53. Thus, WT T-aginduced tumors were fast-growing and aggressive whereas those induced by T-ag
unable to inactivate p53 were slow-growing and apoptotic.
Transgenic mice were produced with the cytoplasmic T-ag mutant of SV40 to
explore the effects of transport-defective T-ag in vivo. A transgenic line was established using the natural viral promoter with the result that animals died regularly
of choroid plexus tumors at an early age (81 days) (Pinkert et al. 1987). T-ag
expressed in the brain tumor cells was localized in the cytoplasm. We can speculate
that cT-ag was able to readily induce brain tumors because it could interact with
p53 (and presumably with Rb proteins) in the cytoplasm, thereby avoiding an
apoptotic response. The rapid tumorigenicity by the cT-ag mutant was somewhat
unexpected as the virus was crippled for transformation of primary cells in culture
(Section The SV40 Viral Genome and Virus-Encoded Proteins). This study
illustrated that transformation assays in vitro may fail to accurately reflect the
oncogenic process in vivo, as different cell types are involved and the surrounding
environments are different.
Transgenic systems have revealed that there are cell types in which inactivation
of p53 is not a requirement for SV40 transformation. These include pancreatic acinar
cells and intestinal enterocytes. In the pancreatic model, full-length T-ag and an
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J.S. Butel
N-terminal fragment of T-ag (T127) unable to bind p53 behaved comparably when
expressed under the control of the rat elastase-1 promoter (Tevethia et al. 1997).
Both WT T-ag and the truncated mutant induced pancreatic acinar carcinomas.
Intestinal enterocytes represent the predominant specialized cell type in the
epithelium of the small intestine. SV40 T-ag expression under the control of the rat
intestinal fatty acid binding promoter results in hyperplasia that over time progresses
to dysplasia (Markovics et al. 2005; Senz-Robles et al. 2007; Rathi et al. 2009).
N-terminal truncated mutants of T-ag (T121, N136) induced hyperplasia similar to
WT T-ag, but there was less frequent progression to dysplasia. In addition, there did
not appear to be stabilization of p53 by full-length T-ag in the enterocytes and
T-agp53 complexes could not be detected in the cells. In contrast, pRb inactivation
was essential for SV40 transformation in this system. T-ag mutants unable to bind
Rb proteins or lacking a functional J domain had no phenotypic effects on the
enterocytes. Finally, recent gene expression studies confirmed that the Rb-E2F
pathway is the primary T-ag target in enterocytes and that the p53 pathway is not
important (Rathi et al. 2009). Using whole-mouse genome arrays to compare mRNA
levels, the N136 truncation mutant was found to upregulate essentially the same
genes as WT T-ag.
Loss of Dependence on T-Antigen in Transformed Cells

Although T-ag is expressed in most tumors, there are indications that the continual
expression of T-ag is not always required to maintain the transformed cell phenotype.
Time-sensitive reversal of T-ag-induced hyperplasia in the salivary gland was
demonstrated in transgenic mice using a tetracycline-responsive gene expression
system (Ewald et al. 1996). When T-ag was expressed, extensive ductal hyperplasia
was evident by 4 months of age. If T-ag expression were silenced at 4 months, the
hyperplasia was reversed. However, if T-ag was not silenced until 7 months of age,
the hyperplasia persisted in the absence of T-ag expression. This demonstrated that
SV40-transformed cells may over time accumulate sufficient genetic changes to
lose their dependence on T-ag expression.
SV40 T-ag targeted to the liver by the regulatory elements of the human a1antitrypsin gene was expressed in nearly all hepatocytes (Sepulveda et al. 1989).
The transgenic animals showed reproducible liver changes with predictable kinetics
(hyperplastic, dysplastic, neoplastic) and developed liver tumors by 10 weeks of
age. The tumors arose as nodules on a background of dysplasia, showing that
additional events beyond T-ag expression were needed for cellular progression to
neoplasia. There was considerable variation in the proportion of T-ag-positive cells
in and among tumor nodules, with some nodules being negative for T-ag. This observation suggested that cellular genetic changes may accumulate and make the continued
presence of T-ag dispensable for tumor progression.
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An SV40 T/t-Antigen Gene Signature

A recent report provided a powerful demonstration of the applicability of SV40
transgenic mouse studies to human cancer (Deeb et al. 2007). An integrated SV40
T/t-antigen gene expression signature was identified from transgenic models of
breast, lung, and prostate cancer. This gene signature reflected primarily the expression
of genes regulating cell replication, proliferation, DNA repair, and apoptosis and
was distinctive from those of several other oncogenes (Ras, Her2/neu, Myc, and PyMT).
This gene signature identified a subset of aggressive human breast, lung, and prostate
carcinomas with poor prognosis and was highly predictive of human breast cancer
prognosis. A comparison of the genes upregulated by T-ag in mouse intestinal
enterocytes found that a majority (78%) overlapped with the SV40 T/t signature
(Rathi et al. 2009). This observation indicates that SV40 T-ag affects the same
molecular pathways in different tissues to mediate transformation.
Other
A transgenic mouse model directed SV40 T-ag expression to mesothelial cells using
the mesothelin promoter. Members of a transgenic line exhibited a low level of
spontaneous tumor development, but when exposed to asbestos developed more
rapidly growing, invasive mesotheliomas than the control nontransgenic mice
(Robinson et al. 2006). This system showed co-carcinogenicity between SV40 and
asbestos in vivo. Those results complemented similar findings of synergism between
SV40 and asbestos in Syrian hamsters in vivo and in tissue culture studies involving
primary human and hamster mesothelial cells (Kroczynska et al. 2006). The results
were supportive also of observations made in humans (Procopio et al. 2000; Cristaudo
et al. 2005). These reports showed that both transgenic and nontransgenic animal
models can be used to examine potential co-carcinogen effects on a virus-induced
cancer, an underexplored area in viral carcinogenesis. The potential role of carcinogens on induction of other human neoplasias linked to SV40 warrants investigation.
Human Infections and Cancer Associations

SV40 was discovered as a contaminant of early poliovaccines in 1960 (Sweet and
Hilleman 1960). The vaccines had been prepared using primary cultures of rhesus
monkey kidney cells, some of which harbored natural persistent infections by SV40.
The virus produced no discernible cytopathic effects in the rhesus cells and its presence went unrecognized. It was only when African green monkey kidney cells were
used that cellular changes (cytoplasmic vacuolization) developed and the virus
was detected. By that time both contaminated inactivated poliovaccines (IPV)
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J.S. Butel
(Salk vaccines) and live, attenuated oral poliovaccines (OPV) (Sabin vaccines),
as well as some contaminated adenovirus vaccines, had been administered to millions of people. Residual infectious SV40 survived the inactivation procedures used
to prepare the killed vaccines, whereas there was no inactivation step to decrease
viral viability in the oral vaccines (Fraumeni et al. 1963; Shah and Nathanson 1976;
Stratton et al. 2003). The presence of SV40 in the human population today, as well
as its potential role as a human pathogen, will be considered here.
Human Exposures to SV40-Contaminated Vaccines

The IPVs were widely used starting in 1955, both in the US and other countries.
The chance of IPV contamination by SV40 depended on several factors, including
the frequency of viral infection in the monkeys, the levels of viral infection in their
kidneys, and whether culture methods for vaccine production used kidneys from a
single monkey or pooled organs from multiple animals. The higher the titer of
contaminating SV40, the higher the likelihood of residual infectious virus surviving
formalin inactivation. Most vaccine lots were not tested for SV40, but in two
vaccine batches that were tested retrospectively live virus was in the range of 103
infectious units per ml. Estimates are that up to 30% of IPV lots contained live
SV40 and that by 1961 approximately 1030 million of the 98 million vaccinated in
the US were exposed to live virus (Shah and Nathanson 1976).
Rhesus macaques, the natural hosts for SV40, were generally used for vaccine
production. Other species, such as cynomolgus macaques, African green, and patas
monkeys, could be readily infected by contact with an infected rhesus. As many
juvenile animals were held in gang cages, this provided opportunities for SV40 to
be transmitted from an infected animal to uninfected ones and productive infections
established before their kidneys were harvested.
The time span during which potentially contaminated poliovaccines were used
was from 1954 to early 1963. Vaccines were supposed to be free from SV40 after
June 1961, but previous lots of vaccine were not withdrawn from the market and
could have been used until 1963 (Shah and Nathanson 1976). However, recent
evidence suggests that the Russian OPV likely was contaminated until the late 1970s
(Cutrone et al. 2005) and there is uncertainty about the status of the OPVs used in
some other countries.
Work was proceeding during the 1950s on derivation of attenuated poliovirus
strains for a live vaccine as debate raged about the relative merits of killed and live
poliovaccines. Large-scale field trials were carried out from 1958 to 1960 for two
candidate live vaccines developed by Dr. Albert Sabin and by Lederle Laboratories,
respectively. These trials were not carried out in the US because widespread use of
the Salk vaccine had produced antibodies in many persons. The candidate OPVs
were grown in monkey kidney cells and were presumably contaminated with SV40.
Virus titers in the oral vaccine were as high as 104106 infectious units per ml (Shah
and Nathanson 1976; Hilleman 1998; Rollison and Shah 2001). In fact, the Baylor
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SV40 and Cancer
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strain of SV40 was originally recovered from a Type II Sabin OPV from 1956 and
other SV40 strains were isolated from monkey kidney cells in use at the time
(Forsman et al. 2004). Sequences of recognized SV40 strains were detected recently
in a Russian OPV seed stock used until the late 1970s (Cutrone et al. 2005). Sabins
vaccines were field tested in Mexico (Ramos-Alvarez and Gomez-Santos 1961) and
very widely in the USSR (Chumakov 1961; Sabin 1985). The Russians prepared
OPV based on Sabins attenuated viral strains and over 100 million persons were
vaccinated in Russia, East Germany, Czechoslovakia, Hungary, Romania, Bulgaria,
Latvia, and other countries in 19591960 (Sabin 1985, 1991). Sabin vaccines were
also tested in Houston, Texas and in Cincinnati, Ohio (Melnick 1960; Sabin et al. 1961;
Sabin 1985).
The Lederle strains were tested in large trials in Central and South America,
including in Costa Rica, Nicaragua, Colombia, and Uruguay (Pan American Sanitary
Bureau 1959, 1960; Cabasso et al. 1960). Those trials involved about one million
people, including many children. In the US in 1958 and 1959, the Lederle vaccine
was tested in Minnesota and New England in about 1,400 people and in a large trial
in Dade County, Florida (~400,000 people) (Pan American Sanitary Bureau
1959, 1960). The Koprowski candidate vaccine, based on the same viral strains as
the Lederle vaccine, was tested in over 12 million people in Poland, Croatia, and the
Belgian Congo (Plotkin 2001; Koprowski 2006).
In 1961, the Type I Sabin vaccine was licensed in the US following an evaluation
that concluded it to be safer and less virulent than the Lederle vaccine. Type II vaccine
was approved later in 1961 and Type III in March 1962. A trivalent vaccine became
available in 1963.
Another source of human exposure to SV40 was contaminated adenovirus vaccines
used to vaccinate military recruits against epidemic acute respiratory disease (Rapp
et al. 1964; Lewis 1998; Rollison et al. 2004). Several different human adenoviruses
(types 15 and 7) had been adapted to grow in rhesus monkey kidney cells in the
1950s for vaccine development purposes before it was known that adenoviruses
grow extremely poorly in those cells unless coinfected with SV40. It was subsequently
discovered that the vaccines were contaminated with live SV40 or with hybrid
adenovirus-SV40 recombinants (Section The SV40 Viral Genome and Virus-Encoded
Proteins). Several hundred thousand military recruits received the contaminated
vaccines between 1957 and 1961 (Shah and Nathanson 1976; Lewis 1998). The widely
studied SV40 strain 776 was isolated from an adenovirus type 1 vaccine seed stock
(Forsman et al. 2004).
Predictions of Contemporary Human Infections by SV40

Among all the potential exposures to SV40-contaminated vaccines, it is unknown
which individuals actually received contaminated vaccines, how much infectious
SV40 was present in each contaminated lot, or who among the exposed was successfully infected by SV40. While there is strong evidence that SV40 human infections
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are occurring today, the distribution and prevalence of those infections are unclear.
There are also numerous findings of the presence of SV40 markers in selected human
cancers, but those positive results are counterbalanced by reports of negative findings.
This inconsistency among studies has inhibited progress in the assessment of SV40 as
a potential human pathogen. A model is proposed here to provide both a rationale for
the maintenance of SV40 infections in restricted geographic locations and a possible
explanation for the conflicting reports in the literature of SV40 detections in humans.
The model is based on virus biology and contaminated vaccine usage. The key
observation relevant to viral transmission is that polyomaviruses, including SV40,
can be found in stool samples, in addition to urine. This suggests that the viruses can
be transmitted by the fecal-oral route, as well as presumably by a urine-oral route
(Vanchiere et al. 2005a, b, 2009; Bialasiewicz et al. 2009; Wong et al. 2009).
A model of SV40 spread among humans can be predicated on the pattern of
poliomyelitis epidemiology before vaccination. The polio system accurately predicted
the epidemiology of hepatitis A virus (HAV) and provided a model for understanding
Helicobacter pylori. In developing countries, polio infections transmitted by the
fecal-oral route were common in infants in whom the infection caused mild or no
disease due to the presence of maternal antibodies; older children and adults were
then immune for life. In regions and populations where standards of sanitation and
household hygiene improved, fewer polio infections occurred in infants and young
children, reducing the overall prevalence of infection and resulting in larger numbers
of susceptible older individuals in whom poliovirus infections were less common
but sometimes caused paralytic poliomyelitis. This led to a change in disease
pattern from endemic to epidemic poliomyelitis (Walton and Melnick 1955; Paul 1971;
Melnick 1985; Sabin 1991). With HAV, also transmitted by the fecal-oral route, the
prevalence of viral antibodies reflects the level of sanitation in the individuals living
environment. Under conditions of overcrowding, lack of clean water, and inadequate
systems for disposal of human waste (situations that are more common in developing
countries), HAV causes infections that occur early in life in most persons and are
usually subclinical (high prevalence rates). In areas with high levels of sanitation,
many individuals reach adulthood without exposure to the virus (low prevalence
rates) and when infection occurs in a susceptible adult, clinically apparent hepatitis
is more common. Even in developed countries, such as the US, there can be pockets
of high infection rates, reflecting local differences in living standards or customs
(Feinstone and Gust 2002). Similarly, a high prevalence of antibodies to H. pylori
in a given population group is a marker for poor sanitation in their living conditions
(Graham 1991; Nurgalieva et al. 2002).
The application of the polio/HAV/H. pylori model to SV40 predicts that human
infections are likely to be maintained by fecal-oral transmission in settings of overcrowding, poor sanitation, and unclean water sources. In regions and populations
with access to clean water and high levels of sanitation, such as in the US and other
developed countries, the prevalence of SV40 infections today would be expected to
be very low, reflecting reduced opportunities for fecal-oral transmission. The model
further predicts that SV40 infections will be distributed unevenly geographically and
among population groups with different standards of living or levels of hygiene.
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It is envisioned that more human infections were seeded by the use of contaminated
OPV as compared to IPV because of a more natural route of exposure (oral rather
than intramuscular) and the higher titers of contaminating infectious SV40. A higher
dose of inoculum would probably have resulted in increased numbers of successful
infections among the vaccinees. As field trials with contaminated OPV in the
Western hemisphere were carried out in locations in Mexico and in Central and
South America rather than in the US, poor sanitation conditions in some areas in
those regions could have provided opportunities for maintenance of recurring cycles
of SV40 infections.
These predictions concerning the importance of sample and subject selection
probably explain, in large part, the inconsistency among reports of SV40 in humans,
with discrepant findings reflecting the demographic backgrounds of the populations
from which specimens were obtained. To analyze SV40 infections or SV40-associated
cancers in humans, studies should involve regions or populations in which the virus
is present. This model provides guidance as to the potential geographic areas and
populations to consider in future study designs.
Evidence of Human Infections by SV40

A review of the evidence involving noncancer samples indicates that SV40 human
infections occur today, at relatively low prevalence, establishing that this monkey
virus has crossed over to humans (Butel and Lednicky 1999; Vilchez and Butel 2004;
Baranova and Carbone 2010; Butel 2010). The earliest indication that SV40 could
establish infections in humans was reported in 1962; 19% of newborns and 15% of
infants 3- to 6-months of age at the time of receiving contaminated OPV were found
to excrete infectious SV40 in their stools for up to 5 weeks (Melnick and
Stinebaugh 1962). Modern studies have employed molecular assays to detect viral
sequences. SV40 excretion was detected in about 8% of pediatric stool samples
from Houston, Texas, and less frequently in adult stools (Vanchiere et al. 2005a, 2009).
SV40 DNA has been found in urine samples from children and, on rare occasions,
in urines from adults (Li et al. 2002a, b; Milstone et al. 2004; Vanchiere et al. 2005b;
Thomas et al. 2009). Viral sequences have also been detected in tissue samples: in
renal biopsies from patients with kidney disease (Butel et al. 1999; Li et al. 2002b;
Milstone et al. 2004), in normal liver tissues from mesothelioma patients (Comar
et al. 2007), and in lymphoid specimens from healthy children (Patel et al. 2008;
Comar et al. 2010).
SV40 DNA has been found at low frequencies in peripheral blood cells from
healthy individuals. Reports have involved subjects from the US (David et al. 2001;
Li et al. 2002a), Europe (Martini et al. 1996, 2002; Paracchini et al. 2005; Heinsohn
et al. 2009; Pancaldi et al. 2009), Japan (Yamamoto et al. 2000; Nakatsuka et al. 2003),
Egypt (Zekri et al. 2007), and Russia (Lapin and Chikobava 2009). In an Italian
study, viral differences in regulatory region sequences were noted across birth cohorts
(Paracchini et al. 2005). Studies (n = 20) using polymerase chain reaction-based
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assays for SV40 sequences in healthy subjects were reviewed recently and the
prevalence rates ranged from 25% to 0% (Paracchini et al. 2006). The studies
were highly heterogeneous, perhaps because those samples often represented convenience controls for cancer studies.
These results establish the presence of SV40 in humans, both in people of an age
who might have received contaminated vaccines and in those born after vaccines were
supposed to be SV40-free. They also demonstrate the nephrotropic, lymphotropic, and
gastrointestinal properties of the virus. It is likely that the kidney serves as a reservoir
of persistent infections, while it is possible that lymphoid and/or gastrointestinal tissues
may be additional sites. However, the DNA-based studies have been too limited in
scope to establish the distribution of viral infections in different areas of the world.
Serology studies are a common approach to establishing the prevalence of a viral
infection, with neutralization assays being the most specific serological measure of
viral antibodies. Studies using plaque reduction or microtiter infectivity assays have
detected SV40 neutralizing antibodies in target groups in the US and the United
Kingdom, with positive results ranging from 2% to greater than 10%. Seroprevalences
in most cases were 5% or less (Butel and Lednicky 1999; Minor et al. 2003; Shah
et al. 2004; Vilchez and Butel 2004; Butel 2010). Samples from two central European
countries, Hungary and the Czech Republic, where early vaccine use was well
documented, also had low overall frequencies of SV40 antibody, although a striking
observation was a higher prevalence of antibodies in females compared to males in
both countries, reaching 15.6% in Hungary (Butel et al. 2003). A study in the central
Asian country of Kazakhstan, known to have used contaminated Russian vaccine,
found an overall seroprevalence of 4.9%, but with higher frequencies among older
ethnic Kazakhs (8.5%) and younger ethnic Russians (9.7%) (Nurgalieva et al. 2005).
Unfortunately, SV40 neutralization assays are time-consuming and labor-intensive
and cannot be applied to large population surveys.
Enzyme immunoassays can be used to test large numbers of samples for serum antibodies. All antibodies that are able to bind to virus particles or soluble capsid proteins
are measured in these assays, including those that are non-neutralizing and those that
recognize cross-reactive epitopes on BKV, JCV, and SV40. Using this methodology,
competition assays with BKV and JCV particles reduced SV40 reactivity in screened
sera to an overall seroprevalence of about 2% (Shah et al. 2004; Kean et al. 2009).
These results provide evidence of SV40 human infections at low rates in the
human groups tested. However, several limitations of the reported studies should be
considered. First is the general lack of knowledge of the human immune response
to SV40 (Section Mouse Model of Cellular Immune Responses to SV40
T-Antigen). Cross-reactive antibodies or cytotoxic lymphocytes to BKV or JCV
may reduce specific responses to SV40 infection. SV40 antibody titers in humans
are usually low, perhaps reflecting a weak specific response. Another factor is that
SV40 antibodies reportedly wane over time, lowering apparent seroprevalences
(Lundstig et al. 2005). Finally, the majority of such studies have been carried out in
countries with high standards of living, some of which had limited exposure to
contaminated OPV. It would be informative to perform serological surveys in populations predicted to be at higher risk of SV40 infections.
15
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405
Association of SV40 with Human Cancer

SV40 is a potent cancer-causing virus with the ability to transform many cell types
from different species, including cells of human origin. There is evidence that SV40
infections occur in humans, but whether the virus may be playing an etiological role
in human cancer remains controversial.
Numerous studies have investigated possible links of SV40 with human tumors
and obtained positive findings. However, other studies have failed to detect evidence of
SV40 in cancer samples. These studies have been reviewed (Butel and Lednicky 1999;
Shah 2000; Arrington and Butel 2001; Rollison and Shah 2001; Stratton et al. 2003;
Vilchez and Butel 2004; White and Khalili 2004; Poulin and DeCaprio 2006;
Rollison 2006; Martini et al. 2007; Baranova and Carbone 2010; Butel 2010). Tumor
types most commonly associated with SV40 markers are mesotheliomas, lymphomas,
brain tumors, and osteosarcomas. Interestingly, these are the most common types of
tumors induced in hamsters by SV40. Controlled studies based on polymerase chain
reaction assays involving the first three tumor types, in which control samples were
analyzed in parallel, are summarized (Table 15.1). Although SV40 was detected
significantly more frequently in cancer than control specimens for all three systems,
the percent positivity among studies ranged from 100% to 0%. This lack of reproducibility has raised questions about the significance of viral detection.
A variety of technical factors have been suggested that might explain the inconsistency among reports, including sample selection, sample processing, laboratory
contamination, DNA quality, DNA quantity, and assay sensitivity. However, a more
important factor may be the geographic source of the specimens analyzed. A recent
study addressed the hypothesis that the presence of SV40 in lymphomas can vary
depending on the populations sampled, even when uniform technical procedures are
applied. Archival specimens from two hospitals in Houston, Texas having patient
populations with significantly different demographics were compared. SV40 DNA
was detected more often in lymphomas from the public hospital (23%) as compared
to those from the veterans hospital (3%; p < 0.0001) (Toracchio et al. 2009). A possible
explanation for those differences is that many immigrants in Houston from Mexico
and Central America access the public hospital for medical care and some individuals
may have emigrated from high SV40 prevalence locales. Studies need to be designed
that focus on regions predicted to be high prevalence areas for SV40 infections.
One study has been described in which samples from Costa Rica, such a predicted
area, were analyzed. SV40 sequences were detected in 24% of non-Hodgkin
lymphomas and not in the control samples (Meneses et al. 2005). SV40 T-ag was
expressed in many of the DNA-positive cancers as detected by immunohistochemistry
(Fig. 15.10). The T-ag reactions in the human lymphomas were less intense as
compared to that of SV40 hamster tumors and a lower proportion of cells in the
lymphomas stained positively. This suggests that T-ag stability or accumulation
differs in the human tumor cells compared to rodent tumors. T-ag expression has
been reported also in studies involving brain tumors (Bergsagel et al. 1992; Zhen
et al. 1999), AIDS-related lymphomas (Vilchez et al. 2005), and mesotheliomas
(Arrington and Butel 2001; Baranova and Carbone 2010).
Table 15.1 Detection of SV40 DNA by PCR in controlled studies of human cancers a
SV40 DNA by PCR
Range in %
No. positive/No. tested (%)
positivity of
Type of cancer No. of studies Cancer
P value cancer specimens
Controlb
Mesothelioma 18
269/671 (40.1)
19/474 (4.0) <0.001 1000
Lymphoma
16
389/2,033 (19.1) 39/1,207 (3.2) <0.001
560
Brain
9
152/912 (16.7)
49/455 (10.8)
0.007 1002
a
Adapted from Butel (2010). See review for original citations
b
Many of the SV40-positive control samples were peripheral blood samples from noncancer
patients or healthy individuals
Fig. 15.10 SV40 T-ag expression in non-Hodgkin lymphomas detected by immunohistochemistry.

(a) T-ag expression in SV40-induced hamster tumor cells. (b and c) T-ag expression in SV40
DNA-positive diffuse large B cell lymphomas from Costa Rica. (d) No T-ag expression detected
in an SV40 DNA-negative lymphoma from Costa Rica. (e and g) T-ag expression in SV40 DNApositive AIDS-related lymphomas from Houston, Texas. (f) No T-ag expression detected in an
SV40 DNA-negative reactive lymph node from an HIV-infected patient. Original magnification
for all panels (except f), 100. Panel f, 40. From Vilchez et al. (2005) and Meneses et al. (2005)
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SV40 and Cancer
407
A recent study compared osteosarcomas from Hungary and from Germany and
found SV40 DNA at high levels in 74% of Hungarian samples and at low levels in 22%
of German tumors (Heinsohn et al. 2009). The authors concluded that SV40 prevalence
varies in different geographical regions.
Complementary lines of evidence have been described, in addition to the polymerase
chain reaction-based surveys of tumors and detections of antigen expression, that
indicated the presence of SV40 in human tumors. Examples include recovery of an
infectious isolate of a novel strain of SV40 from a pediatric brain tumor (Lednicky
et al. 1995a), methylation of specific tumor suppressor genes in SV40 DNA-positive
mesotheliomas and lymphomas (Shivapurkar et al. 2004; Suzuki et al. 2005; Amara
et al. 2007), detection of SV40 sequences in the meningioma of a scientist with a
risk of laboratory exposure identical to those of the exposure source (Arrington
et al. 2004), and derivation of T-ag-positive oligodendroglial cell lines from an
SV40 DNA-positive brain tumor (Kim et al. 2009).
Epidemiological support for a link between SV40 infections and human cancer is
currently lacking (Strickler et al. 1998; Shah 2000; Rollison and Shah 2001; Rollison
et al. 2004; Rollison 2006). However, because of the unique history of human exposures
to SV40, the reported studies have had limitations. These include not knowing how
many in an exposed group were actually infected, the possibility that members of
control unexposed groups were infected, the lack of assays able to reliably identify
those who were infected, and the possible involvement of unknown cofactors
(Vilchez et al. 2003; Dang-Tan et al. 2004; Rollison 2006). Importantly, no epidemiological study has focused on populations exposed to SV40-contaminated OPV in
regions likely to have maintained a high prevalence of SV40 infections.
It is challenging to establish an etiological role for a virus in human cancer, especially when the geographic distribution of human infections is unknown, specific
biomarkers of infection have not been established, only a subset of a given type of
tumor may be involved, unknown cofactors may be contributing factors, and criteria
for causality are not standardized (Butel 2000, 2010; Pagano et al. 2004). It is
possible that the viral presence in tumors reflects persistent infections in those tissues,
rather than a tumor-inducing relationship (a so-called passenger virus). However,
given the wealth of information about the oncogenic capabilities of SV40 and the
fact that human cells can be infected and transformed by SV40, it is difficult to
imagine that humans with SV40 infections would be completely resistant to virusmediated carcinogenesis.
Among the major tumor types linked to SV40, the molecular and biochemical
data from studies of tissue culture cells, animal models, and human specimens, in
aggregate, are strongest for malignant mesotheliomas that SV40 is playing a causal
role in a subset of tumors. In this example, the virus is probably functioning as a
cofactor with asbestos (Baranova and Carbone 2010; Butel 2010).
It is important to determine the biological significance of SV40 association with
human tumors. A causative role in certain cancers would have a significant public
health impact. Research efforts are warranted on a variety of topics, including the
following. Seroprevalence surveys should be conducted to establish the extent of
infections in predicted geographically limited high-prevalence areas. As only subsets
of tumors will presumably be related to SV40, biomarkers need to be developed to
408
J.S. Butel
recognize virus-positive tumors. Investigations should consider novel molecular

mechanisms underlying SV40 effects in human tumors, recalling that exceptions
have been described to conventional principles of polyomavirus transformation
(Sections The SV40 Viral Genome and Virus-Encoded Proteins and Animal Models
and Insights into SV40 Biology). The effect of the immune response on viral
persistence and tumor progression in humans awaits characterization. The documented
synergy between SV40 and asbestos in mesotheliomas suggests that the role of
co-carcinogens in other SV40-associated cancers should be investigated. Virusassociated tumors might respond uniquely to specific therapies, prompting the design
of novel approaches, while a vaccine could be considered to prevent the development
of cancers having a viral component. Finally, the geographic distribution of SV40
infections should be monitored to recognize if some event were to cause the infection
to spread to new populations or regions.
Scientific progress inevitably raises new questions in any biological system.
There is every expectation that SV40 will continue to be an outstanding model
infectious agent of cancer and contribute novel insights into the process of carcinogenesis in the future, just as it has in the past.
Acknowledgments I thank Drs. T.D. Schell and S.S. Tevethia for providing Fig. 15.9. Research
in my laboratory was supported in part by grants CA104818 and CA134524 from the National
Institutes of Health.
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Chapter 16
BK Polyomavirus and Transformation

Tina Dalianis and Hans H. Hirsch
BK Virus Discovery
Polyomavirus (PyV) infections are linked to malignant transformation ever since
the first isolation of the mouse PyV as a transmissible agent causing tumors in newborn or immunosuppressed mice (for review, see [Atkin et al. 2009]). The concepts
have been confirmed by numerous elegant studies for the mouse PyV (MPyV) and
for the simian virus 40 (SV40) that was identified as a contaminant of poliovirus
and adenovirus vaccines in the early 1960s (Stewart et al. 1958; Sweet and Hilleman
1960). Thereby, MPyV and SV40 have become prototypes of DNA tumor viruses
(Atkin et al. 2009; Ramqvist and Dalianis 2009). Given these circumstances, the
detection of cells with atypical nuclei in the urine of a kidney transplant patient with
ureteric stenosis was even more intriguing in 1971 when PyV-bearing intranuclear
inclusions were revealed by electron microscopy (Gardner et al. 1971). This suggested
the hypothesis that a human polyomavirus (HPyV) might play a role in human diseases
and possibly cancer. The viral isolate was called BK virus (BKV) after the patients
initials B.K. and further experimental studies were initiated to characterize its properties in vitro, in animal models, and in human disease.
T. Dalianis
Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska R8:01,
Karolinska University Hospital, 171 76 Stockholm, Sweden
H.H. Hirsch (*)
Department of Biomedicine, Clinical and Transplantation Virology, Institute for Medical
Microbiology, University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland
Infectious Disease and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland
e-mail: hans.hirsch@unibas.ch
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T. Dalianis and H.H. Hirsch
BK Virus Genome and Life Cycle

BKV belongs to genus polyomavirus in the family of polyomaviridae which is today
separated from the previous common category of papilloma, polyoma, and vacuolating viruses summarized as papovaviridae (Johne et al. 2011). The viral particles
are of icosahedral symmetry with a diameter of 4045 nm and contain the circular
double-stranded genome of approximately 5.1 kb. The genome organization of the
polyomaviridae is highly conserved, but there are certain species of this family that
group closer together due to higher homology. Thus, BKV is closest related to simian
SV40 and human JC virus, the etiologic agent of progressive multifocal leukoencephalopathy, a rare, mostly fatal brain disease in immunocompromised patients
(Padgett et al. 1971). The BKV genome can be divided into three regions.
The noncoding control region (NCCR) of approximately 0.4 kb that bears the
origin of replication and the promoter/enhancer sequences controlling viral gene
expression.
The viral early gene region of approximately 2.3 kb encoding the regulatory
proteins called large tumor antigen (LTag) and small tumor antigen (sTag). Both
proteins are generated by alternative splicing of a primary transcript (started
from the NCCR in one direction) and then localize to nucleus and cytoplasm,
respectively.
The viral late gene region of approximately 2.4 kb encoding the capsid proteins
VP1, VP2, and VP3 as well as the BKV agnoprotein. These proteins are also
generated by alternative splicing from a primary transcript (started from the
NCCR in the opposite direction), and then localize to the nucleus for capsid
assembly and the cytoplasm, respectively (Rinaldo et al. 1998).
Initial studies have investigated the BKV life cycle in diverse, mostly nonhuman
cell lines, where the focus was on the characterization of the transforming potential.
The emergence of BKV nephropathy in kidney transplants has generated considerable interest in nontransformed human cells (Hanssen Rinaldo et al. 2005) as well
as in the host cell targeted by this disease (Bernhoff et al. 2008; Gosert et al. 2008;
Low et al. 2004). In the latter cells, BKV are able to replicate with high-efficiency
releasing by host cell lysis, 10,000100,000 viral particles per cell within 4872 h
post infection (hpi).
The primary host cell specificity is mediated by the attachment of the major
capsid protein VP1 to glycosylated surface structures, whereas VP2 and VP3 play a
role in packaging of the viral genome and likely stabilize the three-dimensional
virion structure. The binding sugar moieties have been identified on membrane lipids,
such as gangliosides. BKV capsids interact with an alpha (2,3)-linked sialic acid
linkage present of the gangliosides GD1b and GT1b (Dugan et al. 2006; Low et al.
2006). Additionaly proteinaceous molecules may serve as secondary receptors and
facilitate PyV entry. Thus, BKV appears to interact with a 55-kDa molecule yet to
be identified and is then taken up by endocytosis of caveolae (Dugan et al. 2006;
Neu et al. 2009). From the caveosome, the endoplasmic reticulum (ER) is reached,
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where uncoating and delivery of the episomal viral genome to the nucleus occur
(Neu et al. 2009). The VP1 protein is also the determinant of the six major BKV
serotypes that have been described and is the major target of neutralizing antibody
activities (Jin et al. 1993).
The secondary host cell specificity of BKV is mediated at the level of viral gene
expression from the NCCR (Watanabe and Yoshiike 1986). In line with its predilection for the renourinary tract, BKV infects, and efficiently replicates in, primary
human renal proximal tubular epithelial cells (RPTECs). RPTECs have been wellcharacterized by Imperiale and colleagues (Low et al. 2004) and proved to be an
important model for the study of host cell responses (Abend et al. 2010; Grinde et al.
2007) and antiviral activities (Bernhoff et al. 2008; Bernhoff et al. 2010; Rinaldo et al.
2010; Rinaldo and Hirsch 2007). However, BKV detected in healthy individuals
around the world is characterized by a linear architecture of the NCCR (Flaegstad
et al. 1991; Markowitz et al. 1991; Negrini et al. 1991) and replicates slowly in
RPTECs (Gosert et al. 2008; Olsen et al. 2009). By contrast, BKV variants bearing
rearranged NCCR are rapidly selected in vitro (Johnsen et al. 1995) which includes
the original BKV isolate described by Gardner (Gardner et al. 1971). Thus, the wellcharacterized BKV (Dunlop) is characterized by a rearranged (rr)-NCCR. Recent data
revealed that BKV rr-NCCR variants also emerge in vivo in the blood of kidney transplant patients with prolonged BKV-associated nephropathy. These (rr)- NCCRs confer increased LTag expression and accelerated replication in vitro (Gosert et al. 2008).
Thus, the NCCR is an important determinant of the secondary host cell specificity and
pathology by controlling the expression of the early viral proteins, LTag and sTag, and
by determining the effective switch to late gene expression and lytic replication.
The LTag and sTag are key regulatory proteins of roughly 700 and 170 aa that
permit PyV replication through different direct and indirect mechanisms. The indirect mechanisms involve the interaction with host cell proteins to create an optimal
metabolic environment for the efficient biosynthesis of the viral progeny. The direct
mechanisms consist of binding to the viral genome to recruit the host cell replication complex and to mediate the switch to viral late gene expression. The key elements of the indirect LTag functions have been discussed in great detail elsewhere in
this book. They include the inactivation of the tumor-suppressor protein functions of
the retinoblastoma family pRB, p103 and p107. Thereby, the cell cycle control is
abrogated and the host cell is shifted into a proliferative G2/S state, where enzymes
and building blocks become abundant. Binding of LTag to p53 is thought to counteract apoptosis which may be triggered by accumulating viral DNA fragments and
disintegrating host cell DNA as well as by the metabolic exhaustion. Binding to p53
may also activate cellular genes, like insulin-like growth factor (IGF)-1 expression
and signaling, and potentially affect gene expression by stabilizing regulators, like
p300/CBP and Mdm2 (Reiss et al. 2006). The sTag presumably provides additional
cytoplasmic proliferation signals by inactivating the protein phosphatase 2A (PP2A)
that negatively regulates activating signal transduction along the MAP kinase pathway. The DNA-binding domain and the ATPase/helicase activity of LTag are crucial
for melting the viral DNA and recruiting proteins to replication fork, including the
host cell DNA polymerase. With the switch to late gene expression and capsid
422

BKV Infection
Episomal DNA
Signals
Latency
Early gene expression

small T antigen
Large T antigen
Host cell activation
Cell cycle shift G2/S
Anti-apoptosis
uncoupling
Late gene expression

VP-1,2,3 capsids
Productive BKV infection
Cytopathic cell lysis
No late gene expression

no virion assembly
Abortive BKV infection
Oncogenic transformation
Fig. 16.1 Scheme of BKV infection, replication or transformation
assembly, the late phase of the viral life cycle is initiated. The role of the nonstructural
agnoprotein is presently unresolved. Homologous agno proteins are currently only
found for SV40 and JCV. Its dominant cytoplasmic expression late in the viral life
cycle of BKV suggests a regulatory role in virion assembly and release, but a multitude of other functions including oncogenic transformation have been discussed. The
efficient release of progeny virions from the nucleus requires host cell lysis which
naturally counteracts tumor development. Hence, the uncoupling of early gene
expression from late gene expression must be postulated as a key component of
oncogenic transformation by PyVs including BKV. This can be envisioned on a
functional level, where the NCCR is intact, but late gene expression cannot occur in
a given host cell because of its state or differentiation on a genetic level via mutations, rearrangements, or disruption by genomic integrations that affect VP1 expression or truncate the LTag downstream of the LxCxE domain involved in pB binding
(Fig. 16.1).
Cytopathic BKV Disease

Initial clinical studies in kidney transplant recipients suggested that BKV might
play a role in the stenosis of the alloureter and proposed a role of corticosteroids in
the reactivation of BKV in this setting (Coleman et al. 1978; Gardner et al. 1984).
Although the role of steroids has been confirmed in some later studies (Hirsch et al.
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2002; Hirsch et al. 2010), other patient and immunosuppression-related factors

must be involved as well. Early histopathology studies reported biopsy findings
consistent with virally induced interstitial nephritis that was difficult to be distinguished from acute cellular rejection (Gardner et al. 1984; Mackenzie et al. 1978).
Subsequently, this complication was virtually nondiagnosed (Prince et al. 2008) and
reemerged only when the calcineurin inhibitor cyclosporine-A was widely substituted by tacrolimus in the late 1990s (Binet et al. 1999; Randhawa et al. 1999).
Currently, BK polyomavirus-associated nephropathy (PyVAN) is encountered in
110% of kidney transplant patients, and more than half of the affected patients are
at risk for premature graft failure (Hirsch 2010; Ramos et al. 2009). The patients are
clinically asymptomatic despite a high urine BKV loads of >7 log10 genome equivalents (geq)/mL and the shedding of decoy cells (Funk et al. 2008). High-level
viruria precedes the onset of detectable plasma BKV loads and histological evidence of nephropathy (Hirsch et al. 2002; Nickeleit et al. 2000). With increasing
viral cytopathic damage and denudation of the infected tubuli, inflammation
increases and laboratory signs of progressive graft failure appear in more than 80%
(Drachenberg et al. 2007, 2004; Hirsch et al. 2002). In the absence of effective antivirals, the judicious reduction of immunosuppression at the onset of BKV viremia
has become the treatment of choice in many centers (Hirsch and Randhawa 2009).
Thereby, viremia was cleared in adult and pediatric patients, and the risk of graft
loss was reduced to less than 10% (Ginevri et al. 2007; Hardinger et al. 2010; Schaub
et al. 2010). On the other hand, in patients with persistent, high-level BKV replication, viral variants with rr-NCCR emerged in vivo that were associated with 20-fold
higher plasma BKV loads and more advanced PyVAN in the renal allograft (Gosert
et al. 2008). The rr-NCCRs were shown to consist of different deletions, duplications, and complex combinations in the promoter/enhancer elements (Gosert et al.
2008; Olsen et al. 2006). Reporter gene constructs and recombinant viruses demonstrated that the rr-NCCR variants increased the viral early gene expression the viral
replication capacity in vitro as compared to the naturally found archetype virus
(Gosert et al. 2008; Olsen et al. 2009).
Studies in allogenic hematopoietic stem cell transplant recipients provided
evidence for a role of high-level BKV replication in the late-onset, postengraftment
hemorrhagic cystitis (Arthur et al. 1986; Bedi et al. 1995). The urine BKV loads are
also above 7 log10 geq/mL, but unlike in kidney transplantation the affected patients
are clinically symptomatic with severe signs of cystitis, immobilizing pain, macrohematuria with clots, and even postrenal failure that requires bladder irrigation and
urologic intervention (Hirsch 2010). Although previously seen more frequently,
current estimates suggest that this complication occurs in 515% of patients (Erard
and Hirsch 2009; Hirsch 2010). The risk factors include myeloablative conditioning
often involving cyclophosphamide and total body irradiation, unrelated donors,
graft-versus-host disease, and increasing BKV loads in the blood (Erard et al. 2005;
Giraud et al. 2008a). The pathogenesis seems to involve a combination of factors,
where BKV replication leads to a denudation of the urothelial mucosa predamaged
by the toxic conditioning and an immune reconstitution inflammatory syndrome
(IRIS) response following engraftment of the allogenic stem cells (Hirsch 2010).
424
Transforming BKV Activity in Experimental Systems

A role for BKV in oncogenic transformation has been suggested early after the
reports of its initial isolation in 1971 (Gardner et al. 1971; Padgett et al. 1971; Walker
et al. 1973). Initial in vitro studies indicated that BKV could not be cultured in rodent
cells, but mostly induced an altered phenotype or outright transformation, depending
on the cell type and its underlying lesions (for review, see [Barbanti-Brodano et al.
2006]). Further in vitro studies could link the transforming contribution of BKV to
the viral early gene region encoding the BKV LTag and the sTag (Harris et al. 1996)
and the ability to cooperate with other oncogenic alterations (Harris et al. 1998a, b).
Defective BKV genomes were linked to the transforming potential, and particular
cell types like the pancreas proved to be effectively targeted in rodent- and humanderived cells (van der Noordaa et al. 1986; Watanabe et al. 1979; Yogo et al. 1980).
Animal studies in rodents showed that, diverse tumors could be induced as had
been previously shown for MPyV or SV40 (Shah et al. 1977). Furthermore, animal
studies in rodents also showed that diverse tumors could be induced by BKV, including brain tumors and sarcomas, as had been observed for MuPyV and SV40
(Ramqvist and Dalianis 2009; zur Hausen 2008). Depending on the route of inoculation, the tumor type and penetrance varied considerably. In addition, the immune
system needed to be either immature or experimentally depleted prior to the virus
challenge. In hamsters, for example, little tumorigenicity by BKV was observed in
subcutaneous sites, whereas direct intracerebral inoculation was followed by tumor
formation in more than 80% of the animals (Uchida et al. 1976). Different types of
tumors were observed in these experiments, including neuroblastoma, ependymoma,
pancreatic cancer, and sarcomas of the connective or osteogenic tissues. When an
intravenous application was chosen, prior immunosuppression was needed for
tumor formation in hamsters and this was done by the application of lymphocytedepleting antisera or the use of high-dose steroids (Corallini et al. 1982). However,
primary human cells appeared to be more refractory to the transforming effects of
the BKV early genes (Portolani and Borgatti 1978; Shah et al. 1976). In contrast,
cells with chromosomal abnormalities were more susceptible in line with the presence of cooperative alterations in oncogene or tumor-suppressor gene products.
BK Virus in Human Cancers

Despite the suggestive mechanistic evidence from experimental models, the role of
BKV in human malignancies is controversial (Grossi et al. 1981). The high seroprevalence and frequent detection of BKV in healthy individuals by sensitive
molecular tools is noted (Egli et al. 2008), since this complicates study design and
requires a rigorous reevaluation of the published data. However, the possibility of an
oncogenic role of BKV in human cancer is captured in single case reports of urothelial
malignancies and renal tubular malignancies, typically in the setting of kidney
transplantation as illustrated by the following three cases.
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425
A 53-year-old simultaneous kidneypancreas transplant patient was diagnosed with

metastatic urothelial carcinoma 1 year after losing his kidney transplant to
BK-PyVAN while the pancreas transplant was functioning well (Geetha et al. 2002). In
situ hybridization identified BKV. Immunohistochemistry revealed positive staining
for LTag and co-staining for p53, but adjacent nondysplastic urothelium or adjacent
stroma was negative. LTag and p53 staining was also positive in the metastases.
Sequencing of the p53 gene demonstrated the absence of stabilizing mutations,
implicating the BKV LTag binding in p53 overexpression. The workup of this case
is exemplary and the association of BKV LTag and malignancy is highly suggestive.
However, molecular-genetic analyses are missing, and it is formally possible that
BKV had been infecting the urothelial carcinoma only after its genesis (passenger) rather than contributing to its actual condition. On the other hand, prolonged,
high-level BKV replication and PyVAN in severely immunodeficient patients are
known to allow the emergence of genetic BKV variants with rr-NCCR which may
or may not support lytic replication (Gosert et al. 2008; Myhre et al. 2010). This
may in turn also create opportunities for otherwise rare events and thereby favor
malignant transformation, as proposed earlier (Hirsch and Steiger 2003).
A 40-year-old simultaneous kidney pancreas transplant recipient was diagnosed
with a BKV LTag-positive metastasizing renal cell carcinoma after developing
BK-PyVAN treated with intravenous cidofovir (Narayanan et al. 2007).
A pediatric kidney transplant patient developing a BKV LTagp53 positive
collecting duct carcinoma after BK-PyVAN (Emerson et al. 2008).
While the similarity between these cases is intriguing, there are, however, also
two other reports.
The first is a 10-year-old kidney transplant recipient developing a renal cell
carcinoma of donor origin after BK-PyVAN that was BKV negative by in situ
hybridization (Kausman et al. 2004). This case raises questions whether or not
the hybridization was analytically false negative and should have been complemented by immunohistochemistry for LTag. Alternatively, the results were
conceptually false negative due to a hit-and-run mechanism. Where by BKV
contributed to an earlier stage, but was then lost.
The second case is an 80-year-old man with past colon cancer and significant
steroid exposure for bullous pemphigus (Loghavi and Bose 2010). The patient
was diagnosed with a papillary urothelial carcinoma supposedly superinfected
with BKV (passenger) since only a fraction of cells were LTag positive (Loghavi
and Bose 2010). However, the hit-and-run remains as alternative hypothesis for
the BKV-negative cancer tissue and requires further investigations.
Together, these cases illustrate the caveats which need to be addressed in larger
studies by stringent, quite extensive protocols which best include several complementing diagnostic procedures. Clearly, PCR analysis of tissues may be one element
which can not be sufficient to draw conclusions without ancillary techniques.
The study by Weinreb and colleagues (Weinreb et al. 2006) identified kidney
transplant patients with urine cytology in their pathology database. They found an
426
increased odds ratio of 3.42 (95% confidence interval 1.796.55; p < 0.001) for
urothelial cancers among 123 patients with decoy cell shedding compared to 3,617
patients without decoy cell shedding (Weinreb et al. 2006). Although this data does
not directly implicate a direct contribution of PyV to the malignancy, decoy cells
may serve as noninvasive biomarker of increased risk in patients with high-level replications. Given the high rate of spontaneous BKV shedding in healthy controls, it is
not surprising that smaller studies searching for BKV by more sensitive techniques
like PCR were frequently not conclusive (Fioriti et al. 2003; Knoll et al. 2003). The
study by Herawi and colleagues (Herawi et al. 2006) is an interesting extension of
earlier studies. They identified 732 kidney transplant patients with decoy cell shedding in a retrospective cohort from 1988 to 2003. For 33 of them, biopsies were available which included the diagnosis of urothelial carcinoma in 21 cases. LTag staining
was positive in seven cases (30%) including two cases (10%) with strong signals.
Using tissue microarrays for a random sample of 79 low-grade papillary urothelial
neoplasias, only patchy LTag and p53 co-staining was detected (0.63% or 2 of 316
tumor spots). Similarly, Roberts and colleagues (Roberts et al. 2008) interrogated
the local pathology registry for urothelial cancers in patients without and with a history of renal transplantation. All of the 20 cases without transplantation were negative
for LTag, whereas 1 of 8 transplanted cases had a positive immunohistochemistry for
LTag in tumor cells while adjacent cells were negative (Roberts et al. 2008). Taken
together, urine cytology may identify patients at increased risk for urothelial abnormalities including cancer. A role of BKV in the genesis of urothelial cancers is
possible, and high-level urine viral loads or decoy cells may serve as a risk factor,
but sofar, this association has not been documented with sufficient stringency.
The role of BKV in prostate cancer has also been addressed by several recent
studies. Using broad-range molecular approaches, Sfanos et al. detected BKV in 1%
of prostate carcinoma tissue, EpsteinBarr virus in 8%, and large number of different
bacterial species (Sfanos et al. 2008). The data illustrate that urogenital tissues may
be a habitat of diverse agents and emphasize the need for specific studies to unravel
pathologic contributions (Sfanos and Isaacs 2011). The development of prostate
cancer appears to proceed along several histologically defined presentations called
proliferative inflammatory atrophy (PIA), prostatic benign intraepithelial neoplasia
(PIN), and prostatic carcinoma. Molecular studies suggested that there are low expression levels of relevant tumor-suppressor genes, including the p27 cyclin-dependent
kinase inhibitor and the phosphatase and tensin homologue PTEN, and mutations in
the p53 and pRB1 are generally rare (Gonzalgo and Isaacs 2003). The Imperiale
group investigated the possibility that BKV infection could contribute to prostate
cancer progression by interfering with p53 and pRB1 (Das et al. 2004, 2008). Using
PCR and in situ hybridization on biopsy tissues, BKV DNA was detected in the
epithelium of benign ducts and in PIA lesions in more than 70% of cases. In tissue
sections and by selective laser capture microscopy, LTag was shown to colocalize with
wild-type p53 in the cytoplasm of PIN and PIA cells, whereas a mixture of wild-type
and mutant p53 was found in carcinoma cells. The LTag colocalization in the cytoplasm with the wild-type p53 suggested that sequestration was more important than
the nuclear functions of this interaction, including those required for viral replication
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427
and late gene expression. Expression of VP1 was absent arguing that BKV early
gene expression was indeed uncoupled from late gene expression. However, the
absence of LTag in the carcinoma lesions has been counterintuitive and interpreted
as an indication for a hit-and-run mechanism: LTag exerts its proliferative and
antiapoptotic effects, and spawns further genetic alterations through chromosomal
instability during early stages in transition to cancer. The loss of LTag in the carcinoma indicates that LTag is no longer conferring proliferative advantage, and may
even become a target of cellular immunity. Thus, further independent studies still
need to corroborate the role of BKV in the progression to prostate cancer and to elucidate appropriate interventions, including vaccination and immunotherapy (Abend
et al. 2009; Provenzano et al. 2006).
There is extensive literature on the detection of BKV DNA in other cancers
reviewed recently (Abend et al. 2009). These include colorectal tumors, lymphomas,
pancreatic cancer, brain tumors, and a range of sarcomas, and the interpretation of
the results is controversial. On the other hand, no evidence for BKV sequences was
found in a case series of 38 non-UV light-associated melanomas, thereby arguing
against a role of these PyVs as an alternative genetic insult in this kind of malignancy
(Giraud et al. 2008b).
Conclusions and Outlook

The last decade has witnessed a significant change in our understanding of PyV
prevalence, biology, and pathology, particularly in humans. Thus, the role of BKV in
PyV-associated nephropathy after kidney transplantation is now accepted as a frequent
complication which is being met by national and international recommendations for
screening and early intervention. The role of BKV in human malignancies is suggested
by its oncogenic potential in vitro, in experimental animal models, but only limited
evidence is available from human studies. Lytic BKV replication seems to be a major
biological safeguard against oncogenic disease, but it is conceivable that immunocompromised patients with high-level BKV replication could be at an increased risk for
abortive infection that leads to functional or genetic uncoupling of early and late gene
expression. However, distinguishing a role of BKV as driver versus passenger and
innocent bystander needs to be stringently addressed in future studies.
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Chapter 17
Polyomavirus JC and Human Cancer:

Possible Role of Stem Cells in Pathogenesis
Kamel Khalili, Martyn K. White, Jennifer Gordon, and Barbara Krynska
Introduction
JC virus (JCV) is a ubiquitous human virus and a member of Polyomaviridae family of
DNA tumor viruses, which also includes BK virus and the well-known Simian virus 40
(SV40) (Imperiale and Major, 2007). JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML) (strm et al. 1958; Padgett et al, 1971), most frequently seen in patients with a disrupted immune system. In addition to its causative role
in PML, there is also experimental and clinical evidence for the role of JCV in cancer.
JCV DNA sequences, and expression of the viral oncoprotein T-antigen, were found in
a variety of human neural and nonneural tumors (see Table 17.1). JCV T-antigen, the
main regulatory protein encoded by the virus, has well-established oncogenic potential
in experimental animals and strong cell transforming ability in vitro (Khalili et al. 2003a,
b), suggesting that JCV-mediated cellular transformation may be involved in neoplasia.
However, despite a plethora of data supporting the oncogenic nature of JCV, whether
JCV has a causal role in human cancer remains controversial. Attempts to understand
the latency and dissemination of this virus indicate that the bone marrow plays an essential role in harboring persistent virus (Marshall and Major 2010). Given the growing role
of stem cells in the pathogenesis of human diseases, in particular cancer, an interesting
question is what role stem cells from the bone marrow may have in the pathogenesis of JCV-associated diseases. In this review, we highlight the migratory nature of
K. Khalili (*) M.K. White J. Gordon

Department of Neuroscience and Center for Neurovirology, Temple University School
of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA
e-mail: kamel.khalili@temple.edu
B. Krynska
Center of Neural Repair and Rehabilitation and Shriners Hospitals Pediatric Research Center,
Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA
Department of Neurology, Temple University School of Medicine, 3500 N. Broad Street,
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Table 17.1 Association of JC virus and human cancer

Tumor type
Reference
Oligodendroglioma
Rencic et al. (1996), Caldarelli-Stefano et al. (2000), Del Valle
et al. (2002a)
Astrocytoma
Boldorini et al. (1998), Caldarelli-Stefano et al. (2000),
Del Valle et al. (2001b)
Medulloblastoma
Krynska et al. (1999b)
Ependymoma
Caldarelli-Stefano et al. (2000), Del Valle et al. (2001b)
Glioblastoma
Del Valle et al. (2001b, 2002b)
Colorectal carcinoma
Laghi et al. (1999), Enam et al. (2002), Ricciardiello et al.
(2000), Nosho et al. (2009), Morikawa et al. (2011)
B-cell lymphoma
Del Valle et al. (2004)
Esophageal carcinoma
Del Valle et al. (2005)
Gastric cancers
Shin et al. (2006), Yamaoka et al. (2009), Ksiaa et al. (2010)
Anal carcinoma
Ramamoorthy et al. (2010)
bone marrow-derived stem cells and evidence on the recently discovered broad differentiation potential of nonhematopoietic stem cells from the bone marrow and discuss the
possible relationship between JCV and bone marrow-derived stem/progenitor cells in
relation to viral latency, distribution, and oncogenic capacity.
JCV: Latency, Reactivation, and Cellular Tropism

In PML, JCV replication in the brain causes the cytolytic destruction of oligodendrocytes, the myelin-producing cells, leading to lesions of demyelination and subsequent death of the patient. Antibodies to JCV are highly prevalent in human
populations worldwide indicating that JCV infection is common and widespread
(reviewed in White and Khalili 2011). The virus appears to infect most people during
childhood but may then enter a latent state, where JCV replication is low and asymptomatic. When the immune system is disrupted, most commonly in people with
HIV/AIDS or receiving immunosuppressive drug treatment, JCV emerges from latency
to become reactivated in the CNS leading to PML (Berger 2003). Before AIDS,
PML was very rare (Brooks and Walker 1984), but then increased dramatically
(Berger et al. 1987). PML is characterized by foci of myelin loss, enlarged oligodendrocytes with viral inclusion bodies and bizarre astrocytes, resembling oncogenically transformed cells (strm et al. 1958).
The transmission of JCV from person to person may be fecal/oral and involve an
archetype form of the virus (JCVCY), which is excreted in urine and found in sewage
(Bofill-Mas et al. 2003). Isolates of JCV from PML brain tissue have multiple rearrangements (deletions, duplications, and point mutations) in the control region
(NCCR) and are known as PML-type JCV, e.g., Mad-1. In contrast, archetype JCV
is present in the kidney of normal individuals and may replicate at low levels in renal
tubular epithelial cells. The relationship of archetype to PML type is complex and was
reviewed recently (White and Khalili 2011). Notably, PML-type JCV has recently
17 Tumorigenicity of Human Polyomavirus, JCV
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been detected in bone marrow of individuals without PML (Tan et al. 2009). Many
other tissues are reported to harbor latent JCV, including bone marrow and brain
(reviewed by White and Khalili 2011). Interestingly, while the kidney harbors archetype JCV, latent JCV in the normal brain and bone marrow is of the PML type.
JCV and Oncogenic Potential: Studies on the Molecular

Mechanisms
JCV encodes three capsid proteins, VP1, VP2, and VP3, in the late region, which
are only expressed in permissive cells during viral DNA replication. JCV has two
early proteins, large T-antigen (T-Ag) and small t-antigen (t-Ag), expressed in both
permissive and nonpermissive cells. In nonpermissive cells, these cause cell transformation via their dysregulation of cellular processes. Additionally, another viral
protein, agnoprotein, also dysregulates cellular processes (Khalili et al. 2004). JCV
T-Ag interacts with cellular proteins facilitating transition into S phase, and thus
transformation. T-Ag is a 688 aa nuclear phosphoprotein, which interferes with two
key tumor suppressors, pRb and p53 (Krynska et al. 1997). Aberrant stimulation of
the cell cycle drives oncogenesis (White and Khalili 2004; White et al. 2005; Del
Valle et al. 2008). Interaction of T-Ag with pRb activates E2F, advancing the cell
cycle (White and Khalili 2006), while its interaction with p53 may block p53
protection against DNA damage and transformation.
JCV T-Ag also affects other signaling proteins, including IRS-1 (Lassak et al. 2002;
Khalili et al. 2003a, b) and b-catenin, which it directly binds causing nuclear translocation
and enhanced c-myc and cyclin D1 expression (Enam et al. 2002; Gan and Khalili 2004;
Bhattacharyya et al. 2007). JCV T-Ag also associates with NF2 (Sholler et al. 2004).
The JCV early region also expresses the 172 aa small t-Ag, which shares its N
terminus with T-Ag but has a unique C terminus resulting from alternative splicing.
JCV t-Ag may function through its interaction with phosphatase PP2A (Khalili
et al. 2008; Sariyer et al. 2008). Another regulatory protein, agnoprotein, is encoded
in the late region (Khalili et al. 2004) and is a 71 aa protein with a characteristic
perinuclear subcellular localization as well as a small amount detected in the nucleus
(Del Valle et al. 2000). Agnoprotein dysregulates the cell cycle (Darbinyan et al.
2002) and impairs cellular responses to DNA damage (Darbinyan et al. 2004).
Importantly, JCV induces mutations in cellular DNA, which may contribute to
transformation (White et al. 2006). Thus, JCV infection can induce DNA damage,
impair DNA repair, increase chromosome ploidy, and elevate the levels of gH2AX
and Rad51 (Darbinyan et al. 2007).
Cell Culture and Animal Models of JCV Tumorigenesis

JCV transforms cells in culture, especially glial cells, such as primary cultures of
human fetal glial cells and primary hamster brain cells. JCV-transformed cells
exhibit the so-called transformed phenotype characterized by loss of contact inhibition,
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an ability to grow in soft agar, serum independence, morphological changes,

multinucleation, plasminogen activator production, and enhanced glucose uptake
and are tumorigenic when transplanted in vivo (Del Valle et al. 2001a, 2008).
JCV is highly oncogenic in laboratory animals and was found to induce multiple
types of tumors when injected into the brains of newborn Golden Syrian hamsters
(Walker et al. 1973). Notably, JCV is the only human virus that is able to induce
solid tumors in nonhuman primates and has been found to cause astrocytomas,
glioblastomas, and neuroblastomas in owl and squirrel monkeys, which develop
1624 months after intracerebral inoculation of JCV (Del Valle et al. 2001a, 2008).
The analysis of tumor tissue revealed the expression of the JCV early protein
T-antigen, but no evidence of viral replication or persistent infection could be detected.
Similarly, inoculation of JCV into the brains of newborn rats resulted in induction
of undifferentiated neuroectodermal tumors in the cerebrum of 75% of experimental
animals (Ohsumi et al. 1985, 1986). These studies indicate that expression of
T-antigen in the absence of viral lytic infection may lead to cellular transformation.
Among the most interesting observations on the oncogenecity of JCV are studies on
several lines of transgenic mice which contained the entire gene for JCV T-antigen
under the control of its own promoter. As shown in this animal model system, in the
absence of virus and therefore viral replication, T-antigen is capable of transforming
cells and induces a variety of tumors. Earlier studies by Small et al. (1986) using the
Mad-4 viral promoter driving T-antigen expression reported adrenal neuroblastomas while Franks et al. (1996) generated transgenic mice that developed solid
mesenteric tumors with tumor cells exhibiting characteristics of primitive epithelial/
neuroectodermal origin. Transgenic mice with the JCV early region under the control
of Mad-4 promoter can also develop tumors arising from the pituitary gland (Gordon
et al. 2000) and some of these animals developed solid masses arising from the soft
tissues surrounding the salivary gland, the sciatic nerve, and along the extremities
that are histologically compatible with malignant peripheral nerve sheath tumors,
rare neoplasms that occur in individuals with neurofibromatosis (Shollar et al.
2004). In another series of transgenic animal studies using the archetype or kidneyderived isolate of JCV as a source of transgene, a wide range of tumors, including
primitive neuroectodermal tumors (PNETs), medulloblastomas, pituitary tumors,
malignant peripheral nerve sheath tumors, adrenal neuroblastomas, and glioblastoma
multiforme, were identified (Krynska et al. 1999a; Pina-Oviedo et al. 2007).
Some features of these experimental tumors resemble JCV-associated human cancers
discussed below, i.e., the nonpermissive nature of the JCV infection, high T-Ag expression in tumor cells, and lack of late capsid protein expression or replication.
Association of JCV with Human Malignancies

There are many reports from a number of different laboratories of the association of
JCV with human cancer either by virtue of the occurrence of the cancer concomitantly with PML or by the molecular detection of viral footprints in neoplastic cells
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(see Table 17.1). The first indication for the association of JCV with tumors of the
CNS came from reports of brain tumors being found in patients with coexistent
PML as exemplified by a case reported by Richardson (1961) who first described
PML. A patient with chronic lymphocytic leukemia and PML was found to have an
oligodendroglioma on postmortem examination. Other reported cases have been
reviewed by White et al. (2005). The finding of brain tumors associated with PML
is consistent with a role for JCV in tumor development, but it is also possible that
the cancer develops secondarily to the immunosuppression that leads to PML.
JCV has also been reported to be associated with neural tumors in patients without
PML. Rencic et al. (1996) reported JCV DNA detected by PCR in tumor tissue from
a patient with an oligoastrocytoma. The PCR product was confirmed as JCV by
DNA sequencing and JCV RNA and T-Ag protein were detectable in the tumor by primer
extension analysis, Western blot, and immunohistochemistry, respectively, i.e., JCV
gene expression occurred in the tumor. Del Valle et al. 2001b examined 85 samples
of various tumors of glial origin for JCV DNA and T-Ag expression and found
that 5783% of tumors were positive. In other studies, JCV has been reported to
be associated with CNS lymphoma, glioblastoma multiforme, oligoastrocytoma,
oligodendroglioma, medulloblastoma, and xanthoastrocytoma (reviewed by Del
Valle et al. 2001a; White et al. 2005).
As well as the CNS, JCV has also been reported in tumors outside of the CNS.
JCV DNA sequences have been detected in normal tissue samples taken from the
upper and lower human gastrointestinal tract and in colon cancer (Laghi et al. 1999;
Ricciardiello et al. 2000). In a study of malignant epithelial tumors of the large
intestine, expression of JCV T-Ag and agnoprotein was found in about half of
the samples (Enam et al. 2002) but not in any of the samples of normal gastrointestinal epithelial tissue. Similar results were reported for the upper GI tract. JCV
DNA was found in 11 of 13 normal esophageal biopsies (85%) and 5 of 5 esophageal carcinomas (100%). By immunohistochemistry, JCV T-Ag expression was
detected in 10 of 19 carcinomas (53%) and agnoprotein in 8 (42%) while none of 51
normal, benign, or premalignant esophageal samples expressed viral proteins (Del
Valle et al. 2005). In other studies JCV T-Ag was detected in colon and rectal cancer
cases evaluated by immunohistochemistry (Morikawa et al. 2011), gastric cancers
(Shin et al. 2006; Yamaoka et al. 2009; Ksiaa et al. 2010), and anal carcinoma
(Ramamoorthy et al. 2010). Thus, JCV is associated with tumors throughout the GI
tract. Other studies have begun to define molecular differences between JCV
T-Ag-positive and -negative tumors. For example, Nosho et al. (2009) detected
JCV T-Ag expression by immunohistochemistry in 271 (35%) of 766 colorectal
cancers and T-Ag expression was significantly associated with p53 expression
(P < 0.0001), p21 loss (P < 0.0001), chromosomal instability (P < 0.0001), nuclear
b-catenin (P = 0.006), and microsatellite instability (P < 0.0001), but not with
PIK3CA mutation.
Recently, the association of JCV with tumors was reexamined using immunocytochemistry of commercially available tissue arrays, including colon adenocarcinomas. Expression of both T-Ag and agnoprotein, but not capsid proteins, was found
in some, but not all, tumors of neural and nonneural origin. Notably, more than 40%
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of human glioblastomas and greater than 30% of colon adenocarcinomas were

found to express viral proteins (Del Valle and Khalili 2010).
Despite the accumulating reports of an association of JCV with human cancer,
there have also been reports that failed to detect JCV DNA in panels of human
tumors. For example, Herbarth et al. (1998) failed to amplify JCV DNA from 30
brain tumors classified as oligodendroglioma or astrocytomas and 22 human glioma
cell lines and did not detect T-Ag expression by Northern blot. This and other negative studies and possible technical issues that may be responsible for these disparities
have recently been reviewed (Del Valle et al. 2008). It remains unknown why studies
for JCV DNA in normal and tumor tissues have yielded such variable results and the
extent of association of JCV with human tumors is still an area of controversy.
JCV and Stem Cells: Brain and Bone Marrow

The hypothesis has been proposed that JCV persistently infects hematopoietic stem/
progenitor cells in the bone marrow and circulating B cells, which arise from the
hematopoietic lineage, most likely transport the virus to the sites of latency or lytic
replication in the brain (Marshall and Major 2010). It is believed that in the brain the
hematopoietic cells can infect neighboring oligodendrocytes leading to lytic infection of oligodendrocytes and the development of PML and/or abortive infection of
astrocytes and neurons possibly resulting in the development of tumors (Khalili
et al. 2003a, b). However, recent studies have suggested that non-PML brain tissue
may also harbor the latent virus (reviewed by White and Khalili 2011). The biology
and molecular characteristics of JCV have been the subject of intense research; however, the initiating event, i.e., the clinical status of patients and at what stage of cell
differentiation the virus can infect cells in the brain, is largely unknown. Interestingly,
human fetal brain-derived progenitor cells with the ability to differentiate into neuronal
or astrocytic lineage have been shown to support viral infection in culture (Hou et al.
2006). Additionally, it has been demonstrated that oligodendrocyte progenitors
derived from a human embryonic stem cell line could be infected by JCV
(Schaumburg et al. 2008). However, whether JCV infects progenitor or stem cells in
the brain of PML patients has not been shown. Given the strong presence of JCV in
both the brain and the bone marrow, an interesting question is whether stem cells
play a role in vivo and how the stem cells from the bone marrow and brain may be
involved in the pathogenesis of JCV-associated diseases.
Adult bone marrow contains hematopoietic stem and progenitor cells that generate
all of the major blood cell lineages (Till and McCulloch 1961, 1964; Morrison et al.
1995). The involvement of the hematopoietic lineage in trafficking of the JC virus to
the brain is supported by data showing that infected, circulating B lymphocytes can
carry the virus to the brain and studies showing the presence of JCV DNA in B lymphocytes isolated from a PML patient (Houff et al. 1988; Tornatore et al. 1992).
Furthermore, the infection of CD34-positive hematopoietic cell lines, and primary
CD34-positive hematopoietic progenitor cells isolated from human fetal liver,
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demonstrated the susceptibility of hematopoietic cells to JCV infection (Monaco

et al. 1996). The bone marrow has been reported to be a site of JCV latency, where
neurotropic transformation of the virus could possibly take place, as the regulatory
region of JCV identified in bone marrow aspirates correlates with the PML-type
regulatory region of JCV found in the brain (Tan et al. 2009). However, recent studies
in the context of cases of PML occurring in multiple sclerosis patients show that
CD34-positive cells mobilized to the circulation by natalizumab do not contain JCV
DNA (Warnke et al. 2011). Thus, while JCV DNA can be extracted from bone marrow
aspirates, and bone marrow has been reported to be an important site of JCV latency,
the phenotype of the cell in the bone marrow and the phenotype of the cell in the blood
seeding JCV throughout different organs of the human body remain unknown.
New studies show that in addition to stem/progenitor cells of the hematopoietic
lineage, the bone marrow contains a heterogenous population of nonhematopoietic
stem and progenitor cells that have the ability to differentiate into a variety of nonhematopoietic tissues (Prockop 1997; Jiang et al. 2002; Kucia et al. 2004, 2006a;
Keene et al. 2003). These cells exist during development and persist in small numbers
in adults (Pittenger et al. 1999; Colter et al. 2000; Kucia et al. 2006a). Some of the
nonhematopoietic cells may enter the circulation and traffic throughout different
organs, where they may be involved in the maintenance of homeostasis and/or repair
of solid organs (Prockop et al. 2003; Sasaki et al. 2008; Mezey et al. 2003; Cogle
et al. 2004). These cells can be mobilized from the bone marrow into peripheral
blood and are actively recruited to sites of injury, further suggesting a role for these
cells in tissue maintenance and repair (Rochefort et al. 2006; Sasaki et al. 2008;
Kucia et al. 2006b; Paczkowska et al. 2009). However, in pathological situations,
bone marrow-derived cells can also contribute to the pathogenesis of diseases, such
as atherosclerosis (Sata et al. 2002), gastric cancer (Houghton et al. 2004), osteosarcoma
(Stark et al. 1986; Berger et al. 2008), or childhood leukemia (Greaves 1999).
The presence of JCV in the bone marrow suggests potential scenarios in which
nonhematopoietic bone marrow cells may be involved in infection with JCV. In
pathological situations, bone marrow-derived multipotent nonhematopoietic cells
harboring JCV could be recruited to different tissues, where they potentially could
undergo neoplastic conversion into tumorigenic cells and contribute to the development of tumors. Thus, we propose that in addition to the hematopoietic fraction of
the bone marrow, the nonhematopoietic subpopulation of bone marrow cells can be
another potential source of JCV latency, spreading, and ultimately oncogenic
transformation.
Pathogenesis of JCV: Why Nonhematopoietic

Bone Marrow-Derived Stem Cells?
The role of stem cells in the pathogenesis of human diseases, in particular the development of cancer, has recently gained a lot of attention. Cancer is viewed as a stem cell
disease because of accumulating data suggesting that cancers are maintained by a
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small population of cancer cells functioning as stem-like cells, as well as evidence that
stem cells are a possible origin of some cancer stem cells (Reya et al. 2001; Dontu
et al. 2003; Singh et al. 2004; Xie 2009). There are several indications that some JCV
T-Ag-associated malignancies may in fact originate in primitive nonhematopoietic
bone marrow-derived stem cells that can migrate and are often found in multitude of
tissues. As mentioned above, the nonhematopoietic stem-like cells coexist in the bone
marrow and may be a possible source of neural and nonneural cell types (Lepski et al.
2010 Sasaki et al. 2008). It is well-known that JCV has a strong association with the
bone marrow and the nervous system (reviewed by White and Khalili 2011), and JCV
T-Ag is associated with neural and nonneural tumors (see Table 17.1). Thus, these
unique bone marrow-derived stem cells, which have the ability to migrate and a potential to transition into nonneural and neural-like cells in homed tissues, may be involved
in JCV dissemination and pathogenesis. From the cancer development point of view,
recent studies have shown that tumors may arise from the transformation of normal
stem/progenitor cells into tumorigenic cancer stem cells, which are responsible for the
initiation, maintenance, and recurrence of tumors after therapy (Reya et al. 2001;
Singh et al. 2004; Xie 2009). Given that JCV induces neuronal and glial tumors in
animal models and has been associated with these types of tumors in humans, one may
anticipate that neural stem or progenitor cells may be the target of T-Ag-induced transformation in the brain. However, other evidence demonstrates the association of JCV
with peripheral neuroblastic tumors in animal models and nonneural types of cancers,
including colorectal cancers and cancers of the gastrointestinal tract in humans, which
further suggests alternative models of JCV dissemination throughout different organs
and other cells of origin of T-Ag-induced malignancies. Because of the widespread
nature of JCV, one source of ubiquitous stem cells may be the bone marrow-derived
nonhematopoietic stem cells that can spill into the circulatory system from the bone
marrow and ultimately reside in various tissues (Rochefort et al. 2006; Sasaki et al.
2008; Paczkowska et al. 2009). It is possible that JCV-infected nonhematopoietic stem
cells trafficking through the body could take up residence in the brain and give rise to
cells that can undergo lytic infection, i.e., oligodendrocytes leading to development of
PML, or alternatively can give rise to neurons or astrocytes that undergo transformation and generate astrocytic or neuronal types of brain tumors (Fig. 17.1). The concept
that mobile bone marrow-derived multipotent stem cells, with the ability to give rise
into brain and other cell types (Rochefort et al. 2006; Sasaki et al. 2008; Paczkowska
et al. 2009; Lepski et al. 2010), may be the common cell of origin of T-Ag-related
malignancies perhaps could explain the association of JCV with different types of
tumors found in the brain and non-CNS tissues.
In addition to the above considerations, support for a stem cell origin of T-Aginduced malignancies comes from the fact that signaling pathways (WNT, p53,
pRB) and mechanisms by which T-Ag may lead to cellular transformation and
development of tumors in animal models appear to be essential for the development
and regulation of stem cells (Molofsky et al. 2004; Yang and Hinds 2007; Xie 2009).
Taken together, these data suggest that T-Ag expression may potentially dysregulate
pathways of stem cell self-renewal and contribute to neoplastic transformation of
these cells.
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Fig. 17.1 JCV infection and dissemination from the bone marrow. Hypothesis for JCV infection
of nonhematopoietic stem cells from the bone marrow in dissemination of JC virus in the host and
pathogenesis of JC virus-induced diseases. Bone marrow harbors latent JC virus, where viral DNA
replication may occur sporadically and at a low level establishing a persistent long-lived infection.
Nonhematopoietic cells harboring JC virus can enter the circulation and transport the virus to the
brain and other organs. Once in the brain, the bone marrow-derived stem cells may differentiate
into cells permissive for viral replication, i.e., oligodendrocytes, or can lead to the infection of
native oligodendrocytes, both of which result in lytic infection and the development of PML.
Alternatively, nonhematopoietic stem cells may differentiate into neuronal or astrocytic cells
which are non- or semipermissive for JCV lytic infection, in which initiation of T-antigen expression can induce oncogenic transformation leading to the development of glial or neuronal tumors
in the brain. JC virus can also be transported to other organs via nonhematopoietic stem cells from
the bone marrow leading to the development of non-CNS tumors (not shown)
Transformation of Bone Marrow-Derived Nonhematopoietic

Cells by JCV T-Ag
An important question related to the T-Ag-induced transformation of nonhematopetic bone marrow-derived cells is whether these cells can be transformed by T-Ag
and can generate tumorigenic cells. Recently, new data from our group has shown
that nonhematopoietic cells from the bone marrow, commonly described as mesenchymal stem cells or multipotent stromal cells, can be transformed by JCV T-Ag in
culture. When T-Ag-transformed cells were propagated as xenografts in the flanks
of nude mice, they developed tumors. Histologically, the tumors were heterogenous
with mesenchymal, neural, and neural crest characteristics (Del Valle et al. 2010). It
is assumed that oncogenesis is a reversal of ontogeny in cell development (Rapp
et al. 2008). Thus, our data is particularly interesting as recent reports suggest that
nonhematopoietic mesenchymal cells from the bone marrow may be of neural crest
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origin (Miller 2007). Notably, our data show T-Ag-mediated oncogenic transformation
of nonhematopoietic bone marrow cells with multipotent capacities that are tumorigenic in vivo. Phenotypical heterogeneity of tumors that developed in the flank
suggests that such multipotent cells transformed with JCV T-Ag may be the origin
of some T-Ag-associated neural and nonneural tumors, in particular when influenced
by the microenvironment of surrounding host tissues. Thus, these data provide this
data provides a novel stem cell model of T-Ag-induced oncogenic transformation
that may lend some support for the aforementioned hypothesis that some T-Agassociated tumors may originate from primitive bone marrow-derived multipotent
cells that persist in adults and have the ability to differentiate into variety of nonhematopoietic cell types. Our model is supported by observations made by others, in
which various tumor types developed after subcutaneous injection of transformed
bone marrow-derived cells (Liu et al. 2006), and evidence from animal models of
gastric cancers showing that epithelial tumors may arise from bone marrow-derived
cells during chronic infection (Houghton et al. 2004).
Conclusions
JCV transformation and tumorigenesis are well-established in cell culture and
experimental animal models while their role in human cancer remains contentious.
JCV exerts a variety of molecular effects, including the action of the oncogenic
protein, T-Ag, on the tumor suppressors p53 and pRb as well as the induction of
DNA damage, as described above. The nature of JC viral DNA in tumors is unknown.
JCV may be present in stem cells in the bone marrow and perhaps be carried via the
blood stream to the brain and other organs. Notably, JCV in the bone marrow is
rearranged in the noncoding region, i.e., PML-type configuration, which is in
contrast to the reservoir of JCV in the kidney, which has an archetypal configuration.
Interestingly, some JCV-associated tumors contain viral DNA with PML-type
sequences suggesting that the rearrangements necessary for pathogenesis of PML
may also occur for virus that becomes associated with tumors.
It is important to stress that studies have found that JCV VP1 is not expressed in
JCV-associated tumors, indicating that viral replication is not occurring. Expression
of JCV T-Ag and the more recently identified agnoprotein is usually robust in tumor
cells. In the case of T-Ag, expression was found to occur in some, but not all, of the
tumor cells containing JCV DNA in JCV-associated human medulloblastoma
(Khalili et al. 1999; Krynska et al. 1999b). This is also found in JCV-transgenic
mice, where immunohistochemical analysis of JCV-induced mouse medulloblastomalike tumors showed a heterogenous population of T-positive and T-negative cells
(Krynska et al. 2000). The molecular and cellular processes that drive the heterogeneity
of T-Ag expression among the tumor cells are not yet known. However, this phenomenon is very interesting in the light of the cancer stem cell hypothesis of tumor
443
development and raises questions as to whether JCV T-Ag is expressed in the fraction of cancer stem cells that replicate to drive the growth of tumor.
In view of the reports on the association of JCV with a broad variety of tumors
and the accumulating evidence maintaining that cancer may originate from transformation of stem cells, we raise several important considerations related to the pathogenesis of JCV: (1) stem/progenitor cells may be a target of JCV T-Ag-induced
oncogenic transformation; (2) we envision that ubiquitous nonhematopoietic
bone marrow-derived stem/progenitor cells, which have been postulated to give
rise to various cell types, including brain, may not only be the origin of some of
T-Ag-associated cancers, but also play a role in dissemination of JCV to the brain
and other organs. Therefore, we hypothesize that potential persistent infection of
mobile nonhematopoietic bone marrow cells with JCV can contribute to viral dissemination into the CNS. Notably, JCV in the bone marrow is rearranged in the noncoding
region, i.e., PML-type configuration (Tan et al. 2009). In the brain, nonhematopoietic bone marrow-derived cells harboring JCV may undergo differentiation toward
permissive for viral replication in oligodendrocytes that may result in the development of PML or may undergo differentiation toward cells of neuronal or astrocytic
type, which are non/semipermissive for viral replication, where T-Ag expression
may potentially induce oncogenic transformation and generation of cancer stem
cells leading to the development of tumors in the brain (Fig. 17.1). Another possibility
is that in the brain JCV may infect resident neural stem/progenitor cells, which
differentiate into major cell types in the brain. Infected oligodendrocytes undergo
lytic infection resulting in destruction of the host cell and demyelination seen in
PML while astrocytic cells allow expression of T-antigen but cannot complete the
lytic cycle, leading to cellular transformation and tumor formation (Fig. 17.2).
These two models are not mutually exclusive. However, we are aware that this
hypothesis requires experimental evidence to support our concept. We propose that
the research exploring the oncogenic potential of JCV T-Ag that is focused on novel
in vitro and in vivo models of T-Ag-induced oncogenic transformation with the
contribution of adult stem/progenitor cells from the bone marrow and brain and the
identification of cancer stem cells in T-Ag-associated cancers, in particular human
cancers, is necessary in order to better understand the role of JCV T-Ag in cancer.
In summary, available data suggest that JCV is a ubiquitous virus that usually
exists in a latent state as an asymptomatic infection of the bone marrow and/or
brain and perhaps spreads by intermittent and subclinical infection events. JCV
involvement in tumors may be caused by very rare abortive infection event giving
rise to tumorigenic cells driven to grow progressively by T-Ag expression. A better
understanding of these processes may lead to advances in understanding the role of
JCV in human cancer.
Acknowledgments We wish to thank members of the Department of Neuroscience and Center
for Neurovirology for their continued support, insightful discussion, and sharing of ideas.
444
K. Khalili et al.
Fig. 17.2 JCV infection and transformation of neural stem cells. Bone marrow-derived stem cells
can traffic to the brain, where they infect resident neural stem cells. Neural stem cells infected with
JCV differentiate along the glial lineage into either oligodendrocytes, which are fully permissive
for viral replication, or into astrocytic cells which are semipermissive allowing for T-antigen
expression but not viral replication. Infected oligodendrocytes undergo lytic infection resulting in
destruction of the host cell and demyelination seen in PML while astrocytic cells allow expression
of T-antigen but cannot complete the lytic cycle, leading to cellular transformation and tumor
formation
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Chapter 18
Merkel Cell Polyomavirus

David Schrama and Jrgen C. Becker
Polyomaviruses
The first polyomaviruses have been identified in mice in the early 1950s, i.e., the K
virus [now known as the murine pneumotropic virus] and the murine polyomavirus
(Kilham 1952; Gross 1953). The name polyomavirus, however, was first given in
1958 due to its ability to produce a variety of solid tumors (Stewart et al. 1958).
In the species list 2009 version 7 of the International Committee on Taxonomy of
Viruses (ICTV), 13 different species are listed. Among them, simian vacuolating
virus 40 (SV40) might be the most prominent member. SV40 was found infecting
rhesus monkey kidney cells used for the production of polio vaccines (Sweet
and Hillemann 1960) raising health concerns due to the widespread administration
of the polio vaccine and the tumorigenicity of SV40 in experimental models.
Notably, despite intense scientific effort, a causal relationship between SV40 and
human cancers could not yet been established. However, these studies on SV40
contributed largely to our understanding of viral oncogenesis and cellular biology
(Cheng et al. 2009).
Of the 13 species of polyomavirus listed by ICTV in 2009, 4 are human species.
Two of these are the JC polyomavirus (JCV) and BK polyomavirus (BKV) associated
with progressive multifocal leukoencephalopathy in immunocompromised hosts
and nephropathy in renal transplant recipients, respectively. Two others were isolated
from respiratory samples in symptomatic pediatric patients and named after the
institutions where they were identified, i.e., the Karolinska Institute virus (KIV) and
Washington University virus (WUV). Within the list of the ICTV, however, the
newest human members are not yet included. Indeed, in the last 2 years, four new
D. Schrama J.C. Becker (*)

Division of General Dermatology, Department of Dermatology, Medical University of Graz,
Auenbruggerplatz 8, A-8036 Graz, Austria
e-mail: juergen.becker@medunigraz.at
449
450
D. Schrama and J.C. Becker
Fig. 18.1 Appearance and structure of a polyomavirus icosahedral capsid and MCV genome.
(a) The viral capsid is composed of 72 pentamers of the major capsid protein VP1 that contacts 72
copies of the minor capsid proteins VP2/3 arranged on an icosahedral lattice. The appearance of
SV40 serves as an example. (b) Map of MCV genome organization. VP3 is not depicted, since
currently there is no indication that VP3 is generated by internal translation from the MCV genome.
However, the MCV genome encodes a pre-miRNA (MCV-mir-M1) which is expressed from
the late strand, MCV-mir-M1 lies antisense to the early transcripts, and can negatively regulate
early transcripts expression. The mature sequences are MCV-mir-M1 5p and MCV-mir-M1 3p.
In MCC, the MCV miRNA probably plays no role as the tumors are likely only competent to
express early gene products, and the miRNA is presumably only expressed during lytic infection
when the late genes are expressed (Seo et al. 2009)
species were reported (Feng et al. 2008; Schowalter et al. 2010; van der Meijden
et al. 2010): Merkel cell polyomavirus (MCV or MCPyV), human polyomavirus-6
(HPyV6) and HPyV7, and trichodysplasia spinulosa-associated polyomavirus
(TSV). Thus, currently, eight human polyomaviruses have been described.
Polyomaviruses are nonenveloped, 4045-nm icosahedral capsids containing
approximately 5 kb of double-stranded, circular DNA. The DNA closely associated
with histones is packaged into chromatin-resembling cellular genomes (minichromosomes). The polyomavirus genome is almost evenly divided into an early and a
late region encoded on opposite strands (Fig. 18.1). Gene expression is regulated by
a noncoding, transcriptional control region, which also harbors the origin of DNA
replication. Transcription of the early region from the early promoter starts immediately upon entry and uncoating of the genome. Early message is differentially
spliced to encode at least two and up to five proteins in the respective polyomaviruses; the large tumor (T) antigen (LT) and small T antigen (sT) are invariantly
expressed in all polyomaviruses. The T antigens are the oncogenically transforming
proteins as they initiate viral DNA replication. After the onset of viral DNA replication, the late region is expressed from the late promoter. From the late message,
three to four capsid proteins VP1, VP2, VP3, and VP4 are generated by differential
splicing and internal translation. In this regard, VP3, and VP4 when present, is generated by internal translation of VP2 from an in-frame methionine (Met) codon.
Since MCV lacks the conserved Met-Ala-Leu motif that forms the amino-terminus
of all previously described polyomavirus VP3 proteins, MCV may encode only
VP1 and VP2, but not a functional VP3 protein (Pastrana et al. 2009) (Fig. 18.1).
18
451
Polyomaviruses replicate their genomes in the nucleus of the host cell. Since they
encode only a few proteins, polyomaviruses heavily rely on cellular proteins for
both DNA replication and gene transcription.
Merkel Cell Carcinoma and MCV Detection

In 1972, Toker described five patients with unusual skin tumors characterized by
histologically anastomosing trabeculae and cell nests (Toker 1972). In 1982, Rywlin
suggested to name this tumor Merkel cell carcinoma (MCC) after the possible cell
of origin, the Merkel cell, which functions as slowly adapting mechanoreceptor in
the basal layer of the epidermis (Rywlin 1982; Maricich et al. 2009). For a long
time, Merkel cells were believed to be derived from the neural crest. Thus, the
hypothesis from several groups that MCC may arise from primitive totipotent epidermal stem cells, able to differentiate along different cell lines, was contradictory
to the initial hypothesis (Albores-Saavedra et al. 2009). Recently, however, the
report that mammalian Merkel cells do not develop from neural crest progenitors
but rather from epidermal stem cells conciliated these two hypotheses (Van
Keymeulen et al. 2009; Morrison et al. 2009). Notably, MCCs express both
neuroendocrine and epithelial markers.
MCC is an uncommon cutaneous malignancy, with age-adjusted incidence rates
of 0.180.41 per 100,000 persons. MCCs are most often localized on the sun-exposed
skin of Caucasians older than 50 years. The mean age of patients at the time of initial
diagnosis is about 75 years (Albores-Saavedra et al. 2009). In a study of 6,700 MCC
patients, 66% were diagnoses with local, 27% with nodal, and 7% with distant metastatic diseases (Lemos et al. 2010). The initial stage largely affects 5-year survival
rate with 64% for patients with local, 39% for patients with regional, and 18% for
patients with distant metastatic disease (Lemos et al. 2010).
MCC occurs much more frequently in severely immunosuppressed populations
caused, for example, by immune-suppressive drugs in organ transplant patients,
lymphoma, or HIV infection (Rollison et al. 2010). Noteworthy, many cancers with
infectious etiologies are more often observed in the context of immunosuppression,
such as Kaposis sarcoma, lymphomas, cervical, oropharyngeal, and penile cancers
in HIV-infected individuals (Engels et al. 2008) and nonmelanoma skin cancers,
lymphomas, and cancers of the oral cavity, vulva, and vagina in organ transplant
recipients (Adami et al. 2003). Thus, the observation that MCC is also more
common in immunocompromised individuals first raised the hypothesis that MCC
may also have an infectious origin. Indeed, Feng and coworkers were able to provide
evidence for a possible viral oncogenesis of MCC (Feng et al. 2008). They applied
the digital transcriptome subtraction (DTS) technique to specifically seek for an
infectious agent in MCC. DTS involves transcriptome sequencing followed by in
silico subtraction to exclude human from candidate viral transcripts (Feng et al.
2007). Pooling cDNA libraries made from four MCC lesions, a DTS candidate was
detected demonstrating a significant degree of similarity to the LT of the African
452
green monkey lymphotropic polyomavirus (LPV). The complete genome of 5,387 bp

was subsequently sequenced by PCR walking using primers designed from the DTS
viral transcript [Figure 1; (Feng et al. 2008)] After their initial identification of the
MCV genome, they verified MCV presence in eight of ten MCCs. In six of them,
they even could detect the viral genome integrated into the human genome.
Importantly, the patterns of integration suggest that MCV infection/integration
occurs before the clonal expansion of the tumor cells. Meanwhile, MCV presence
in MCCs has been generalized to geographically diverse populations with about
80% of MCCs positive for MCV (reviewed in Rollison et al. 2010). The fraction of
MCV-negative MCCs suggests that MCC is a heterogenous disease having at least
two pathoetiologies. Interestingly, until recently, MCVs closest relatives known to
date were LPV from some African green monkeys and ChPyV from one chimpanzee.
This was fortified by a report of Leendertz et al. identifying two new groups of
polyomaviruses in chimpanzees and gorillas, demonstrating that the great apes the
closest phylogenetic relatives of humans are infected with such polyomaviruses,
which are by far the closest known relatives to MCV (Leendertz et al. 2010).
Several serological and molecular studies have meanwhile established that MCV
is prevalent in the general population (Schowalter et al. 2010). Indeed, in a recent
study of Weiland et al., MCV was found in anogenital, oral samples (31%), and
eyebrow hairs (50%) of HIV-positive men as well as in 8 of 13 forehead swabs
(62%) of healthy controls suggesting a widespread distribution of MCV (Wieland
et al. 2009). Moreover, infection with MCV is common: 4277% of subjects from
the general population have antibodies to capsid proteins of MCV. For example,
among 166 healthy blood donors from the USA, IgG antibodies against MCV VP1
and VP2 proteins were observed in 107 (64%) (Kean et al. 2009). Interestingly, the
anti-VP1 antibody titers were significantly higher in patients with MCV-positive
MCC as compared to patients with MCV-negative tumors or healthy controls. In a
separate study of 1,501 healthy adult blood donors, the prevalence of IgG antibodies
against MCV VP1 was 46% (Tolstov et al. 2009). Although MCV prevalence
increases in an age-dependent manner, initial exposure to MCV seems to occur already
at very young ages, with seroprevalences of 20, 30, and 40% for children aged 15,
510, and 1015 years, respectively (Kean et al. 2009). In contrast to VP antibodies,
antibodies recognizing MCV T antigens are relatively specifically associated with
MCC and almost not detectable in healthy individuals. Moreover, although they do
not effectively protect against disease progression, they may serve as a clinically
useful indicator of disease status (Paulson et al. 2010).
Given the widespread presence of MCV, its mere detection in most of the MCC
cases is certainly only a first indication for the involvement of this virus in the
etiology of these cases. It could also be just a bystander virus. For example, MCV
presence has been infrequently observed in other, non-MCC cancers albeit often
with a lower copy number of virus per genome (Becker et al. 2009a; Murakami
et al. 2011; Andres et al. 2009). In experimental models, polyomaviruses induce
tumors upon integration of the viral genome into the host genome (Poulin and
DeCaprio 2006). Integration might render the viral infection oncogenic by disrupting
viral genes, by creating viruscell fusion proteins, or by dysregulating expression of
18
453
cellular oncogenes or tumor-suppressor genes. Therefore, it is important that the

initial report of MCV by Feng and colleagues not only noted an integration in six of
the eight MCV-positive MCC samples, but that the banding pattern on Southern
blots also indicated a monoclonal integration (Feng et al. 2008). Subsequent studies
confirmed clonal integration in ten out of ten MCV-positive tumors examined
(Sastre-Garau et al. 2009). Notably, integration sites varied between different MCC
tumors suggesting that this event is likely to occur at random sites in the host
genome. Since primary and metastatic tumor samples of one MCV-positive MCC
patient exhibited an identical integration pattern, MCV integration most likely
occurs before tumor metastasis. Thus, these observations suggest that infection and
subsequent integration precede clonal expansion of tumor cells (Fig. 18.2). This
assumption should translate into at least one MCV copy per cell. Indeed, several
studies reported average copy numbers of more than one MCV genome per cell
(Shuda et al. 2009; Laude et al. 2010; Houben et al. 2010a). However, Bhatia et al.
reported that many of the MCV-positive tumors had less than 1 virus copy per 300
cells (Bhatia et al. 2010b), and certainly in most studies there are at least some
samples with a low copy number. Future studies have to clarify whether those samples
lost the virus, are only contaminated by MCV, or that an infected cell can contribute
to transformation of neighboring uninfected cells by paracrine mechanisms as
suggested by Bhatia et al. (2010b).
Small and Large T Antigens, the Potential Oncogenic Proteins

Since MCV integrate just like SV40 in experimental models (Pipas 2009) at different
sites into the host genome, it seems unlikely that destruction of certain host genes
by integration contributes to the etiology of MCC. Indeed, the prime candidates for
a transforming activity of MCV are the early proteins, i.e., small and large T antigens,
which are referred to as tumor antigens because they were originally detected using
antibodies from tumor-bearing animals. The functions of MCVs sT and LT have
not yet been unraveled, as only a short period has passed since the discovery of
MCV. In contrast, much time and effort have been spent on the characterization of
SV40 T antigens. Thus, the following functional data refers mostly to findings on
SV40 biology. The involved mechanisms, however, are probably similar for MCV,
especially as MCV T antigens contain most of the respective domains (Fig. 18.3).
As mentioned before, polyomaviruses rely heavily on the cellular replication
machinery to replicate their genome because of their small genome size. Indeed, by
reprogramming the host cell cycle, they abrogate the quiescent state and induce
progression into S phase. Thereby, they create a suitable environment for viral replication. Importantly, the molecules targeted by the virus to promote unscheduled
DNA replication or to inhibit innate immune signaling in the setting of viral replication
are often the same as those involved in oncogenesis. In this regard, viral infection
leads to transformation of a range of cultured rodent and human cells and induces
tumors in newborn hamsters (Pipas 2009).
454
Fig. 18.2 Proposed viral carcinogenesis of Merkel cell carcinoma. GT1b has been identified as a
putative host cell receptor for MCV (Erickson et al. 2009). Polyomaviruses must enter host cells
by endocytosis and navigate through various intracellular compartments, where they undergo
sequential conformational changes which enable them to uncoat and deliver the DNA genome into
the nucleus (Tsai and Qian 2010). In MCCs, the MCV genome might get integrated into the host
genome, cause transformation, and is then found clonally integrated in all MCCs of one patient.
The interference of TA function might be a future target of MCC therapy, since knockdown in
MCV-positive MCC cell lines caused growth arrest and cell death
18
455
Fig. 18.3 MCV small and large T antigen domains. The T antigen region of MCV is depicted
(upper row). Through alternative splicing, the LT (middle row) and sT proteins are generated.
Integrated MCV genomes generally harbor a mutation or deletion truncating the LT (truncated area
is indicated by arrow). Only those domains likely to be involved in carcinogenesis are depicted
with the respective cellular protein partners
Only SV40 constructs containing the early region, i.e., sT and LT, are sufficient
for transformation while those encoding only the late region do not transform (Pipas
2009). By binding to the viral origin of DNA replication and recruiting cellular
replication factors, LT promotes DNA synthesis and hence plays a key role in regulating the viral life cycle. As a multifunctional nuclear phosphoprotein, LT also
prepares the cell for replication by stimulating cell cycle progression from G0/G1
into S phase. The latter function is likely to be the main contributor to oncogenic
transformation. Notably, transfection of origin-deficient SV40 genomic DNA
significantly enhances in vitro transformation of human cells, suggesting that viral
replication presents an obstacle to stable transformation (Small et al. 1982).
Importantly, in nearly all tumor-derived MCV genomes sequenced, missense
mutations or deletions in the early region result in the expression of truncated LT
antigens while MCV strains derived from non-MCC tissues encode full-length LT
(Shuda et al. 2008; Sastre-Garau et al. 2009). These mutations truncating either the
origin-binding domain (OBD) or the helicase domain eliminate the ability of MCV
to replicate. Until recently, these MCC LT mutations have not been detected in
MCV present outside of MCC tumors, and thus are signature mutations (zur Hausen
2008). In a recent publication analyzing 70 chronic lymphocytic leukemia (CLL)
cases, however, an LT deletion in the helicase gene has been described in 6 of 19
MCV-positive CLLs. This observation suggests that MCV might have a role of MCV
in a subset of CLL cases, although the integration of this virus into the host genome
has yet to be proven (Pantulu et al. 2010). Thus, the loss of replication activity of
MCV LT integrated in Merkel cell tumor cells provides further evidence that MCV
is not simply a passenger virus which happens to grow well in MCC cells.
Besides the OBD and helicase/ATPase domains required for viral replication,
MCV LT contains additional conserved features of other polyomavirus LT proteins,
456
the conserved region 1 (CR1), the DnaJ domain, and the retinoblastoma (RB)
tumor-suppressor protein family-binding motif LXCXE (Fig. 18.3, Shuda et al.
2008). In addition, for the p53/DNA-binding region, there is also a homology of
about 50% with the other polyomaviruses (Johnson 2010).
In the adenovirus 1A, CR1 is required for the cell-transforming E1A region to
bind RB (Berk 2005). In SV40, however, crystallographic data suggest that the
CR-1-like region is buried in the hydrophobic core indicating that the function of
CR1 might be different in polyomaviruses LT (Sullivan and Pipas 2002).
The DnaJ domain, present in both sT and LT, is a molecular chaperone which
recruits DnaK family chaperones (i.e., the heat-shock protein family) to perform
functions, such as protein folding, protein transport, or remodeling of protein
complexes. In this regard, polyomavirus DnaJ domain binds the constitutively
expressed HSC70 (Whalen et al. 2005). It seems that the DnaJ domain is critical for
stimulation of viral replication and to enhance oncogenic transformation by functionalinactivating RB members (Campbell et al. 1997; Pipas 1998). Notably, the contribution
of the DnaJ domain to LT transformation seems variable. For example, DnaJ
activity is unnecessary for anchorage-independent growth and for immortalization
of mouse embryo fibroblasts, but required for promotion of growth in low serum
and high saturation density (Stubdal et al. 1997).
One major domain for the transformation activity of LT is the RB family-binding
motif LXCXE. Indeed, interaction of T antigen with the RB family of proteins is
essential for transformation. It is currently believed that LT must block the growthsuppressive functions of RB proteins in order to induce transformation. RB is a
negative regulator of cell proliferation by repressing the E2F transcription factor
which in turn regulates the expression of genes required for entry and progression
of the cell cycle. By binding to the hypo- or underphosphorylated form of RB, LT
disrupts pRB/E2F complexes (Fig. 18.4a). As a consequence, many E2F target
genes are activated or derepressed leading to cell cycle progression (Pipas 2009).
For MCV, cotransfection of LT mutants with RB in 293 cells followed by immunoprecipitation confirmed MCV LT binding to RB through the LXCXE motif (Shuda
et al. 2008). The RB/E2F signaling pathway is of such central significance for the
control of cell proliferation that it is assumed that its regulation is disturbed in practically all tumors. In MCC, however, loss of RB seems to be a rare event (Becker
et al. 2009b). Thus, MCV present in the majority of MCC cases might contribute to
a disturbed regulation of this important pathway.
To elicit transformation, LT may also act on another critical cellular target, i.e.,
the tumor suppressor p53, for which a multitude of functions have been described.
For example, the antiproliferative activity of p53 plays a significant role in the
avoidance of tumor development. Probably, the most important mechanism to control
p53 in normal proliferating cells involves the control of protein stability via ubiquitination by the p53-specific ubiquitin ligase mdm2 and subsequent proteasomal
degradation; oncogenic stress results in the induction of the tumor-suppressor
protein p14ARF, which binds to mdm2, inactivates it, and thus prevents degradation of p53. The p21 protein is a critical downstream target of p53 that mediates
cell cycle arrest by inhibiting cyclin-dependent kinases, RB phosphorylation, and
18
457
Fig. 18.4 Possible mechanisms by which MCVs T antigens might contribute to transformation.
(a) Large T antigen (LTA) might exert transforming capacity by binding RB in combination with
the chaperone protein hsc70 leading to the release of E2F. Unbound E2F transcription factor mediates cell cycle progression. Another mechanism might be by binding and inactivating the tumor
suppressor p53. However, since this domain is often lost in MCV integrated into the host genome,
this pathway might be negligible. (b) Small T antigen (sT) binds to PP2A displacing the regulatory
B subunit. This modified sT/PP2A complex might act on many different pathways. For example, it
prevents the dephosphorylation and inactivation or degradation, respectively, of AKT and Myc
cell cycle progression. Experiments with SV40 have demonstrated that LT/p53
complexes can be observed. LT binds p53 within its core DNA-binding domain
leading to loss of target gene activation. Notably, mutated LTs not capable of binding
p53 were defective in transformation (Peden et al. 1998). As mentioned before,
458
MCV LT demonstrates only limited homology to the other polyomavirus in the p53
DNA-binding region. Moreover, LT-truncating mutations often affect the p53-binding
region (Shuda et al. 2008). Thus, p53 binding capacity might be not very relevant
for MCV in the etiology of MCC. In accordance, alterations of the p53 pathway
independent of the MCV virus have been observed (Becker et al. 2009b).
In contrast to LT, for sT, only a few domains have been described. Moreover, sT
cannot transform cells by itself; however, it can cooperate with LT to fully transform
cells. sT codes for the same DnaJ domain as LT, but its function or target has not
been elucidated nor has it been implicated in sT-mediated transformation (Boyapati
et al. 2003). Indeed, sT activities can be largely attributed to the binding of the serine
threonine protein phosphatase PP2A, which appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases (Fig. 18.4b)
(Millward et al. 1999). PP2A is a heterotrimeric enzyme family composed of a scaffold
A subunit, a regulatory B subunit, and a catalytic C subunit. There are 2 different A
subunits, 2 C subunits, and at least 17 B subunits that can assemble together into
>100 different holoenzyme complexes (Sablina and Hahn 2008). sT interacts with
PP2A by either preventing the binding of B subunits or replacing them leading in
most cases to an inhibition of enzyme activity (Cho et al. 2007). Since PP2A regulates
many different kinases, probably only a fraction of the targets of PP2A potentially
involved in sT-induced tumorigenesis have been identified to date. For example,
SV40 sT can activate AKT signaling through activation of PI3K, contributes to
stabilization of Myc by inhibiting PP2A-dependent dephosphorylation, and inhibits
cap-dependent translation (Cheng et al. 2009).
For MCVs role in the etiology of MCC, only indirect evidence is available.
Indeed, results of classical transformation assays for MCV T antigens have not yet
been published. Certainly, given the time since the discovery of MCV and the
impact it had on the scientific community, it seem conceivable that the transforming
capacity of the T antigens might be not very high in standard assays. One source of
indirect evidence for involvement of MCV in MCC is studies comparing MCVnegative and -positive MCC cases. In this regard, two groups have demonstrated
that p53 and KIT expression in tumors was associated with the absence of MCV
DNA or lower copy number, respectively. Moreover, higher copy numbers of MCV
DNA was directly associated with the presence of RB suggesting that the molecular
pathogenesis of MCC is multifactorial (Bhatia et al. 2010a; Waltari et al. 2010).
In contrast, we and others did not observe significant differences between viruspositive and -negative MCC cases (Handschel et al. 2010; Houben et al. 2010a).
However, we recently demonstrated that expression of the oncogenic T antigens is
mandatory for the maintenance of MCV-positive MCC cell lines; upon knock down
of T antigens by shRNA, MCV-positive MCC cells displayed growth arrest and
partially died (Houben et al. 2010b). Since the knockdown did not differentiate
between sT and LT, their exact roles have still to be untangled. Nevertheless, this
observation is the first direct experimental evidence that TA expression is necessary
for the maintenance of MCV-positive MCC and provides a further line of evidence
that MCV is the infectious cause of MCV-positive MCC.
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459
Conclusion
The recent findings that (a) MCV is integrated into the host genome in the majority
of MCC cases, (b) integrated MCV genome encode truncated LT, and (c) MCVpositive cell lines are dependent on T antigen expression strongly imply that MCV
is the etiological agent in a major subset of MCC cases. The exact mechanism,
however, has still to be unraveled. But since the discovery of MCV has boosted the
research activity on MCC, this will probably sooner or later translate into the knowledge
of the signaling pathways involved in the pathogenesis of this tumor.
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Zur Hausen H (2008) A specific signature of Merkel cell polyomavirus persistence in human
cancer cells. Proc Natl Acad Sci USA 105:1606316064
Chapter 19
Human Papillomaviruses and Cancer

Jianxin You and Susanne Wells
Introduction
Papillomaviruses (PVs) are small DNA tumor viruses that induce a diverse range of
benign and malignant epithelial lesions in the infected host (Howley and Lowy
2001). Over 140 human PV (HPV) types have been identified to date. Depending on
the potential for inducing malignant transformation, these viruses are further classified
into high-risk and low-risk HPVs (Howley and Lowy 2001). Persistent infection
with high-risk HPVs are recognized as the major cause of cervical cancer (zur Hausen
2002), which is the second most common cancer among women worldwide and
the leading cause of death from cancer among women in developing countries;
approximately 500,000 cases and 275,000 deaths are reported annually. Over 97%
of cervical cancers contain high-risk HPV genomic DNA and express the viral
oncogenes E6 and E7, thus providing a direct link between HPV infection and carcinogenesis. In this chapter, we summarize the HPV life cycle and oncogenic events
that contribute to the role of HPV in carcinoma development. Most of our current
knowledge derives from studies on cervical cancers, but HPV has also been implicated in other anogenital cancers, as well as head and neck cancers (HNCs), and
skin cancers (Giuliani et al. 2007; McKaig et al. 1998; Pfister 2003; Vernon et al.
1998). In the last section of the chapter, we discuss emerging developments on the
understanding of the role of HPV in HNCs.
J. You (*)
Department of Microbiology, University of Pennsylvania School of Medicine,
3610 Hamilton Walk, Philadelphia, PA 19104, USA
e-mail: jianyou@mail.med.upenn.edu
S. Wells
Division of Hematology/Oncology, Cincinnati Childrens Hospital, 3333 Burnet Avenue,
MLC 7013, TCHRF, Room S7-206, Cincinnati, OH 45229, USA
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The Differentiation-Dependent Papillomavirus Life Cycle

Productive Viral Life Cycle
PVs contain circular double-stranded DNA genomes of approximately 8,000 bp
within an icosahedral capsid. The viral genome encodes eight open reading frames
(ORFs), including six early genes (E1, E2, E4, E5, E6, and E7) and two late genes
(L1 and L2) (Doorbar 2006) (Fig. 19.1). HPVs have a specific tropism for squamous
epithelial cells and must infect cells within the dividing basal layer. The productive
life cycle of HPV is intimately tied to the differentiation status of the host squamous
epithelium (Howley and Lowy 2001) (Fig. 19.2). Various phases of the HPV life cycle
Fig. 19.1 HPV16 genome organization and functions of HPV gene products. The HPV16 genome
contains 7,904 bp and encodes eight open reading frames (ORFs). The six early ORFs, E1, E2, E4,
E5, E6, and E7, are expressed from either p97 (early promoter) or p670 (late promoter) at different
stages of host epithelial cell differentiation. The late ORFs, L1 and L2, are expressed from p670.
All the viral genes are transcribed from only one strand of the double-stranded circular DNA
genome. Functions of the viral gene products are also shown
19 Human Papillomaviruses and Cancer
465
Fig. 19.2 The productive HPV life cycle. HPVs have specific tropism for squamous epithelial
cells. They infect cells within the dividing basal epithelial layer through microabrasions. The HPV
life cycle is strictly tied to the differentiation program of the host keratinocyte. After infection,
viruses establish themselves as episomes at 50100 copies per infected basal cell and replicate
with the host DNA once per cell cycle. The viral oncoproteins E6 and E7 expressed in the basal
cells modify the cell cycle to retain the differentiating host keratinocytes in a state that allows viral
genome replication. Expression of low levels of E1, E2, E4, and E5 allows maintenance of the viral
genome in the suprabasal cells. Viral genome amplifies in the upper epithelial layers, where the
expressed late genes L1 and L2 serve as structural proteins to encapsidate the amplified viral
genomes. Virions can then be sloughed off the surface of the host epithelium
are controlled through tightly regulated activation of the early and late viral promoters
as the infected basal cell migrates toward the epithelial surface (Doorbar 2006; Hebner
and Laimins 2006). Productive infection is achieved through different viral proteins
playing specific roles at distinct phases of the viral life cycle (Fig. 19.2).
Within infected basal cells, viral genomes are established and maintained as
extrachromosomal elements (episomes) that replicate along with host DNA
(Fig. 19.2). In the basal epithelium where initial stages of the viral life cycle takes
place, the early viral promoter is transcribed to express the viral E1, E2, E6, and E7
genes (Hummel et al. 1992; Ozbun and Meyers 1998). The establishment of the
viral genome as a stable episome in the basal epithelia cells requires expression of
the viral E1 and E2 proteins. The papillomavirus E2 is a sequence-specific DNAbinding protein involved in viral DNA replication, episome maintenance, and viral
transcription (Hebner and Laimins 2006). It consists of an N-terminal transcriptional activation domain linked to a C-terminal DNA-binding/dimerization domain
by a flexible hinge (McBride et al. 1991). The C-terminus of E2 is involved in interactions with viral replication factor E1 and also recognizes a palindromic motif
[AACCg(N4)cGGTT] in the noncoding region of the viral genome (Dell et al. 2003).
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The multiple functions of E2 rely on its sequence-specific recognition of a number

of E2-binding sites (E2BSs) within papillomavirus genomes. E2 initiates viral
genome replication by loading the viral helicase E1 onto the origin of replication,
which in turn recruits cellular proteins necessary for DNA replication (Conger et al.
1999; Han et al. 1999; Howley and Lowy 2001; Loo and Melendy 2004; Masterson
et al. 1998; Mohr et al. 1990). This allows the viral episome to be maintained at low
copy numbers in the basal epithelium. During mitosis, E2 ensures accurate partitioning of the replicated viral genomes to daughter cells by tethering them to host
mitotic chromosomes (Bastien and McBride 2000; Ilves et al. 1999; Lehman and
Botchan 1998; Piirsoo et al. 1996; Skiadopoulos and McBride 1998). In the differentiating host epithelium, E2 also contributes to the tight regulation of the viral
oncogene transcription to create a cellular environment conducive to completion of
the productive viral infectious cycle. E2 binds to four E2BSs in the long-control
region (LCR) of the HPV genome in a cooperative manner that results in either
activation (at low levels of E2) or repression (at high concentrations of E2) of E6
and E7 expression from the early promoter (Bouvard et al. 1994; Doorbar 2006;
Steger and Corbach 1997). E6 and E7 are the primary viral oncogenes, which
promote cell growth through a variety of mechanisms, including inactivation of the
p53 and pRb tumor suppressors, respectively (Doorbar 2006; Dyson et al. 1989;
Huibregtse et al. 1991; Munger et al. 1989; Scheffner et al. 1993). The dose-dependent
ability of E2 to either repress or activate early viral gene expression is thought to
result from differential affinities of E2 for its various binding sites (Hines et al.
1998). At low concentration, E2 binds the primary binding site distal to the HPV
promoter and leads to promoter activation (Steger and Corbach 1997). As E2 level
increases, occupancy of the remaining sites proximal to the promoter leads to transcription repression of the early promoter through displacement of other cellular
transcription factors (Demeret et al. 1994; Dong et al. 1994; Steger and Corbach
1997; Tan et al. 1994).
Suprabasal cells normally exit the cell cycle and differentiate to produce the
protective barrier of the skin (Madison 2003). The strict control of the viral early
promoter allows the minimum expression of E6 and E7 that is needed to drive cells
into S phase for viral genome replication while preventing the inopportune overexpression of these viral oncoproteins that leads to dysplasias and carcinomas.
As discussed below, the cellular factors that contribute to the tightly regulated viral
transcription are not very well-understood. As HPV-positive cells differentiate, the
late promoter is activated leading to the expression of the two late-gene products,
capsid proteins L1 and L2, and the high-level amplification of the viral genome
(Doorbar 2006; Hebner and Laimins 2006). In the highly differentiated suprabasal
cells, the replicated viral DNA genomes are packaged into newly formed capsids
and infectious progeny virions are released from the cell (Howley and Lowy 2001)
(Fig. 19.2).
Besides E1, E2, E6, and E7 proteins, the viral E5 protein also plays important
role in the productive stage of the viral life cycle (Fehrmann et al. 2003; Genther
et al. 2003). HPV E5 is a transmembrane protein that resides predominantly in the
467
endoplasmic reticulum. Association of E5 protein with the vacuolar proton ATPase

delays the process of endosomal acidification (Disbrow et al. 2005; Hwang et al.
1995; Straight et al. 1995), contributing to an increase in epidermal growth factormediated receptor signaling and the maintenance of a cellular environment for viral
replication in the upper epithelial layers (Crusius et al. 2000). In addition, E4
accumulates in the cells at the time of viral genome amplification and contributes to
multiple facets of the papillomavirus life cycle (Nakahara et al. 2005; Peh et al.
2004; Wilson et al. 2005). The E4 proteins encoded by several HPV types have
been shown to cause cell cycle arrest in G2 and to antagonize E7-mediated cell
proliferation (Davy et al. 2002; Knight et al. 2004; Nakahara et al. 2002). However,
the role of E4 in genome amplification is poorly understood (Doorbar 2006). E4 can
also disrupt the keratin network to facilitate the efficient release of the assembled
virions from the cornified envelope at the cell surface (Bryan and Brown 2000;
Doorbar et al. 1991; Wang et al. 2004).
Abortive Viral Infection

The incidence of cervical cancer development from an HPV-infected lesion is low,
with most infections limited by the host immune system (Parkin et al. 2005). Highgrade cervical neoplasia usually arises in individuals who maintain persistent active
infection for years or decades following initial exposure and fail to resolve their
infection. In these situations, the continuous stimulation of S-phase entry and cell
proliferation by E7, coupled with the loss of p53-mediated DNA repair pathways
resulting from E6 expression, leads to the accumulation of additional genetic
mutations in the cellular genome that eventually lead to cancer (Doorbar 2006).
The identification of cervical lesions as flat condyloma, low-grade squamous intraepithelial neoplasia lesions (LSIL), high-grade squamous intraepithelial neoplasia
lesions (HSIL), or invasive cervical cancer has allowed the detection of molecular
changes in the normal epithelial cell differentiation that occur after viral infection
(Doorbar 2006). In productive lesions, cells are driven into S phase only in the
lower epithelial layers and virus production at the epithelial surface relies on the
ordered and timely expression of viral gene products (Middleton et al. 2003; Peh
et al. 2002). The viral protein expression patterns in low-grade cervical lesions
resemble the patterns found in productive lesions with viral coat proteins usually
detected in cells at the epithelial surface (Middleton et al. 2003). However, the
timing of viral gene expression becomes progressively disturbed during neoplastic
progression. In high-grade neoplasia, the proliferative phase becomes more
extensive with viral genome amplification occurring closer to the epithelial surface,
and with viral capsid protein synthesis abolished (Middleton et al. 2003). These
changes reflect an abortive infection in which viral gene expression becomes deregulated
and the productive stages of the virus life cycle can no longer be properly supported
(Doorbar 2006).
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HPV-Persistent Latent Infection

High-risk HPV-infected cells progress to invasive cancer only after they have
accumulated substantial cytogenetic changes needed for malignant progression.
The cellular events that cause this transition do not occur until many years after the
initial infection, supporting the concept that persistent infection with highrisk HPVs is required for the malignant progression of HPV pathogenic lesions.
HPVs establish persistent infection by maintaining their genomes as episomes
that replicate along with host DNA in infected basal epithelial cells (Howley and
Lowy 2001).
The functions of the high-risk HPV E6 and E7 oncoproteins are essential for
the viral genome to be stably maintained in the infected cells (Thomas et al. 1999).
It was postulated that these oncoproteins may create a suitable cellular environment for HPV maintenance through abrogating the cellular checkpoints that block
the long-term retention of viral episomes (Kadaja et al. 2009b). In addition, to
establish persistence in host cells, some PVs have evolved a strategy for hitchhiking
on cellular mitotic chromosomes. This mechanism ensures that the replicated
viral episomes are retained inside the nuclei of dividing host cells and faithfully
partitioned to the daughter cells during mitosis. For many papillomaviruses,
this noncovalent association of viral DNA with chromosomes is mediated by the
viral E2 protein. Bovine papillomavirus type 1 (BPV1) genomes persist as stable
episomes in transformed rodent cells, and have been used as a model system to
establish the role of E2 in viral genome maintenance (Bastien and McBride
2000; Ilves et al. 1999; Law et al. 1981; Lehman and Botchan 1998; Skiadopoulos
and McBride 1998). E2 and BPV1 episomes are closely associated with mitotic
chromatin in dividing cells (Ilves et al. 1999; Lehman and Botchan 1998; Skiadopoulos
and McBride 1998). Long-term maintenance of viral episomes requires BPV1 E2
binding to the multiple E2BSs within a cis-minichromosome maintenance element
(MME) of the viral genome (Piirsoo et al. 1996). These studies demonstrate that
BPV1 E2 facilitates viral genome segregation by anchoring viral episomes to
mitotic chromosomes.
The cellular bromodomain protein Brd4 has been postulated to serve as a host
chromatin adaptor for BPV1 E2 (You et al. 2004). Brd4 is a member of the BET
family proteins that harbor double bromodomains (Dey et al. 2000). It binds to
acetylated histone H3 and H4 on host chromatin through its bromodomains and
becomes associated with mitotic chromosomes (Dey et al. 2003). Brd4 binds BPV1
E2 through its C-terminal domain and tethers the E2/viral episome complex to host
mitotic chromosomes to ensure the faithful partitioning of replicated viral episomes
during cell division (You et al. 2004, 2005). Further studies demonstrated that the
Brd4E2 interaction bridges a number of animal and human PV episomes onto
mitotic chromosomes for viral genome maintenance (Abbate et al. 2006; Baxter
et al. 2005; Brannon et al. 2005; Cardenas-Mora et al. 2008; Ilves et al. 2006;
McBride et al. 2004; McPhillips et al. 2005), establishing this complex as a potential
469
therapeutic target for PV infections. Through these interactions, Brd4 also contributes
to E2-mediated transcriptional activation and repression of the viral oncogenes
(Ilves et al. 2006; Lee and Chiang 2009; McPhillips et al. 2006; Schweiger et al.
2006; Senechal et al. 2007; Wu et al. 2006). The structure of the N-terminal domain
of HPV16 E2 in complex with the C-terminus of Brd4, thus, offers a molecular
platform for developing antiviral compounds to block E2Brd4 interaction during
HPV latent infection (Abbate et al. 2006).
The molecular mechanism involved in maintaining E2 and papillomavirus
episome persistence in dividing host cells appears complex. A number of additional
mitotic cellular factors have been identified as interacting proteins for E2. Among
them, ChlR1, a DNA helicase that functions in sister chromatid cohesion, appears
essential for loading the papillomavirus E2 protein onto mitotic chromosomes
during early mitosis (Parish et al. 2006). Other proteins, such as MKlp2 and TopBP1,
have been shown to interact and colocalize with E2 during different phases of mitosis
(Donaldson et al. 2007; Yu et al. 2007). It is conceivable that Brd4 and the identified
additional cellular receptors may interact either sequentially or simultaneously with
E2 and the viral episome to constitute a tethering cascade/complex for viral genome
maintenance and segregation in latently infected cells.
Different modes of papillomavirus anchoring on mitotic chromosomes have also
been reported. A wide range of papillomaviruses have adopted the strategy of tethering their genomes to host chromosomes, but individual viruses appear to use different chromosomal targets. A recent localization analysis of 13 different animal
and human E2 proteins from 7 papillomavirus genera have shown that E2-mediated
tethering of viral genomes to mitotic chromosomes is a common strategy shared by
these papillomaviruses. However, different viruses bind to different regions of the
host chromosomes during mitosis. Unlike BPV1 E2 whose nonspecific binding
to all mitotic chromosomes is reflected by small speckles associated with Brd4,
several other E2 proteins appear to localize to more specific regions of mitotic
chromosomes (Oliveira et al. 2006). For instance, the alpha-papillomavirus E2
proteins only closely associate with prophase and telophase chromosomes while
binding to the pericentromeric region of metaphase chromosomes (Oliveira et al.
2006). In contrast, the HPV8 E2 binds as large speckles at the pericentromeric
regions of chromosomes (Oliveira et al. 2006). Further analysis indicates that the
HPV8 E2 protein targets the short arms of acrocentric mitotic chromosomes and
interacts with the repeated ribosomal DNA loci of host mitotic chromosomes. In
addition to mitotic chromosomes, some high-risk HPV type E2 proteins have been
shown to associate with mitotic spindles to enable the partitioning of HPV origincontaining plasmids as minichromosomes in transfected cells (Van Tine et al. 2004).
The molecular mechanisms by which high-risk HPVs adopt the cellular machinery
to maintain persistent infection in host cells remain an interesting topic for future
research. A complete understanding of the HPV tethering mechanism is bound to
uncover new targets for the development of novel therapeutic strategies to cure
latent HPV infection.
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Molecular Events Contributing to Malignant Progression

of HPV-Positive Lesions
HPV Integration and Cervical Cancers
HPV DNA is maintained episomally in benign, precancerous lesions, but integration
into the cellular genome is frequently observed in cervical cancers and effectively
eliminates the viral life cycle (Pett and Coleman 2007). It has been suggested that
integration of the HPV genome into the host cell chromosome may be a critical
event in the development of most cervical cancers (Peitsaro et al. 2002). Although
HPV integration can occur randomly throughout the genome, several studies have
indicated a preference for integration occurring within common fragile sites
(Thorland et al. 2000, 2003). Variable portions of the viral genome are present in the
integrants, but common features include sustained expression of E6/E7 under the
control of their viral promoter as well as loss of the viral E2 gene (Baker et al. 1987;
Corden et al. 1999; Durst et al. 1985; Howley and Lowy 2001; Jeon et al. 1995; Jeon
and Lambert 1995b; Park et al. 1997; zur Hausen 2000) (Fig. 19.3). As described
above, HPV E2 is also a transcription repressor that can directly bind to and repress
the viral E6/E7 promoter of high-risk HPVs (Demeret et al. 1997; Romanczuk et al.
1990; Tan et al. 1992; Thierry and Howley 1991). The loss of E2 expression derepresses
the viral oncogene E6 and E7 expression to reduce p53 and pRb expression, which
in turn stimulates cellular proliferation (Dyson et al. 1989; Gonzalez et al. 2001;
Munger et al. 1992, 2004; Scheffner et al. 1990). As such, disruption of the E2 gene
has been mechanistically linked to malignant progression of HPV-associated cancers (Howley and Lowy 2001). In cervical cancer cell lines containing integrated
HPV DNA and a disrupted E2 gene, reintroduction of E2 represses the HPV E6/E7
promoter and reverses cellular immortality (Dowhanick et al. 1995; Francis et al.
2000; Goodwin and DiMaio 2000; Hwang et al. 1993; Thierry and Yaniv 1987)
(Fig. 19.3). It has been suggested that E2 represses the E6/E7 promoter by binding
its cognate sites proximal to the promoter and displacing other cellular transcription
factors (Demeret et al. 1994; Dong et al. 1994; Tan et al. 1994). In addition, E2
could function as an active repressor by preventing the assembly of a functional
preinitiation complex on the viral chromatin template (Dostatni et al. 1991; Wu and
Chiang 2007; Yan et al. 2010).
A small proportion of human cervical cancers harbor exclusively episomal viral
DNA (Guo et al. 2007; Hudelist et al. 2004; Matsukura et al. 1989; Park et al. 1997),
suggesting that viral integration is not necessarily required for cancer development.
A postulated intermediate state in which transcriptionally silent integrants coexist
with episomal genomes has been described in clinical lesions. Because E2 expressed
from episomal genomes can repress oncogene expression from the integrant
(Bechtold et al. 2003; Demeret et al. 1997; Romanczuk et al. 1990; Tan et al. 1992),
episome loss and clonal selection for integrants are likely important steps during
cervical carcinogenesis (Pett and Coleman 2007; Pett et al. 2004). HPV integration
has been shown to confer a selective growth advantage compared to cells with
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Fig. 19.3 Repression of HPV oncogenes by the E2 protein. Shown is a schematic representation
of a fragment of high-risk HPV genome integrated into the host genome. Integration of HPV DNA
into the host genome frequently results in disruption of the E2 gene, which normally inhibits the
production of E6 and E7 proteins of high-risk HPVs. Loss of E2 expression derepresses the expression of E6 and E7, which function as oncogenes to promote tumor growth and malignant transformation by inhibiting p53 and pRb, respectively. E6 protein binds p53 via E6-associated protein
(E6AP), which ubiquitinates p53, targeting it for degradation by proteasome. The binding of E7 to
pRb releases E2F for the transcription of its responsive genes to promote cell cycle progression. In
cervical cancer cell lines containing integrated HPV DNA and a disrupted E2 gene, reintroduction
of E2 represses E6 and E7 expression, thereby inhibiting their effects on p53 and pRb and reversing cellular immortality [Modified from Thierry (2009)]
episomal HPV copies (Jeon and Lambert 1995b) perhaps at least in part due to the
stabilization of E6 and E7 mRNAs expressed from the viral integrants (Jeon and
Lambert 1995a).
The Contribution of HPV Oncogenes to Human Cervical Cancers

High-risk HPV E6 and E7 proteins are universally expressed in human HPV-positive
cancers in vivo and function cooperatively in vitro, findings that have defined them
as the primary viral oncogenes. The viral E5 protein can further contribute to HPV
malignancy (DiMaio and Mattoon 2001), and carries independent carcinogenic
potential in transgenic mouse models (Maufort et al. 2007, 2010). Viral oncogenic
potential is also reflected in the ability of high-risk, but not low-risk, HPV genomic
DNA to immortalize primary human keratinocytes in vitro (zur Hausen 2000). In
order to progress to transformed phenotypes, either the coexpression of defined
oncogenes or extended passaging in tissue culture is required (DiPaolo et al. 1989;
Durst et al. 1989; Hurlin et al. 1991; Pei et al. 1993).
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Since the specific activity of high-risk HPV E6 and E7 has consistently been
associated with cervical cancers, deregulated expression of the viral oncogenes is
considered a predisposing factor in the development of HPV-associated cervical
cancers.
The E7 oncogene appears to play a dominant role in cell culture studies; highrisk HPV E7, but not E6, is capable of immortalizing primary human keratinocytes
on its own. A critical role for E7 has also emerged in E6 and E7 transgenic mouse
models of cervical cancer (Jabbar et al. 2009; Riley et al. 2003) and HNC (Strati and
Lambert 2007). Many proteinprotein interactions have been reported between
the high-risk HPV oncoproteins and host cellular factors, at least some of which
contribute to oncogenic E6 and E7 activities (Jones and Wells 2006).
E7 binds and degrades Rb-related pocket proteins and neutralizes the cyclindependent kinase inhibitors p21 and p27 through direct interactions (Funk et al.
1997; Jones et al. 1997; Zerfass-Thome et al. 1996). Deregulation of these molecules is critical for HPV-mediated S-phase induction in terminally differentiated
cells. One important consequence of these E7 functions is the activation of E2F
transcriptional activity, the uncontrolled expression of genes that regulate G1/S
cell cycle progression and subsequent inappropriate entry of differentiating
keratinocytes into S phase (Banerjee et al. 2006; Cheng et al. 1995). The maintenance of an S-phase milieu conducive to viral replication is further supported
by the binding of E7 to a transcriptionally repressive E2F family member E2F6
(McLaughlin-Drubin et al. 2008) and to chromatin-modifying histone deacetylases (HDACs) independent of Rb association (Longworth and Laimins 2004b;
Longworth et al. 2005).
The function of the viral E6 protein complements that of E7 with regard to cancer progression. To counteract the growth arrest or apoptosis that would normally
occur in response to E7-mediated inappropriate cell proliferation, the high-risk
HPV type E6 forms a tripartite complex with p53 and the cellular ubiquitin ligase
E6AP (E6-associated protein), which causes proteasome-mediated p53 degradation (Huibregtse et al. 1991; Kao et al. 2000; Scheffner et al. 1990). This compromises the p53-mediated DNA damage response and allows the accumulation of
secondary mutations in the host genome that eventually lead to the development
of cervical cancers. In addition, in vitro studies establish that E6 inhibits p300mediated acetylation on both p53 and nucleosomal core histones, thereby converting
p53p300 from an activating complex to a chromatin repressor for p53-dependent
transcription (Thomas and Chiang 2005). E6 also transcriptionally and posttranscriptionally upregulates the human telomerase catalytic subunit, an enzyme that is
frequently activated in human tumors (Galloway et al. 2005; Katzenellenbogen
et al. 2007, 2009; Liu et al. 2005, 2009). The high-risk E6 proteins further contribute to the development of metastatic tumors through binding and stimulating the
degradation of several cellular targets that contain PDZ motifs, which are thought
to be involved in the regulation of cell growth and attachment (Nguyen et al. 2003a, b;
Zeitler et al. 2004).
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HPV Oncogenic Activities, Host DNA Damage Response,

and Genomic Instability
Deregulated expression of the high-risk HPV oncogenes is a critical event for the
progression of HPV-positive lesions. High-risk HPV E6/E7 activities fulfill some,
but not all, classical criteria for cellular transformation as described by Hanahan
and Weinberg (2000). These include inactivation of the p53 and Rb tumor suppressors and elevated telomerase activity. Various approaches to specifically inhibit
E6 and E7 have resulted in cellular growth arrest of HPV-positive cancer cells
(Alvarez-Salas et al. 1998; Hall and Alexander 2003; Hu et al. 1995; Venturini et al.
1999; von Knebel Doeberitz et al. 1992; Watanabe et al. 1993). However, in order
to achieve bona fide transformed potential, E6/E7 immortalized cells require additional hits. Similarly, in high-risk HPV-infected cells, substantial cytogenetic
changes are needed for progression to invasive tumors. The acquisition of additional
oncogenic events is greatly stimulated by genome instability, which occurs upon the
individual and particularly the joint expression of high-risk E6 and E7 by a multitude
of molecular mechanisms. Over 20 years ago, expression of high-risk HPV E6 and
E7 protein was shown to stimulate integration of exogenous DNA (Kessis et al.
1996). Such cells harbor DNA breaks, which can ultimately result in the acquisition
of anaphase bridges and catastrophic breakage-fusion-bridge cycles. Doublestranded HPV DNA intermediates might arise from integrated HPV DNA and
during the normal life cycle as a result of onion-skin type and/or rolling circle
DNA replication (Flores and Lambert 1997; Kadaja et al. 2009a; Mannik et al.
2002). Additionally, DNA damage responses arise as a result of expression of the
HPV replication machinery and the high-risk HPV oncoproteins (Kadaja et al.
2007). For example, HPV16 gene expression has been shown to induce both numerical and structural chromosome instability during HPV-associated carcinogenesis
(Duensing and Munger 2002). Genomic instability and the resulting multipolar
mitoses, aneuploidy, and chromosomal rearrangements are early hallmarks of HPVassociated cancers, which are specific to the high-risk HPV types. In fact, they occur
even in the presence of viral episomal HPV genomic DNA and prior to integration
(Duensing et al. 2001a), suggesting that they play a causal role in the development
of malignancies.
Molecular cross talk between DNA strand breaks, DNA damage signaling, HPV
gene products, and transformation is likely complex and occurs at multiple levels.
The detection and repair of cellular DNA strand breaks depend upon three major
kinases and members of the phosphoinositide 3 kinase-like kinase (PIKK) family:
ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), and DNAdependent protein kinase (DNA-PK). Each can phosphorylate a histone H2A
variant H2AX on serine 139 rapidly following DNA damage. The resulting gH2AX
is a marker of damaged sites, which recruits the DNA repair machinery and further
amplifies the DNA damage signal. ATM is best known for its ability to respond to
double-strand breaks and to phosphorylate itself on serine 1981 as well as downstream targets, such as the Chk2 kinase. ATR is largely activated by single-stranded
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DNA breaks, such as from UV exposure and replication stress, and phosphorylates
Chk1 and other targets. High-risk E6 protein interferes with the recovery of stalled
replication forks in a p53-independent manner and through the deregulation of Chk1
(Chen et al. 2009). E6 expression also leads to abnormal centrosome numbers after
prolonged cell passaging likely through the inactivation of p53. High-risk HPV E7
independently causes centrosome abnormalities (Duensing et al. 2001b; Duensing
and Munger 2004; Schaeffer et al. 2004). These occur rapidly as a result of centrosome
overduplication within a single S phase. In contrast to normal centrosome duplication, E7-deregulated centrosome duplication is dependent upon high CDK2 activity
and is at least partially independent of the retinoblastoma protein family (Duensing
et al. 2000, 2001a, 2008; Duensing and Duensing 2008; Duensing and Munger
2002, 2003). Expression of high-risk, but not low-risk, HPV E7 additionally stimulates
the activation of the Fanconi anemia pathway which is crucial for the maintenance
of genome stability (Hoskins et al. 2008; Spardy et al. 2007), and forces the degradation
of claspin, an important regulator of the upstream ATR DNA damage sensor of
replication stress (Spardy et al. 2009). High-risk HPV E6 and E7 also cause polyploidy
through bypassing mitotic and postmitotic checkpoints (Heilman et al. 2009; Liu
et al. 2007; Thomas and Laimins 1998; Thompson et al. 1997). These events may
cooperate to further stimulate cellular genome instability, viral integration and
rearrangement, and ultimately progression to malignancy.
Virally induced activation of cellular DNA damage responses has emerged as a
common theme in the literature, and can serve to either support viral replication
as in the case of SV40, HSV, or HIV or to inhibit replication as in the case of adenovirus (Lilley et al. 2007; Sinclair et al. 2006). ATM is a particularly interesting
molecular modifier of viral DNA replication: its activity is stimulated by both HSV
and adenovirus. However, while HSV replication exploits ATM activation, ATM
activity obstructs adenoviral replication and the virus thus evolved to degrade specific
DNA repair components to circumvent the cellular DNA damage response. With
regards to the HPV life cycle, however, our understanding of the effects of specific
DNA damage-signaling responses has lagged behind. This is likely due to technical
reasons that relate to the difficulties of studying the HPV life cycle, which is tightly
linked to the keratinocyte differentiation program (Doorbar 2005; Longworth and
Laimins 2004a). At least some aspects of the viral replicative cycle must be analyzed
in 3D models of differentiated epidermis, such as organotypic epithelial rafts.
Manipulation of DNA damage responses and viral replication, then, need to be
interpreted in the context of geographically distinct keratinocytes, which maintain
varying degrees of endogenous proliferation and HPV replication. An elegant recent
report highlights the specific importance of ATM activation by HPV31 episomes in
the productive HPV replicative cycle (Moody and Laimins 2009). ATM, Chk2, and
gH2AX phosphorylation were found elevated in HPV31-positive keratinocyte
populations and organotypic epithelial rafts. The specific inhibition of either ATM
or Chk2 did not affect viral maintenance over time, but greatly reduced the assembly
of viral replication centers as well as productive viral amplification in calciumdifferentiated cells. Interestingly, the authors further showed that Chk2 was required
for caspase 7 cleavage, which together with caspase 3 was previously implicated in
475
cleavage of the viral E1 protein and productive replication. These observations link
the HPV-activated DNA damage response directly to the viral replication machinery,
and represent the first step in elucidating the role of DNA damage signaling in the
HPV life cycle. Whether specific ATM inhibitors might then be useful clinically for
the treatment of HPV infections remains to be determined.
The Role of HPV in Head and Neck Cancer

High-risk HPV infection is the primary risk factor for cervical cancer. Studies also
suggest that HPVs may play a role in many other types of anogenital cancers and
skin cancers (Giuliani et al. 2007; Parkin 2006; Pfister 2003; Vernon et al. 1998).
The implication of HPV in these other types of cancers has been covered in great
details in these reviews (Giuliani et al. 2007; Pfister 2003; Vernon et al. 1998).
Studies have also found that oral HPV infection is a strong risk factor for HNCs
(McKaig et al. 1998). In this section of the chapter, we focus on the emerging development on the molecular understanding of HPV in HNCs.
HNC comprises the sixth most common malignancy worldwide, with an incidence
of approximately 500,000 and an associated mortality of over 300,000 per year
(Parkin et al. 2005). Treatment regimen often approximate the limits of patient
tolerance, and more than half of patients who receive radiation therapy suffer from
debilitating acute toxicity in the form of mucositis, pharyngitis, and/or severe dermatitis.
HNC survivors often experience poor quality of life from disfiguring surgeries and
permanent tissue damage following radiation and chemotherapy. Despite advances
for locoregional control, overall cure rates have shown little improvement in
decades. Early detection, targeted delivery of radiation and chemotherapy, and therapy
de-escalation based on biomarkers need to be pursued for improved outcomes (Corry
et al. 2010). These renewed efforts must be accompanied by molecular research into
the mechanisms of HNC development, metastasis, and treatment response.
HNCs arise from the oral cavity, oro-, naso-, and hypopharynx as well as the
larynx. The majority of HNCs are squamous cell carcinomas (SCCs), which originate
from keratinocytes in the pluristratified squamous epithelium lining of the upper
aerodigestive tract. Alcohol and tobacco consumption have long been recognized as
major risk factors. Additionally, an etiological association with HPV was first
experimentally suggested by Syrjanen et al. (1983), and has been further substantiated and reported for approximately one quarter of HNSCCs, predominantly those
originating in the oropharynx (Braakhuis et al. 2004; DSouza et al. 2007a, b; Dai
et al. 2004; Gillison et al. 2000; Gillison and Shah 2001, 2003; Herrero et al. 2003;
Kreimer et al. 2004a, b; Schwartz et al. 1998; Smith et al. 2004; van Houten et al.
2001; Wiest et al. 2002; zur Hausen 2009). Transmission of carcinogenic HPV types
occurs predominantly via sexual contact, including oral sex and open-mouthed kissing
(DSouza et al. 2009). Approximately 90% of HPV-positive HNCs harbor viral
sequences encoded by the high-risk HPV16 type (Capone et al. 2000; Gillison et al.
2000; Kreimer et al. 2005; Munoz et al. 2003; Nasman et al. 2009), a finding that is
476
J. You and S. Wells
in contrast to a broader spectrum of HPV types in cervical cancer, such as 18, 31,
and 45. HPV-positive tumors can be considered a distinct disease when compared
to HPV-negative HNCs. In agreement with sexual transmission as the predominant
route of HPV infection, HPV-positive tumors are strongly associated with sexual
behavior. In contrast, HPV-negative HNCs are linked to alcohol and tobacco use.
Recent clinical evidence additionally suggests that HPV-positive compared to
HPV-negative tumor status is associated with good prognosis in patients treated
with radiotherapy and perhaps even in patients that received surgery alone (Ang
et al. 2010; Fischer et al. 2010). It is likely that incomplete inactivation of p53 and
Rb pocket proteins in HPV-positive cells as opposed to their deletion or mutation in
HPV-negative cells confers an inherently less-malignant and more therapy-responsive
phenotype. Viral protein immunogenicity may further contribute to a positive
outcome (Lowy and Munger 2010; Spanos et al. 2009). However, precise molecular
mechanisms mediating differential biologies and outcomes are likely diverse and
remain to be defined. Molecular comparisons of HPV-positive and -negative HNCs
should also consider differences at the level of the originating cell status, expression,
and activity of specific viral proteins, including but not limited to E6 and E7 and
unique microRNA, transcriptome, and proteome profiles.
As discussed earlier, in general, viral integration stimulates but is not necessarily
required for cervical cancer development. The physical state of HPV DNA in HNSCCs
has not been extensively examined yet, but may depend upon the site of infection
and transformation (Koskinen et al. 2003; Mellin et al. 2000; Venuti et al. 2000).
Integrated genomes have been detected in SCCs from oral, tonsillar, and laryngeal
carcinomas, but usually at a frequency of less than 50%. This indicates that, similar
to cervical cancers, HPV integration into host cell DNA may not be a necessary
requirement for malignant transformation but that the proportion of SCCs harboring
exclusively viral episomes may be greater in HNC compared to cervical cancer.
It is tempting to speculate that HPV infection and transformation in the head and
neck region occur by mechanisms similar to that in the cervix. However, the preponderance of HPV16 and a lesser contribution of HPV to the overall disease burden
might suggest tissue-specific differences which could in turn affect HPV infection,
maintenance, replication, and/or virus production in a virus type-specific manner.
This could be due to intrinsic differences between cervical versus oropharyngeal
keratinocytes or the result of differential immunological control of virus infection in
the cervical versus oropharyngeal tract. Models of HPV transformation in the human
head and neck region are now needed to compare and contrast molecular mechanisms that drive HNC malignancy. The results promise to yield new and exciting
insights into the biology of HPV.
477
Concluding Remarks
HPVs establish persistent infection by maintaining their genomes as episomes in
infected cells. Papillomavirus hitchhikes on host mitotic chromosomes for transmitting
its genetic materials across the cell cycle to ensure accurate segregation of the replicated
viral episomes to the daughter cells during host cell division. In the meantime, the
virus hijacks the cellular machinery to facilitate viral transcription and efficient
propagation of viral genomes. Through its association with a diverse range of host
cellular factors, papillomavirus subverts the normal cellular control of proliferation
to benefit its own survival and to induce malignant progression of host cells. Studies
from recent years have greatly improved our understanding of HPV productive
infection and associated cancer progression. However, how different cellular
environmental and signaling events influence the viral gene expression during
epithelial differentiation remains an important question for future study in order to
fully understand the consequences of HPV infection in humans. The application of
HPV vaccine in cervical cancer prevention has been discussed in depth elsewhere
(Marra et al. 2009). It is worth noting that currently available HPV vaccines protect
against up to four major types of cancer-causing strains. However, the vaccines do
not protect against many other HPV types, are costly, and are not useful for those
who are already infected with the virus. As described above, besides the deregulated
expression of high-risk HPV oncogenes, substantial cytogenetic changes in host
genome are needed for high-risk HPV-infected cells to progress to invasive tumors.
Accumulation of secondary genetic changes usually occurs during the years or even
decades of a precancerous state after the initial infection. Alternative therapeutic
approaches are, therefore, needed to cure ongoing infections. A greater understanding
of the cellular factors and events that regulate persistent latent papillomavirus
infection provides a point of departure for developing new therapeutic compounds
to abrogate critical virushost interaction and viral presence. Mechanistic insights
into the functions of the viral oncogenes that account for the tumorigenic nature of
HPV-associated diseases also offer new strategies to prevent papillomavirusinduced human cancers. Comparing and contrasting these oncogene activities in
cervical cancer to other HPV-associated tumors determine whether similar or distinct
treatments are ultimately needed.
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Chapter 20
Tumorigenesis by Adenovirus Type 12 E1A

Hancheng Guan and Robert P. Ricciardi
Introduction
All human adenoviruses, of which there are 51 known serotypes, are capable of
transforming mammalian cells including those of human and rodent origin. Two
viral immediate early gene products E1A and E1B, which are encoded on the far left
end of the linear viral genome, are responsible for cell transformation. E1A stimulates
the cell cycle via its binding and interference of key cellular regulators including
retinoblastoma protein (Rb) and p300/CBP, thus leading to constitutive cellular DNA
synthesis and the loss of cell cycle regulation. In turn, through its interaction with the
tumor suppressor p53, E1B serves to block cell growth arrest and apoptosis incurred
by E1A-stimulated unconstrained cellular DNA synthesis and damage. The net result
is immortalization and continued cell proliferation, i.e., transformation. In adenovirustransformed cells, E1A and E1B genes are normally integrated into the chromosomes.
Transformation by adenovirus can be fulfilled directly by transfection of E1A and
E1B into mammalian cells in vitro or by infection of nonpermissive cells (e.g., those
of rodents) with the virus. It is noted that adenovirus transformation requires the two
viral genes to be expressed above a certain threshold. Transformation by human
adenoviruses has been the subject of several recent reviews (Ricciardi 1999;
Gallimore and Turnell 2001; Williams et al. 2004; Hohlweg et al. 2004; Endter and
Dobner 2004; Chinnadurai 2004) and is not discussed in detail here.
H. Guan
Department of Microbiology, School of Dental Medicine, University of Pennsylvania,
R.P. Ricciardi (*)
Department of Microbiology, School of Dental Medicine, University of Pennsylvania,
Levy Research Building, 4010 Locust Street, Philadelphia, PA 19104, USA
e-mail: ricciard@upenn.edu
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H. Guan and R.P. Ricciardi
While all human adenoviruses have the ability to transform cells in culture as
described above, only a small group of serotypes have the additional capability to
generate tumors in immunocompetent adult rodents. Among the tumorigenic strains
of human adenovirus, adenovirus type 12 (Ad12) is the most intensively studied.
An important feature related with Ad12 tumorigenesis is the ability of the viral transformed cells to escape immune surveillance and destruction (reviewed in Williams
et al. 2004; Hohlweg et al. 2004; Ricciardi et al. 2006). Ad12 E1A oncoprotein
(E1A-12) has been shown to be the key determinant of tumorigenicity (Bernards et al.
1982; Sawada et al. 1988). As reviewed below, Ad12 tumorigenesis relies on the ability
of E1A-12 to mediate evasion of cytolysis by CTLs and NK cells. Some of the mechanisms for these immune escape strategies mediated by E1A-12 have recently become
disclosed. Moreover, a new insight into the involvement of E1A-12 induced expression
of neuronal and tumor-related genes in Ad12 tumorigenesis has been revealed.
Ad12 Tumorigenesis Requires MHC Class I to Be Shutoff:

A Means for CTL Avoidance
About five decades ago, Ad12 was first found to generate tumors in newborn Syrian
Hamsters (Huebner et al. 1962; Trentin et al. 1962). However, many other human
adenoviruses including the well-studied Ad5 were shown to be nontumorigenic.
It was later discovered that adenoviruses that were classified as nontumorigenic
could be made to induce tumors in immunosuppressed rats or mice (Gallimore 1972;
Bernards et al. 1983). These studies suggested that Ad12 generates tumors in immunocompetent adult rodents by providing the tumor cells with a means of avoiding
cytolysis by CTLs. This notion was supported by the finding that cell surface expression
of major histocompatibility complex (MHC) class I molecules is dramatically
reduced on Ad12 transformed cells (Schrier et al. 1983; Eager et al. 1985).
MHC class I molecules are glycoproteins belonging to the immunoglobulin superfamily. MHC class I molecules are expressed on the surfaces of most mammalian cells
and play an essential role in tagging cells that express foreign (e.g., viral) proteins for
immune destruction by CTLs. The class I molecules accomplish this by presenting
nonself proteins, in the form of processed peptides, to the cell surface where they can be
recognized by CTLs to trigger lysis of the target cell. However, cells that express very
low surface levels of MHC class I molecules are able to escape from recognition and
lysis by CTLs (Zinkernagel and Doherty 1979; reviewed in Garcia-Lora et al. 2003).
As stated above, reduced expression of MHC class I molecules on the cell surface
provides Ad12-transformed cells with a powerful strategy for immune evasion.
Significantly, this reduction in MHC class I surface expression occurs with all of the
haplotypes such as H2-K, -D, and -L in mouse (Eager et al. 1985). By contrast,
substantial levels of each of the class I glycoproteins are expressed on the surfaces
of nontumorigenic Ad5-transformed cells (Eager et al. 1985). It is noted that as with
Ad5-transformed cells, Ad12 tumorigenic cells have intact MHC class I genes, since
stimulation of the interferon inducible element in their promoters by interferon-gamma
20
491
(IFN-g) can activate MHC class I expression in these tumorigenic cells (Eager
et al. 1985; Kimura et al. 1986).
It is well documented that reduced cell surface levels of MHC class I antigens
contribute to CTL escape and tumorigenesis by Ad12-transformed cells. A study
in which influenza virus-infected Ad12-transformed cells were challenged with
influenza-specific CTLs revealed resilience of the cells to lysis, whereas Ad5transformed control cells were susceptible to the lysis (Yewdell et al. 1988).
Significantly, treatment of the influenza virus-infected Ad12-transformed cells
with IFN-g induced an increase of surface MHC class I molecules and renewed
CTL lysis of the cells (Yewdell et al. 1988). This demonstrates that downregulation of MHC class I surface antigen expression on Ad12-transformed cells
enables evasion of cytolysis by self-restricted CTLs. As a consequence, Ad12transformed cells can continuously proliferate to generate tumors in rodents with
intact immune systems. Consistent with these results, another study showed that
expression of an exogenous MHC class I gene in Ad12-transformed cells could
abrogate tumorigenicity (Tanaka et al. 1985). It was also shown that IFN-g
induced transient expression of MHC class I antigens in Ad12-transformed cells
is sufficient to reduce their tumorigenicity in immunocompetent rodents (Hayashi
et al. 1985). In addition, subcutaneous administration of IFN-g following the
introduction of a tumorigenic dose of Ad12-transformed cells completely hindered
the development of tumors in rodents (Hayashi et al. 1985). Importantly, cells
from freshly excised tumors generated by Ad12-transformed cells were found to
exhibit diminished MHC class I antigens (Eager et al. 1985). These data clearly
demonstrate a direct association between Ad12 tumorigenesis and diminution of
MHC class I surface expression.
E1A-12 Is Solely Responsible for MHC Class I Shutoff

Although cell transformation by Ad12 requires both E1A-12 and E1B-12 genes,
only the E1A-12 gene is responsible for cell surface MHC class I reduction
(Schrier et al. 1983; Vasavada et al. 1986). This was first demonstrated by
expressing E1A-12 protein as the only product of Ad12 in a human cell line
(Vasavada et al. 1986). This E1A-12 expressing cell line exhibited dramatic
reduction in class I HLA levels of each haplotype (HLA, A, B, and C) compared
with a matched control cell line. Another study using hybrid cell lines of Ad12and Ad5-transformed cells also revealed that MHC class I surface antigens of the
hybrid cells were reduced to the same extent as that of their parental Ad12transformed cells (Ge et al. 1994). This indicates that E1A-12 is able to reduce
the high surface class I levels of Ad5-transformed cells. Indeed, a direct stable
introduction of E1A-12 into Ad5-transformed cells gave rise to reduced expression of MHC class I antigens on the cell surface (Guan et al. 2008). Together,
these findings demonstrate that E1A-12 actively mediates the dramatic reduction
of MHC class I surface antigens.
492
E1A-12 Mediates MHC Class I Shutoff

at the Transcription Level
The low amount of MHC class I surface antigens on Ad12 transformed cells were
found to be directly related to a drastic reduction in the class I mRNA levels (Schrier
et al. 1983; Eager et al. 1985; Vasavada et al. 1986; Liu et al. 1996). Significantly,
as revealed by nuclear run-on experiments, diminished transcription is responsible
for these reduced levels of MHC class I mRNAs (Ackrill and Blair 1988; Friedman
and Ricciardi 1988). This suggested that the class I promoter is negatively regulated
in Ad12 tumorigenic cells. Extensive mutational analyses identified that the class
I promoter enhancer region (located between nucleotides 205 and 159 upstream
of the transcription start site) is the targeted element responsible for diminished
MHC class I transcription (Ge et al. 1992). This enhancer, when appended to the
class I basal TATA box promoter (located between nucleotides 37 and +1), strongly
stimulated transcription in Ad5-transformed cells, but not in Ad12-transformed cells
(Ge et al. 1992). As discussed below, modulation of transcription factor binding to
the class I enhancer region by E1A-12 accounts for transcription diminution of all
MHC class I genes.
The MHC class I enhancer comprises two DNA regulatory elements, referred to
as R1 and R2 (Kimura et al. 1986). The R1 site contains a recognition sequence for
the transcription activator NF-kB, whereas the R2 sequence has a recognition
sequence for nuclear hormone receptors including the transcription repressor COUPTFII. As detailed below and depicted in Fig. 20.1, E1A-12 prevents NF-kB binding
and transactivation activity at the class I enhancer R1 site, while concurrently mediating
the binding of the nuclear hormone repressor COUP-TFII to the R2 site.
E1A-12 Prevents NF-kB (p50/p65) Binding to the Class I

Enhancer R1 Site as well as Its Transactivation Activity
by Blocking Phosphorylation of p50 and p65
NF-kB is a transactivator composed predominantly of two subunits p50 and p65.
The p50 subunit is largely responsible for DNA binding, whereas the p65 subunit is
accountable for transactivation through its C-terminal activation domain. NF-kB is
the master regulator of immune responsive genes including the class I alleles
(reviewed in Xiao and Ghosh 2005). Under normal circumstances, NF-kB is retained
in the cytoplasm via its association with inhibitor IkB. Upon stimulation by cytokines
such as TNF-a, IkB is phosphorylated by IkB kinase (IKK) and subsequently
ubiquitinated and degraded by the 26S proteasome (reviewed in Baldwin 1996;
Pahl 1999; Scheidereit 2006). This leads to the release and translocation of NF-kB
to the nucleus where it binds to target genes and activates transcription.
In both Ad5 nontumorigenic and Ad12 tumorigenic cells, NF-kB constitutively
translocates to the nucleus and is similarly abundant (Liu et al. 1996). Yet, only
20
Ad5 non-tumorigenic cells
493
E1A-5
PKAc
50 65
P-S337 P-S276
R2 SITE
50 65
R1 SITE
Class I ON
Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac
Ad12 Tumorigenic cells

PKAc
E1A-12
E1A-12
E1A-12
50 65
NCoR HDAC1/8
COUP -TFII COUP -TFII
R2 SITE
Class I OFF
R1 SITE
Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac
Fig. 20.1 Model of E1A-12-mediated repression of MHC class I transcription. Upper panel: Active
MHC class I transcription in Ad5 nontumorigenic cells. The ability of NF-kB (p50/p65) to bind
DNA and stimulate transcription requires phosphorylation of each subunit. Specifically, NF-kB is
phosphorylated by the catalytic subunit of PKA (PKAc) at serine-337 of p50 and serine 276 of p65.
It is noted that E1A-5 can bind PKAc but not NF-kB. The R2 site of the class I enhancer is not
occupied by the COUP-TFII repression complex and the chromatin is acetylated (Ac), indicative of
active transcription. Lower panel: Downregulated MHC class I transcription in Ad12 tumorigenic
cells. Binding of E1A-12 to both NF-kB and PKAc prevents access of PKAc to p50 and p65,
thereby prohibiting phosphorylation and binding of NF-kB (p50/p65) to the R1 site. By contrast,
E1A-12 mediates the binding of the COUP-TFII repression complex to the R2 site. This repression
complex includes homodimers of COUP-TFII, which directly bind the R2 site, the nuclear corepressor
NCoR, the histone deacetylases HDAC1 and HDAC8, and E1A-12. The HDACs act to deacetylate
chromatin (Ac), indicative of inactive transcription. The recruitment or stabilization of HDAC1
and HDAC8 to the repression complex is dependent on the presence of E1A-12
nuclear NF-kB in Ad12 tumorigenic cells fails to bind the R1 site of the class I
enhancer (Liu et al. 1996). Exchange of NF-kB p50 and p65 subunits between Ad5and Ad12-transformed cells clearly revealed that the p50 subunit is largely responsible
for the NF-kB DNA binding deficiency in Ad12 tumorigenic cells (Kushner and
Ricciardi 1999). Essentially, p50 proteins from Ad12 tumorigenic cells fail to support
DNA binding when reconstituted with the p65 subunit from Ad5 nontumorigenic cells.
By contrast, NF-kB composed of p50 from Ad5-transformed cells and p65 from
494
either cell strongly binds DNA. It was discovered that p50 from Ad12 tumorigenic
cells is hypophosphorylated, whereas this subunit is highly phosphorylated in Ad5transformed cells (Kushner and Ricciardi 1999). This finding supports the notion
that phosphorylation of p50 is critical for DNA binding (Li et al. 1994). Indeed,
phosphatase treatment of p50 from Ad5-transformed cells abolished p50 DNA binding activity (Kushner and Ricciardi 1999).
As indicated above, due to the hypophosphorylation of p50, NF-kB fails to bind
to the class I enhancer even though it is abundant in nuclei of Ad12 tumorigenic
cells (Kushner and Ricciardi 1999). To address how phosphorylation of p50 affects
its DNA binding activity, extensive mutagenesis of p50 was conducted to define
which phospho-residues of p50 are essential for DNA binding. This led to the finding
that serine residue 337 is critical for DNA binding of p50 (Hou et al. 2003). This
serine residue was determined to be phosphorylated by the protein kinase A catalytic subunit (PKAc) and its phosphorylation by this kinase is required for p50 DNA
binding (Guan et al. 2005). It is interesting to note that this discovery provides
another example by which viral related studies often lead to understanding natural
host mechanisms, in this case, the manner by which DNA binding of NF-kB is regulated by phosphorylation.
The major question regarding the mechanism of how E1A-12 prevents DNA
binding of NF-kB is becoming clearer. It has been recently discovered that while
both E1A-12 and E1A-5 can physically interact with PKAc, only E1A-12 is able to
bind p50 (Guan et al. 2008; Guan and Ricciardi, unpublished). Significantly, the
binding of E1A-12 to p50 prevents PKAc from phosphorylating serine-337, whereas
the kinase activity of PKAc is not affected by E1A-12 (Guan et al. 2008). These data
suggest that E1A-12, PKAc and p50 could form a tri-complex, in which E1A-12 may
block p50 from gaining access to the catalytic site of PKAc. Since PKAc is essential
to cell survival, then E1A-12 in this context would target only a single substrate of
the kinase, i.e., serine-337 of p50. Interestingly, by binding to the p65 subunit, E1A12 also prevents PKAc from phosphorylating serine-276 in a similar manner (Guan
et al. 2008; Jiao et al. 2010). Phosphorylation of p65 serine-276 by PKAc is important for NF-kB transactivation (Zhong et al. 1997, 1998, 2002). It has been proposed
that phosphorylation of p65 serine-276 by PKAc induces a conformational change
that releases an intramolecular masking of the C-terminal transactivation domain by
the N-terminal region, thus leading to enhanced binding by the coactivator CBP/
p300 (Zhong et al. 1998). E1A-12 deletion analyses demonstrate that the N-terminal
40 residues are sufficient to bind p65 and block serine-276 phosphorylation (Jiao
et al. 2010). This very N-terminal region is also capable of binding the p50 subunit
(Jiao et al., unpublished data), which is likely to block p50 phosphorylation as well.
In summary, one mechanism employed by E1A-12 to downregulate transcription
from the class I promoter is to prevent NF-kB from binding to the enhancer R1 site
and stimulating transcription. Specifically, E1A-12, especially the N-terminal
40-residue region, impedes the ability of PKAc to phosphorylate NF-kB at serine-337
of p50 and serine-276 of p65 (Fig. 20.1). As a consequence, NF-kB DNA binding
and transactivation activities are inhibited, MHC class I transcription is downregulated, and MHC class I antigen expression on the cell surface is diminished.
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495
E1A-12 Mediates Repression of MHC Class I

Transcription by Recruiting COUP-TFII/HDAC
to the R2 Site of the Class I Enhancer
The R2 site of the MHC class I enhancer contains a recognition sequence for
nuclear hormone receptors (Kimura et al. 1986). Ad12 tumorigenic cells were
shown to display a strong binding activity to the R2 site, whereas such binding
activity is absent in Ad5-transformed cells (Ge et al. 1992; Kurshner et al. 1996).
In Ad12 tumorigenic cells, this strong binding activity to the R2 site is correlated
with repression of the class I promoter (Ge et al. 1992; Kralli et al. 1992). It was
determined that the nuclear hormone orphan receptor COUP-TFII is responsible
for the R2 site binding activity (Liu et al. 1994; Smirnov et al. 2001). COUP-TFII,
which is known to function mainly as a transcriptional repressor (Zhou et al.
2000), occupies the R2 site as a homodimer in Ad12 tumorigenic cells (Liu et al.
1994). Interestingly, this is the only known instance in which COUP-TFII binds
to the well-studied MHC class I enhancer to repress transcription. It was discovered that COUP-TFII, via its C-terminal domain, forms a repression complex at
the R2 site with HDAC (histone deacetylase) and N-CoR (Smirnov et al. 2000).
Histone deacetylation by HDAC in the class I promoter causes chromatin condensation, which results in transcription repression of the target gene. Since there is
COUP-TFII binding to the R2 site only in Ad12 tumorigenic cells, this explains
why the R2 site exerts its repression on MHC class I transcription in Ad12 tumorigenic cells, but not in Ad5 nontumorigenic cells.
In Ad12 tumorigenic cells, what accounts for COUP-TFII binding to the R2 site
of the class I enhancer? It was shown that Ad12 tumorigenic cells contain elevated
levels of COUP-TFII mRNA and protein when compared with Ad5 nontumorigenic
cells (Smirnov et al. 2001). E1A-12 is involved in upregulating COUP-TFII expression. Obviously, this explains in one way how E1A-12 mediates repression at the
R2 site of the class I enhancer. Significantly, in addition to upregulating COUPTFII, E1A-12 was found to be an integral component of the COUP-TFII/HDAC
complex (Zhao et al. 2003). Essentially, the physical presence of E1A-12 in this
complex is necessary for transcriptional repression of MHC class I (Zhao and
Ricciardi 2006). It is noted that E1A-12 does not bind to DNA directly, but functions
through proteinprotein interactions (reviewed in Flint and Shenk 1997). A recent
ChIP (chromatin immunoprecipitation) assay, a method used to determine specific
proteins that bind to targeted DNA region, revealed that HDAC1 and/or HDAC8
reside in the COUP-TFII repression complex at the class I enhancer R2 site (Zhao
and Ricciardi 2006). Elimination of E1A-12 from this COUP-TFII repression
complex by antisense oligonucleotides also removes HDAC1 and HDAC8 from the
complex, which results in an increase in both chromatin acetylation (a hallmark of
active transcription) of the class I enhancer and expression of MHC class I antigens
on the cell surface (Zhao and Ricciardi 2006). These data strongly indicate that
E1A-12 mediates MHC class I repression via recruiting (or stabilizing) HDAC1 and
HDAC8 to the COUP-TFII repression complex.
496
In summary, E1A-12 mediates MHC class I repression via the enhancer R2 site.
E1A-12 not only upregulates COUP-TFII expression and binding activity to the R2
site but also acts to recruit (or stabilize) HDAC1 and HDAC8 to the COUP-TFII
repression complex, thus resulting in chromatin condensation of the class I gene
(Fig. 20.1). As a consequence of these E1A-12-mediated activities at the enhancer
R2 site, MHC class I transcription is repressed and the cell surface levels of MHC
class I antigens are dramatically diminished.
E1A-12-Mediated Regulation of Both NF-kB and COUP-TFII/

HDAC Binding Provides a FAIL-SAFE Mechanism to Ensure
MHC Class I Repression
What is the advantage for E1A-12 to employ two distinct mechanisms, i.e., inhibition of NF-kB DNA binding and transactivation plus enhancement of COUP-TFII/
HDAC repression, for MHC class I shutoff? It is likely that E1A-12 uses these two
mechanisms to ensure that MHC class I antigens on the cell surface do not become
induced by physiological fluctuations. For example, under normal cell growth conditions, E1A-12 is able to prevent nuclear NF-kB DNA binding and transactivation by
blocking phosphorylation of both p50 and p65 as described above (Hou et al. 2003;
Guan et al. 2005, 2008; Jiao et al. 2010). However, cytokines such as TNF-a and
IL-1b are able to induce a new pool of nuclear NF-kB that is capable of DNA binding
in Ad12 tumorigenic cells, while the preexisting pool of hypophosphorylated NF-kB
remains unable to bind DNA (Hou et al. 2002). This indicates that while E1A-12
constitutively blocks phosphorylation of the preexisting pool of NF-kB, this mechanism is not always sufficient to completely prevent phosphorylation of a massive
new pool of cytokine-induced NF-kB. Nevertheless, the binding of COUP-TFII/
HDAC repressor complex to the R2 site should be able to override the cytokineinduced NF-kB DNA binding and transactivation activity, thus keeping MHC class
I shutoff. Indeed, TNF-a and IL-1b fail to enhance MHC class I transcription as long
as the COUP-TFII/HDAC complex binds to the class I enhancer (Hou et al. 2002).
Only when the repressive effect of COUP-TFII/HDAC is relieved, a pronounced
activation of MHC class I transcription by NF-kB following cytokine treatment is
observed (Hou et al. 2002). As expected, treatment of Ad12 tumorigenic cells with
both cytokines and HDAC inhibitor TSA leads to a significant increase in the level
of cell surface MHC class I antigens (Hou et al. 2002). Further evidence that COUPTFII repression dominates over NF-kB activation in MHC class I transcription
regulation is obtained from NF-kB knockout cells, in which activation by exogenous
NF-kB is completely blocked by exogenous COUP-TFII (Smirnov et al. 2001).
In summary, Ad12 tumorigenic cells apparently benefit from the collateral effects
of COUP-TFII/HDAC repressor complex and hypophosphorylated NF-kB in downregulating MHC class I transcription (Fig. 20.1). These dual functions provide a
stringent FAIL-SAFE mechanism to ensure that transcription of MHC class I genes
remains shutoff under oscillating physiological conditions. In the event that NF-kB
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497
is activated by cytokines and other stimulators, COUP-TFII/HDAC will still maintain

repression of MHC class I transcription, thus ensuring immune escape and survival
of tumorigenic Ad12 cells. Alternatively, if COUP-TFII/HDAC repressor complex
loses its binding to the class I enhancer or the repression activity under certain
physiological conditions, then hypophosphorylation of NF-kB will ensure that this
activator fails to bind DNA and stimulate MHC class I transcription. We propose
that to stringently protect cells from lysis by CTLs, E1A-12 coevolved dual functions
that serve to block NF-kB DNA binding and transactivation at the class I enhancer
R1 site, while simultaneously recruit the COUP-TFII/HDAC repressor complex to
the R2 site.
E1A-12 Mediates Escape from Natural Killer Cells

by Downregulating NK Activating Ligands
E1A-12 Confers Resistance to NK Cell Lysis
NK cells of the innate immune system serve as the first line of defense. These effectors
of innate immunity respond to the integration of inhibitory and activating signals
from target cells (Garcia-Lora et al. 2003; Makrigiannis and Anderson 2003; Lodoen
and Lanier 2005; reviewed in Lanier 2005). It is well known that loss of MHC class
I antigen expression on target cells relieves the NK inhibitory receptor and leads to
lysis of target cells. Yet, surprisingly, Ad12 tumorigenic cells that have diminished
MHC class I surface antigens are also resistant to lysis by NK cells (Sawada et al.
1985; Kenyon and Raska 1986). The E1A-12 gene of tumorigenic Ad12 specifically
confers this resistance to NK lysis (Sawada et al. 1985; Kenyon and Raska 1986).
Could it be that E1A-12 has found a way to downregulate cell surface expression of
both activating ligands and MHC class I surface antigens as a means of avoiding
NK and CTL lysis? Recent evidence (below) suggests that E1A-12 does employ
this strategy to escape both innate (NK) and cell-mediated (CTL) immunity.
E1A-12 Reduces Expression of Activating Ligands

that Trigger NK Lysis
NKG2D is a major activating receptor expressed on the surface of murine NK cells.
The NKG2D receptor on NK cells recognizes activating ligands (RAE1-a, -b, -g, -e,
MULT1, and H60) that are upregulated on infected and transformed target cells.
By contrast, MHC class I molecules expressed on target cells engage inhibitory
receptors on NK cells. Because MHC class I molecules function as recognition
elements for CTLs but as ligands for inhibitory receptors on NK cells, they play
diametric roles in CTL versus NK cell-mediated lysis (Kenyon and Raska 1986;
498
NK
inhibitory
receptor
Target
Cell
A
NK Cell
NKG2D
activating
receptor
NK Cell
MHC class I
inhibitory
ligand (-)
Activating
ligand (+) Lysis depends
on balance of
+/- ligands
Target
Cell
B
LYSED
NK Cell
Ad12
Tumorigenic
Cell
NOT LYSED
Fig. 20.2 Mechanism by which Ad12 tumorigenic cells escape lysis by NK cells. Top panel:
Modulation of NK lysis of target cells depends on the balance of inhibitory (e.g., MHC class I) and
activating ligands. Middle panel: Loss of inhibitory MHC class I favors lysis. Bottom panel: Ad12
tumorigenic cells have diminished MHC class I expression yet escape NK cells due to E1A-12mediated reduction of all NKG2D activating ligands. See text for details
reviewed in Lanier 2005). Ultimately, the deciding factor as to whether the NK cell
should kill the target cell is somewhat complex in that lysis is modulated by the
balance of inhibitory and activating signals (Fig. 20.2, top panel). Indeed, when
inhibitory MHC class I molecules are downregulated, the engagement of NKG2D
receptor and activating ligands enables lysis (Fig. 20.2, middle panel).
Sometimes, however, the activating signals are sufficiently elevated to cause
lysis, even though surface MHC class I is expressed. This apparently is the case in
nontumorigenic Ad5-transformed cells that express MHC class I antigens. Here, E1A-5
has been shown to sensitize the nontumorigenic Ad5 cells to NK lysis, by binding
to p300 and upregulating the RAE-1 activating ligand that is recognized by the
NKG2D activating receptor (Cook et al. 1996; Routes et al. 2005). Presumably, in
Ad5 nontumorigenic cells, the activating ligand expression (e.g., RAE-1) dominates
the inhibitory MHC class I signal. Contrary to expectations, the almost complete
diminution in MHC class I expression on Ad12 tumorigenic cells does not make
them sensitive to NK lysis. Rather, Ad12 tumorigenic cells are highly resistant to
NK lysis and E1A-12 has been shown to be responsible for conferring this resistance
(Sawada et al. 1985; Kenyon and Raska 1986; Heyward et al., unpublished).
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499
Recent findings now reveal that E1A-12 mediates resistance to NK lysis by inhibiting
expression of all of the NKG2D activating ligands on the cell surface (Heyward
et al., unpublished). This paninhibition is depicted in Fig. 20.2 (bottom panel),
which indicates that on Ad12 cells, there is lack of both inhibitory MHC class I ligand
as well as all of the NKG2D activating ligands. Further studies demonstrated that
the level of mRNA for each NKG2D activating ligand is also greatly reduced
(Heyward et al., unpublished). Thus, the diminished levels of NKG2D activating
ligands on the surface of Ad12 tumorigenic cells directly correlate with the dramatically downregulated mRNA expression of NKG2D activating ligands. Based on
general knowledge that E1A proteins regulate gene expression of multiple promoters,
it is likely that E1A-12 may target regulatory transcription factor complexes that are
common to all of the NKG2D activating ligands. Currently, the regulatory domains
of the NKG2D activating ligand promoters are poorly characterized (Nomura et al.
1996; Nausch et al. 2006). However, a comparison of the NKG2D activating ligand
promoter sequences revealed binding sites for NF-kB and COUP-TFII (Heyward
and Ricciardi, unpublished), which, of course, are the targets of E1A-12-mediated
repression of the class I promoter as described above. Intriguingly, should the binding
activities of NF-kB and COUP-TFII in the NKG2D activating ligand promoters
also be affected, this would suggests that E1A-12 uses a single mechanism to
coordinately enable escape of Ad12 tumorigenic cells from both innate (NK) and
cell-mediated (CTL) immunity.
E1A-12 Induces Expression of Neuronal

and Tumor-Related Genes
While E1A-12-mediated downregulation of MHC class I and NK ligands essentially
contributes to viral tumorigenesis via enabling immune escape from CTLs and NK
cells, other cellular genes mediated by the E1A-12 protein likely also play a critical
role in Ad12 tumorigenesis. Therefore, a broader view of Ad12 tumorigenesis may
be better addressed by understanding of E1A-12-mediated global changes in gene
expression. Recent studies using microarray have revealed that E1A-12 is involved
in modulating numerous genes that are implicated in various cell functions including
the cell cycle, transcriptional regulation, signal transduction, immune response, and
tumor invasiveness (Guan et al. 2003, 2009). Of special interest among those genes
regulated by E1A-12 are a number of neuronal and tumor-related genes whose
expression is induced by E1A-12 in Ad12 tumorigenic cells (Guan et al. 2009).
Compared with E1A-5 and the counterparts of other nontumorigenic adenoviruses, E1A-12 contains a unique Spacer region that is composed of 20 amino
acids between the conserved regions CR2 and CR3 (Fig. 20.3). This Spacer region
is essential for Ad12 tumorigenesis (reviewed in Ricciardi 1999, 2006; Williams
et al. 2004). Deletion of the Spacer or alteration of even a single amino acid in the
Spacer can abolish Ad12 tumorigenesis (reviewed in Williams et al. 2004; Ricciardi
et al. 2006). However, Ad12 Spacer mutants retain the ability to repress MHC
CR1
CR2 S CR3
MHC class I repression

124
(Tumorigenesis)
Transformation
Transactivation
Tumorigenesis
Transformation

Transformation
500
266
CR4 C
143
Spacer region
DENGMAHVSASAAAAAADRE
Fig. 20.3 E1A-12 oncoprotein. Depicted is the largest spliced form of the E1A-12 protein
(266 residues) showing four conserved regions (CR1, CR2, CR3, CR4), and the unique 20-residue
Spacer (S) region. Indicated are the functions encoded by each domain
class I expression as well as to transform cells in culture (reviewed in Ricciardi 1999,

2006; Williams et al. 2004). A recent microarray study has revealed that the Spacer,
along with other regions in E1A-12, plays a critical role in upregulating a number
of neuronal and tumor-related genes including N-MYC, ROBO1, and neuronal
protein 3.1 (Guan et al. 2009). Specifically, this microarray compared gene expression profiles between Ad12 cell lines with only a single amino acid difference in
E1A-12: one cell line expressed wt E1A-12 and was tumorigenic, whereas the
other cell line expressed a point-mutant of E1A-12 and was nontumorigenic. Thus,
the differential expression in cellular genes could be accounted for by a singleamino acid substitution in E1A-12. The fact that the E1A-12 spacer is essential for
Ad12 tumorigenesis suggests that some neuronal genes it upregulates are likely
involved in the viral tumorigenic process. For example, N-MYC is known to possess cell-transforming and oncogenic functions (Ingvarsson 1990; Kawagoe et al.
2007); moreover, the transmembrane receptor ROBO1, whose normal function
involves axon path finding and neuronal migration (Kidd et al. 1998; Zallen et al.
1998; Brose et al. 1999), has been reported to participate in mediating glioma cell
migration (Mertsch et al. 2008). Importantly, ROBO1 has been found to be highly
expressed in several nonneuronal tumors including hepatocellular carcinoma
(Ito et al. 2006) and colorectal cancer (Grone et al. 2006). In accord with a potential
nexus between neuronal gene expression and E1A-12-mediated tumorigenesis is the
reduction of MHC class I surface antigens on neuronal cells as well as a requirement
of COUP-TFII for neuronal development (Qiu et al. 1994). Particularly relevant to
these molecular studies is the observation that Ad12-induced tumors exhibit mesenchymal and neuronal characteristics (reviewed in Hohlweg et al. 2004). It is, thus,
intriguing to speculate that Ad12 may usurp the functions of certain neuronal genes
to promote tumorigenesis.
It is noted that the induction of neuronal gene expression is not limited to
Ad12 tumorigenic cells, but is relatively common in tumor cells. For example, it
has been found that several nonneuronal tumors including breast, ovarian,
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501
colorectal, and pancreatic cancers all display aberrant expression of neuronal

genes (Chen et al. 1997; Garber et al. 2001; Albert and Darnell 2004; Zhang et al.
2005; Grone et al. 2006). While the real functions and the involvement of tumorassociated neuronal genes in cancer formation has yet to be further elucidated,
it is possible that the neuronal gene expression may provide the tumor cells with
some survival advantage, e.g., immune privilege and relatively high motility as
found with neuronal cells.
Neuronal Gene Induction by E1A-12 Involves the Inhibition

of the Neuronal Repressor NRSF and the Stimulation
of Certain Neuronal Transactivators
Like many other cellular genes, neuronal genes are regulated by both transcription
repressors and activators. In nonneuronal cells, however, neuronal genes are normally
repressed by the master neuronal regulator NRSF (neuron-restrictive silencer factor),
also known as REST (repressor element 1-silencing transcription factor) (reviewed
in Coulson 2005; Majumder 2006). NRSF consists of two N- and C-terminal repressor
domains (RD1 and RD2) and nine C2H2 (Cys2His2) zinc-finger motifs, as well as
lysine- and proline-rich domains (Fig. 20.4). The two repressor domains serve as
scaffolds for formation of distinct, large repressor complexes via recruitment of
multiple corepressors such as Sin3, HDAC, and CoREST (reviewed in Coulson 2005;
Majumder 2006). NRSF recognizes and binds to cis-acting DNA sequences called
RE1 (repressor element 1) found in over a thousand neuronal genes. NRSF is rarely
or not expressed in neuronal cells, but is widely expressed in nonneuronal cells.
NRSF was highly expressed in both Ad5- and Ad12-transformed rat and mouse
kidney cells. Surprisingly, NRSF was barely present in the nuclei of these cells, but
located mainly in the cytoplasm (Guan et al. 2009). This indicates that the neuronal
repression function of NRSF is compromised in the viral transformed cells.
Interestingly, NRSF was able to efficiently translocate into the nucleus in these
cells, but was unable to accumulate in the nucleus, even when the cells were treated
with the nuclear export inhibitor leptomycin B (Guan and Ricciardi, unpublished
data). By contrast, treatment of the adenovirus-transformed cells with the proteasome
inhibitor MG-132 significantly increased nuclear accumulation of NRSF (Fig. 20.5).
These data indicate that in the viral transformed cells, the loss of NRSF in the
nucleus is not due to its nuclear entry blockage or enhanced nuclear export, but
rather rapid degradation by proteasomes.
Proteasomal degradation first requires the target protein to be covalently modified
by ubquitination (reviewed in Finley 2009; Schrader et al. 2009). Our most recent
data indicated that NRSF is ubiquitnated in the nuclei of both Ad5- and Ad12transformed cells, and this ubiquitination can be blocked by PYR-41, an inhibitor of
ubiquitin-activating enzyme E1 (Guan and Ricciardi, unpublished data). Importantly,
E1A is likely responsible for promoting NRSF ubiquitination, since knockdown of
E1A in the viral transformed cells lessened the nuclear presence of ubiquitinated
502

DNA binding domain
(8 zinc-fingers)
RD1
N
Lys-rich
region
Pro-rich
region
RD2
9
Fig. 20.4 Schematic representation of NRSF. NRSF consists of two N- and C-terminal repressor
domains (RD1 and RD2), nine zinc-finger motifs, and lysine- and proline-rich domains. RD1 and
RD2 can recruit distinct corepressors to form large repressor complexes for transcription silencing.
The nine zinc fingers are numbered. Zinc fingers 18 form the DNA binding domain, whereas zinc
finger 9 is required for corepressor recruitment by RD2
Fig. 20.5 Degradation of NRSF by proteasomes in the nucleus. Ad12-transformed cells were either
left untreated or treated with proteasome inhibitor MG-132, followed by confocal immunofluorescent microscopy using an antibody against NRSF (green). Nuclei were stained with DAPI (blue)
NRSF, which is much larger in size than the cytoplasmic un-ubiquitinated form
(Guan and Ricciardi, unpublished data). Recently, two research groups have identified two adjacent, but distinct degrons near the C-terminal RD2 (Guardavaccaro
et al. 2008; Westbrook et al. 2008). Both degrons are recognized by the ubiquitin E3
ligase SCFb-TRCP and can elicit NRSF degradation by proteasomes. We found that
deletion or mutation of either of the two degrons eliminated NRSF degradation in
the nuclei of Ad12-transformed cells, showing that both degrons are required for
E1A-mediated proteasomal degradation of NRSF (Guan and Ricciardi, unpublished).
These data strongly indicate that E1A is involved in stimulating ubiquitination and
proteolysis of NRSF in the nucleus, thus relieving NRSF silencing effect on neuronal genes.
The rapid proteasomal degradation of NRSF in both Ad5- and Ad12transformed cells should serve as a means by which the viral oncoprotein E1A
20
503
overcomes repression of neuronal genes. It is noted that by interacting with the

NRSF corepressor CoREST, the ICP0 protein of herpes simplex virus 1 is able to
mediate nuclear export of NRSF to the cytoplasm (Gu et al. 2005; Gu and Roizman
2007). While the relief of NRSF repression should enable both Ad5- and Ad12transformed cells to express neuronal genes, neuronal gene expression was found
to occur only in Ad12 tumorigenic cells. What accounts for this difference? In
addition to the relief of NRSF repression, the induction of neuronal genes by
E1A-12 would further require the activation of neuronal promoters. Based on sitedirected mutagenesis analysis in the promoter region of neuronal gene a-internexin, our data clearly demonstrate that neuronal gene induction by E1A-12 (but
not E1A-5) is dependent on the binding of certain nuclear factors especially basic
helix-loop-helix (bHLH) transactivators to the promoter (Guan et al. 2009). In
strong contrast, neuronal promoter binding by these factors was not found in nontumorigenic Ad5-transformed cells (Guan et al. 2009). Yet, further study is needed
to identify these nuclear factors and how their DNA binding activities are differentially regulated by E1A12 and E1A-5.
Involvement of E1A-12-Mediated NRSF Degradation

and Neuronal Gene Induction in Ad12 Tumorigenesis
Apart from being a key neuronal repressor, NRSF has recently been identified as a
tumor suppressor in nonneuronal cells (Westbrook et al. 2005, 2008). Significantly,
knockdown of NRSF in epithelial cells was able to cause cell transformation
(Westbrook et al. 2005). In accordance with this, NRSF dysfunction and/or deregulation with concurrent aberrant neuronal gene expression have been implicated in
human cancers (reviewed in Coulson 2005; Majumder 2006). The dual role of
NRSF in repressing both neuronal genes and tumor formation in nonneuronal cells
implies that there is a connection between aberrant neuronal gene expression and
tumor formation. However, it has yet to be determined that repression of neuronal
genes by NRSF in nonneuronal cells really contributes to tumor suppression. In terms
of Ad12 tumorigenesis, a related question that remains to be addressed is whether
E1A12-mediated NRSF proteasomal degradation or neuronal gene induction or
both are essential for cell transformation and tumorigenesis by the virus. In light
of the fact that the nontumorigenic E1A-5 can also induce NRSF proteasomal degradation but not neuronal gene expression, it is intriguing to speculate that the loss of
NRSF repression is implicated in viral transformation, whereas the neuronal gene
induction is likely a forward step involving tumorigenesis.
In summary, E1A-12, as with its nontumorigenic adenovirus counterparts including
E1A-5, is capable of reprogramming cellular gene expression and transforming
cells. Uniquely, E1A-12 is able to mediate MHC class I shutoff, which enables
Ad12 tumorigenic cells to escape from CTL-mediated cytolysis. Also, E1A-12 is
likely involved in mediating evasion of cytolytic killing by NK cells through the
downregulation of NKG2D activating ligands in Ad12 tumorigenic cells. Importantly,
504
Ad12 tumorigenesis requires the function encoded by the unique 20-amino acid
Spacer between the conserved regions CR2 and CR3. This Spacer, together with
other regions in E1A-12, plays a critical role in inducing neuronal and tumor-related
genes. Neuronal gene induction by E1A-12 requires not only the relief of NRSF
repression via ubiquitin-mediated proteolysis in the nucleus but also the activation
of neuronal promoters by certain transactivators. The E1A-12-mediated proteasomal
degradation of NRSF, as well as the induction of neuronal and tumor-related genes
by the oncoprotein, likely plays an important role in Ad12 tumorigenesis.
Acknowledgment We wish to acknowledge Grant CA29797 from the National Institutes of
Health (to R. P. R.).
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Chapter 21
Overview of Hepatitis Viruses and Cancer

Timothy M. Block, Jinhong Chang, and Ju-Tao Guo
Introduction
The liver is one of the bodys largest organs, is part of the digestive system, and is
heavily vascularized, as illustrated in Fig. 21.2. Primary cancer of the liver and bile
duct is increasing in incidence in the world and in the USA. Hepatocellular carcinoma
(HCC), the most common primary liver cancer, is increasing in incidence both worldwide and in the USA. In the world, HCC is now responsible for an estimated 600,000
deaths annually and, as indicated in Table 21.1, is now the third or fourth most common
cause of cancer death in the world, accounting for as much as 13% of all cancer
mortality. In the USA, HCC is the fastest rising cancer in annual incidence and
accounts for approximately 19,000 deaths per year and is now the tenth most common
cause of cancer death (Howlader et al. 2011). Thus, while many cancers are declining in incidence and mortality, HCC is on the rise in the developed countries
(Fig. 21.1).
Despite the overwhelming evidence supporting that chronic infections with HBV
and HCV are the primary cause of HCC (Block et al. 2003; Tsai and Chung 2010),
the molecular pathways by which the viral infections lead to the development of
HCC remain largely elusive.
Curiously, although both HBV and HCV infect hepatocytes, the parenchyma
cells of the liver, and establish life-long persistent infections under certain circumstances, they are virologically distinct, in many aspects. Most obviously, HBV is a
T.M. Block (*)

Department of Microbiology and Immunology, Drexel University College of Medicine,
Pennsylvania Biotechnology Center, Doylestown, PA, USA
Hepatitis B Foundation, Pennsylvania Biotechnology Center, Doylestown, PA, USA
e-mail: timothy.block@drexelmed.edu
J. Chang J.-T. Guo
Department of Microbiology and Immunology, Drexel University College of Medicine,
Pennsylvania Biotechnology Center, Doylestown, PA, USA
509
510
T.M. Block et al.
Table 21.1 Leading causes

of cancer death in the world
Organ
Lung
Stomach
Colorectal
Liver
Breast
Deaths per year

1,300,000
803,000
639,000
610,000
519,000
The five leading causes of death due to

cancer, worldwide. Note that there is significant geographic variation for liver,
stomach, and lung. Based on World
Health Organization statistics (2010),
Parkin (2006), and El-Serag et al. (2001)
HCC Etiology Worldwide

Other
22%
HBV
53%
HCV
25%
HCC Etiology
in the US (2004)
Alcohol,
No HBV,
HCV 9%
HBV+HCV
4%
HBV
16%
HCV
55%
Fig. 21.1 Etiologies of hepatocellular carcinoma (HCC). The percentage of HCC estimated to be
attributed to each of the indicated, major, etiologies: alcohol, hepatitis B virus (HBV), hepatitis C
virus (HBV), and other in the world (a) and the USA (b) is shown. References: Howlader et al.
(2011), Miller et al. (2008), El-Serag et al. (2001), and Parkin (2006)
pararetrovirus that harbors a DNA genome, but replicates via reverse transcription
of an RNA intermediate. Although it is not essential for viral replication, HBV DNA
often integrates into host chromosomes, which may directly transform hepatocytes
or induce instability of chromosomal DNA (Seeger and Mason 2000). On the contrary,
HCV is an RNA virus that belongs to the hepacivirus genus of the family Flaviviridae
(Tellinghuisen et al. 2007).
21
511
Multiple HBV and HCV proteins have been implicated in the disruption of cellular
signaling pathway that lead to unchecked cell growth (Table 21.3). However,
the most prominent driving force for the development of HCC in HBV and HCV
chronically infected livers is thought to be the sustained cycles of hepatocyte
necrosisinflammationregeneration, driven by interaction between virus infection
and host antiviral immune response (Nakamoto et al. 1998). This chapter highlights
the biological property of HBV, HCV, and HCC from the virological and molecular
cell biological perspectives. In-depth discussion on HBV and HCV biology is provided
in subsequent chapters.
Epidemiology
As summarized in Table 21.1, HCC is the fifth most common cancer and the
third leading cause of cancer death worldwide (El-Serag and Rudolph 2007). Its
incidence and geographic distribution are largely governed by its prominent etiology,
the chronic infections with HBV or HCV. Thus, HCC is most common in the areas
where HBV (and to a lesser extent, HCV) are epidemic, especially East and SouthEast Asia, and sub-Saharan Africa (Davis et al. 2008).
In addition to chronic HBV and HCV infections (Marrero 2006), there are other
risk factors for HCC development, most importantly, a family history of HCC,
chronic alcohol consumption, and ingestion of aflatoxin B1-contaminated food.
There is an increasing appreciation for the fact that combinations of these risk
factors, when present in the same individual, greatly increase the probability of
HCC occurrence. For example, those who are chronically infected with HBV have
a lifetime risk of HCC of between 15 and 20%, with an odds ratio of ~13.7, and the
HCC usually occurs after the fourth or fifth decade (Donato et al. 1998). However,
those with chronic hepatitis B who are either alcoholic or coinfected with HCV
have a lifetime risk of HCC of greater than 20%, and the HCC appears to declare
itself much earlier in life (Aravalli et al. 2008; Davis et al. 2008).
Table 21.2 HCC incidence distribution by age in the USA

Age at diagnosis
Percentage of all diagnosed at that age
Under 20
0.3
2034
0.7
3544
2.4
4554
15
5564
21.7
6574
23.5
7584
25.8
85 and older
10.5
Age at which HCC is diagnosed, as a percentage of all HCC
diagnosed, in the USA (Howlader et al. 2011 Miller et al. 2008)
512
T.M. Block et al.
As shown in Table 21.2 and illustrated in Fig. 21.3, the majority of HCC cases occur
in older people. This observation reflects the fact that HCC is often associated with
several decades of HBV/HCV chronic infection. However, HCC occurs occasionally
in young people and children, usually in a setting of chronic HBV infection (Table 21.2).
In contrast to adult HCC, which is usually developed in the cirrhotic liver, the pediatric
HCC is commonly developed in liver that is in the absence of cirrhosis. This observation
implies that pediatric and adult HCC may develop through distinct mechanisms.
Alternatively, perhaps childhood HCC may occur in individuals with specific genetic
(or environmental) factors that accelerate HCC development.
Hepatitis B Virus
HBV is the prototype member of Hepadnaviridae family and contains relaxed
circular (rc) partially double-stranded DNA (3.2 kb in length) genome with its DNA
polymerase protein covalently attached to the 5 terminus of minus strand DNA.
The HBV genome contains four open reading frames. Because of overlapping
coding regions and proteolytic processing reactions, the virus specifies a total of seven
viral proteins from these ORFs, which include DNA polymerase, nucleocapsid
(core) protein and a secreted viral protein HBeAg, three envelope proteins (surface
antigens), and the X protein (Seeger and Mason 2000).
One of the most intriguing biological features of HBV is that the viral genomic
DNA is replicated via protein-primed reverse transcription of an RNA intermediate
called pregenomic (pg) RNA in the cytoplasmic nucleocapsids (Summers and
Mason 1982). However, unlike classical retroviruses, the integration of hepadnavirus
genomic DNA into host cellular chromosomes is not an obligatory step in its life
cycle. Instead, a nuclear episomal covalently closed circular (ccc) DNA is formed
from the rcDNA genome in nucleocapsids, either from incoming virions during
initial infection or from the pool of progeny nucleocapsids formed in the cytoplasm
during replication (Tuttleman et al. 1986; Wu et al. 1990). Those two pathways
culminate in the formation of a regulated steady-state population of 1050 cccDNA
molecules per infected cell (Beck and Nassal 2007; Newbold et al. 1995; Seeger and
Mason 2000). The cccDNA exists as a minichromosome in the nucleus and serves
as the template for the transcription of viral RNAs (Zoulim 2005). The stability of
this key replication intermediate is still in debate, but a continued productive hepadnavirus infection clearly requires a persistent population of cccDNA as the source
of viral RNAs for viral replication and production of virions (Moraleda et al. 1997;
Tuttleman et al. 1986; Wu et al. 1990; Zhang et al. 2003).
Concerning the molecular mechanisms by which chronic HBV infection causes
HCC, the following gene products or virological property of HBV have been implicated in the hepatocarcinogenesis process.
21
513
Viral Load
Virion DNA is what is conventionally detected by serum assay and reported in
laboratory assessments of people chronically infected, and referred to as viral
load. Approximately 1540% of all people with chronic HBV infection eventually
develop liver cirrhosis, hepatic decompensation, and HCC. An elevated serum
HBV DNA level (10,000 copies/mL) is a strong risk predictor of HCC independent
of HBeAg, serum alanine aminotransferase level, and liver cirrhosis (Chen et al.
2006a, b). Consistently sustained suppression of HBV replication with antivirals
can significantly reduce the likelihood of cirrhosis and HCC development (Yuen
et al. 2007). These observations imply that high level of HBV replication is one of
the dominant driving forces for HCC development.
Integration of HBV DNA into Host Chromosomes

Despite the fact that HBV DNA integration into host chromosome is not required
for viral replication, it does occur randomly in infected hepatocytes. The frequency
of duck hepatitis B virus (DHBV) DNA integration has been estimated to be at least
one viral genome per 103104 cells by 6 days postinfection of ducklings (Yang and
Summers 1999). Interestingly, the frequency of integrated woodchuck hepatitis
virus (WHV) DNA in chronically infected woodchucks was found to be one or
two orders of magnitude higher than that in transiently infected woodchucks,
implying that integration and other genomic damage accumulate over the duration
of infection (Summers and Mason 2004). Analysis of integrated viral DNA sequences
and genetic studies indicated that the most likely precursor of integrated viral DNA
is the double-stranded linear (dsl) DNA, a replication product of in situ priming of
plus strand DNA (Bill and Summers 2004). Owing to the disruption of the circular
genome, integrated HBV DNA is able to be transcribed into functional mRNAs for
envelope proteins, but not 3.5 kb precore mRNA and pgRNA, and thus would be
unable to support production of infectious virus.
HBV DNA integration has been reported to occur in a large fraction of HCC
tumors derived from people chronically infected with HBV, most notably in clonal
patterns. It was hypothesized that HBV DNA integration could activate cellular
protooncogene by insertion activation mechanism and thus contribute to the development of HCC. However, this hypothesis has been confirmed only in WHV-infected
woodchucks, in which WHV DNA were found to integrate close to members of the
myc oncogene family (Fourel et al. 1990). In human HCC, HBV DNA integration is
largely random and only in a few cases, close to important cell growth regulatory
genes (Dejean et al. 1986; Wang et al. 1990). Despite this observation, viral
DNA integration may play a role in HCC tumor genesis by promoting instability of
chromosomal DNA in virally infected cells.
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T.M. Block et al.
X Protein
HBV X is a 17 kDa, 155-amino-acid protein. It is called X protein or HBx because
of uncertainty about its function in a natural viral infection. WHV X protein is not
essential for WHV DNA replication in cultured cells, but is required for its infectivity
in vivo (Chen et al. 1993; Zoulim and Seeger 1994). Recent studies reported by
Schneider and colleagues indicated that HBx protein promotes viral DNA synthesis
in HBV genome transfected HepG2 cells via immobilizing intracellular calcium
and thereby activating Pyk and FAK-mediated signaling pathways (Bouchard et al.
2001, 2003). The role of the HBx protein in HCC development has been suggested
by several studies, but the molecular mechanism remains controversial (Bouchard
and Schneider 2004).
Truncated Forms of Pre-S2/S Proteins

and Large Envelope Protein
HBV specifies three envelope glycoproteins called large (L, LHBs), middle (M, MHBs)
and small (S, SHBs or more commonly abbreviated as HBsAg) surface proteins. The
molecular weights of the three envelope proteins as found in the infectious virion and
subviral particles, are 23 and 26 kDa (for SHBs), 30, 33, and 36 kDa (for MHBs) and
36 and 39 kDa (for LHBs), the size varying with the degree of glycosylation (40).
While LHBs is translated from 2.4 kb mRNA, MHBs and SHBs are translated from
2.1 kb mRNA by using different starting codons (Seeger and Mason 2000).
The truncated form of the pre-S2/S proteins are occasionally found to be
expressed in HCC from rearranged integrated viral genomes. It had been shown that
these proteins were able to transactivate cellular oncogenes, such as c-myc and c-fos
(Schluter et al. 1994) and activate PKC and c-Raf-1/MEK/ERK pathway (Hildt
et al. 2002). In addition, it is well documented that overexpression of LHBs
activates endoplasmic reticulum (ER) stress (Xu et al. 1997), which can further
induce oxidative DNA damage and genomic instability and thus promotes hepatocarcinogenesis (Hsieh et al. 2004). Indeed, it had been elegantly demonstrated in a
transgenic mice model that overexpression of LHBs in hepatocytes induced HCC
(Chisari et al. 1989).
Hepatitis C Virus
HCV is a member of the family Flaviviridae, which also includes some well-known
human and animal pathogens, such as yellow fever virus, West Nile virus, Dengue
virus, and bovine viral diarrhea virus (Choo et al. 1989). These viruses have in common
a single-stranded, positive-sense RNA genome carrying one long open reading frame
that is flanked by nontranslated regions (NTR). The HCV genome has a length of
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515
about 9,600 nucleotides and encodes an approximately 3,000-amino-acid-long

polyprotein that is proteolytically processed into ten polypeptides (Reed and Rice 2000).
Three of them are structural proteins required for capsid formation (core) and
assembly into enveloped viral particles (E1 and E2). Four virus-encoded proteins are
enzymes including cysteine and serine proteases (NS2 and NS3), an ATP-dependent
helicase (NS3), and a RNA-directed RNA polymerase (NS5B). The functions of the
remaining three polypeptides, p7, NS4B, and NS5A, for viral replication are not yet
known. The 5 NTR spans about 340 nucleotides and harbors the cis-elements for
viral RNA replication and an internal ribosomal entry site (IRES) directing the translation of viral polyprotein. The 3 NTR has a highly conserved sequence element
(3 X) that is essential for viral RNA replication (Tellinghuisen et al. 2007).
HCV replication begins with sequential interactions of the virion particle with its
cellular receptor and coreceptor molecules on cell surface, including CD81, class B
scavenger receptor (SRBI), claudin-1 and occludin, followed by entry into hepatocyte via endocytosis (Evans et al. 2007; Ploss et al. 2009). Viral genomic RNA is
released into the cytoplasm upon fusion of viral envelope and endosomal membrane
and serves as a template for translation of viral polyproteins. Accumulation of
viral proteins induces rearrangements of ER membranes that form the locales for
replication of viral RNA. Minus strand RNA synthesis leads to the formation of a
double-stranded RNA molecule that bears a promoter at the 3 end of minus strand
RNA required for amplification of plus strands by a semiconservative mechanism
(Tellinghuisen et al. 2007). During the early phase of the infection, the progeny plus
strand RNA is transported to ribosomes for additional rounds of translation and
replication of viral RNA. But late in infection, the RNA is packaged into virion
particles by viral core and envelope proteins on the surface of lipid droplets and
secreted out from infected cells (Miyanari et al. 2007).
Apparently, unlike HBV, HCV is unable to reverse transcribe its RNA genome
and thus to integrate it into the host chromosome. However, there is ample evidence
suggesting that multiple HCV proteins, particularly core and NS5A protein, evoke
host cellular responses that may contribute to HCC development.
Core Protein
The mature form of HCV core contains approximately 174aa that is separated into
two structural domains. The domain I, encompassing N-terminal 122aa, is highly
basic and responsible for RNA binding and capsid assembly. The domain II encompasses the C-terminal part of HCV core protein that is hydrophobic and mediates
interactions with lipids and membrane proteins. Besides to serve as an essential
structural component of HCV, the core protein appears to have diverse functions and
interact with many cellular proteins that may lead to hepatocarcinogenesis. For
examples, HCV core has been demonstrated to bind to tumor suppressor proteins,
such as p53 and pRb (Ray et al. 1997). In addition, HCV core is able to upregulate
the expression of TGF-b and VEGF and activates multiple signal transduction
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T.M. Block et al.
pathways, including PKC, RB/E2F1, ASK1-JNK/p38, and ERK (Hassan et al. 2009).
Consistent with its pleiotropic function on cellular signal transduction pathways,
overexpression of HCV core protein, alone, in Huh7 cells induces the expression of
more than 300 cellular genes with most of the induced genes involved in cell growth
and oncogenic signaling (Fukutomi et al. 2005).
NS5A Protein
Two forms of NS5A protein, termed p56 and p58, can be distinguished by their
electrophoretic mobility. While the p56 is a basally phosphorylated protein, the p58
is hyperphosphorylated within a serine-rich region in the center of the protein. The
hyperphosphorylation of NS5A has been demonstrated to regulate HCV RNA replication (Evans et al. 2004). Besides being an essential component of HCV RNA
replication complex and playing a critical role in directing viral RNA onto the surface
of lipid droplets for virion assembly, NS5A protein has been attributed to an array
of cellular functions, including inhibition of interferon response and apoptosis,
modulation of signal transduction and gene transcription, induction of cell transformation, and ROS production (Macdonald and Harris 2004). Like the core protein,
NS5A can also directly bind to p53 and inhibit its transcriptional transactivation
activity (Majumder et al. 2001). Moreover, our own study reveals that NS5A can
activate PI3K/Akt/mTOR signal transduction pathway and promote HBV replication
in HBV and HCV coinfected hepatocytes (Guo et al. 2007).
Steatosis and Oxidative Stress

Chronic HCV infection is characterized by prevalence of steatosis and increased
oxidative stress. Steatosis is independently associated with the development of
HCC in patients with HCV-related cirrhosis (Pekow et al. 2007). A recent transgenic
mice study demonstrates that overexpression of HCV core protein in hepatocytes
induces severe steatosis in a PPAR-alpha-dependent manner and results in HCC
development in 35% of HCV core transgenic mice bearing homozygote wild-type
PPAR-alpha genes (Tanaka et al. 2008).
Increased oxidative stress in chronically HCV-infected liver may result from HCV
induced steatosis, ER stress and immune-cell-mediated oxidative bursts. Both HCV
core and NS5A proteins have been implicated in induction of oxidative stress and ROS
production. ROS has been shown to be able to activate multiple signal transduction
pathways, such as MAPK, PI3K, NFkB, and Wnt/b-catenin, and thus modulate many
cellular functions, including gene expression, cell adhesion, cell metabolism, cell proliferation as well as apoptosis. Most importantly, ROS can induce oxidative DNA damage,
which in turn increases chromosomal aberrations associated with cell transformation.
21
517
Liver
Hepatic
Bile Duct
Stomach
Gall
Bladder
Pancreas
Common
Bile Duct
Small
Intestine
Fig. 21.2 The digestive system with a focus on the liver. Illustration of the liver, its two lobes, and
how it resides within the human digestive track
Molecular Pathogenesis of HCC

Molecular Pathways of HCC
Hepatocarcinogenesis is a complex multistep process involving a number of genetic
and epigenetic alterations, which result in the activation of cellular oncogenes
and/or the inactivation of tumor suppressor genes, and dysregulation of multiple
signal transduction pathways (Farazi and DePinho 2006; Tsai and Chung 2010).
As indicated by the age distribution of the disease (Fig. 21.2 and Table 21.2), when
it occurs, it is usually after decades of chronic infection and often preceded by
necroinflammatory liver disease (Fig. 21.3).
However, it has been difficult to identify common genetic changes in more
than 2030% of tumors, suggesting that HCCs are genetically heterogeneous
(Branda and Wands 2006). As discussed in the above sections, although cell culture
studies have suggested that multiple HBV and HCV proteins are able to interact
with key components of cellular signal transduction pathways and lead to unchecked
cell growth, their roles as viral oncogenes to promote HCC development in vivo
have not been firmly established. Instead, as illustrated in Fig. 21.4, regardless the
etiology, malignant transformation of hepatocytes most likely results from the sustained
518
T.M. Block et al.
HCC, in US, Incidence

Distribution by Age
30.00%
25.00%
20.00%
15.00%
Series1
10.00%
5.00%
0.00%
Under 20-34 35-44 45-54 55-64 65-74 75-84 85 and
older
20
Relative amount of
Morbidity/Mortality
Fig. 21.3 Distribution of the incidence of HCC by age in the USA. The percentage of ~18,000
cases of HCC diagnosed at each of the ages indicated by histograms is shown (Miller et al. 2008)
Time of infection
HCC
Active hepatitis
Inactive hepatitis
Acute
hepatitis
10
20
30
40
50
Years of chronic infection
60+
Fig. 21.4 Relative progression of different clinical conditions associated with chronic hepatitis virus,
as a function of decades of infection. Dramatization of the likelihood of moving from one clinical
state to another increases with period of time of infection (based on Block et al. 2003). Progression
from one step to the other is not necessary, but is the most common, natural history of the disease
cycles of increased liver cell death induced by chronic liver injury and regeneration
in the condition of inflammation and oxidative DNA damage over a long period of
time, usually several decades (Nakamoto et al. 1998).
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519
In case of chronic hepatitis B and hepatitis C, as illustrated in Fig. 21.4, the

virally infected hepatocytes can be targeted and killed by viral specific cytotoxic T
lymphocytes (CTLs) that recognize epitopes derived from various viral proteins
and presented by the MHC I molecules on the surface of infected hepatocytes.
However, such immune response is insufficient to eliminate viral infection and
killed hepatocytes are replaced by proliferation of residual hepatocytes (Chisari and
Ferrari 1995). This cycle of immunorecognition-killing (liver destruction) and
regeneration repeats itself over years. Indeed, there is well-documented evidence
suggesting that the rate of hepatocyte turnover is accelerated in HBV and/or
HCV-infected individuals. Proliferation of hepatocytes in an environment of
inflammation is prone to accumulate mutations, which ultimately leads to transformation and hepatocarcinogenesis.
Inherited and Acquired Mutations

After chronic infection with HBV or HCV, having a close family history of HCC is
probably the greatest risk factor for developing the disease (Hoofnagle 2004; Zhang
et al. 2010). HCC susceptibility genes have been elusive, but there is a growing
interest in pursuing genes whose heritable lesions may be associated with HCC risk
(Zhang et al. 2010).
Indeed, multiple genetic and epigenetic alterations have been documented to
associate with HCC. So far, comparative genomic hybridization studies have revealed
frequent chromosomal gains in 1q, 6p, 8q, 11q, and 17q, and losses in 1p, 4q, 8p,
13q, and 17p (Farazi and DePinho 2006). Interestingly, a recent genome-wide
association study identified one intronic SNP (rs17401966) in KIF1B on chromosome
1p36.22 that is highly associated with HBV-related HCC. In addition to KIF1B, the
association region also encodes two other potential tumor-related genes, UBE4B
and PGD, and thus confers susceptibility to HBV-related HCC (Zhang et al. 2010).
Furthermore, p53 inactivation and mutation seems to be a common pathogenic
pathway in HBV- and HCV-induced HCC. HBx, HCV core, and NS5A proteins
have all been shown to bind p53 and inhibit its function. Analyses of HBV and
HCV-related HCCs have also shown that p53 mutations can be found in 43% of
patients with advanced HCC (Minouchi et al. 2002).
It has been shown by biochemical analysis and genome-wide gene expression
studies that multiple signal transduction pathways are altered in HCC by viral
proteins, virus-induced cellular responses, or unidentified mechanisms, and thus
may contribute to hepatocarcinogenesis. These pathways include Insulin/IGF-1/
IRS-1/MAPK, Wnt/Frizzeled/b-catenin, Ras/Raf/MAPK, Janus kinase (JAK)/ signal
transducer and activator of transcription (STAT), phosphatidylinositol 3-kinase
(PI3K)/Akt, Hedgehog and growth factors such as epidermal growth factor, and
transforming growth factor-b (TGF-b) pathways. Interestingly, a recent integrative
transcriptome analysis stratifies HCC into three subclasses, each correlated with
clinical parameters as well as biological mechanism known to be operative in the
Table 21.3 Selected pathways and molecular targets associated with HBV and HCV-induced
HCC
Target
HBV
HCV
References
Wnt/b-catenin
Yes
Yes
Colnot et al. (2004), Hickman and Helin (2002),
Taniguchi et al. (2002)
P53
Yes
Unknown Azechi et al. (2001)
pRB
Yes
Unknown Satoh and Kaziro (1992), Yoshida et al. (2006)
MAP kinases
Yes
Yes
Bai et al. (2003)
Cytokines
Yes
Yes
Budhu et al. (2006)
P16
Yes
Yes
Satoh and Kaziro (1992)
Frizzled
Unknown
Yes
Colnot et al. (2004)
Pathways and molecular targets that have been reported to be altered (in some cases, mutated, in
some cases upregulated, in other cases downregulated, depending on the pathway or target) in people
with HBV and HCC-associated HCC are shown. For most of the targets, alterations have been
reported for both HBV and HCV-associated HCC. Citations supporting the associations are shown
T
a
Cancer
b
Biological
Event
Infection,
antigen
presentation
Clinical
Event
In-apparent infection
Chronic liver
damage,
hepatocyte
regeneration
Hepatitis, fibrosis, cirrhosis
Genetic
alterations
Transformation
Pre-malignant masses, then HCC
Fig. 21.5 Idealization of the molecular and cellular events occurring which are thought to lead to
the pathogenesis of chronic viral infection and, ultimately, HCC. (a). Events thought to happen at
the cellular level, where (a): T lymphocytes attack infected hepatocytes expressing viral antigens,
followed by (b): destruction of the infected cells which are replaced by scar tissue, leading to
fibrosis and cirrhosis accompanied by continuous regeneration of new hepatocytes (c): some of
which make acquire mutations, leading to transformation and cancer. (b): The progression of pathogenesis, explaining the natural history of HCC, from the biological perspective and the clinical
manifestation of these events, is shown (based on Block et al. 2003)
21
521
pathogenesis of HCC (Hoshida et al. 2009). For examples, the subclass 1(S1) was
characterized by TGF-b induced activation of the WNT signaling pathway, S2
was characterized by high level alpha-fetoprotein expression as well as MYC and
AKT activation, and S3 was associated with hepatocyte differentiation, featured by
hepatocyte-like gene expression profile, smaller tumor and better prognosis
(Fig. 21.3). Such a molecular classification of HCC is helpful in understanding the
HCC pathogenesis and development of targeted therapeutic intervention of HCC.
A summary of some of the prooncogenes disrupted by HBV and HCV is shown
in Table 21.3 and Fig. 21.5.
Role of MicroRNA in Hepaocarcinogenesis

MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nts in
length. They are derived from cellular or viral transcripts and bind to their target
mRNAs in a sequence-specific manner, resulting in either mRNA cleavage or translational repression (Ambros 2004; Bartel 2004; Jackson and Standart 2007).
MicroRNAs have been shown to play a fundamental role in regulating gene expression and thus modulating multiple cellular functions. Aberrant expression of several
miRNAs was found to be involved in human hepatocarcinogenesis. For examples,
upregulation of mir-221 and mir-21 could promote cell cycle progression, reduce
cell death and favor angiogenesis and invasion (Meng et al. 2007; Pineau et al.
2010). Moreover, the most abundant miRNA in the liver, miR-122, is involved in
cellular stress response, regulation of cholesterol metabolism and required for HCV
replication in hepatocytes in vitro and in vivo (Bhattacharyya et al. 2006; Chang
et al. 2004; Esau et al. 2006; Jopling et al. 2005; Lanford et al. 2010). miR-122 is
also considered to have the potential as tumor suppressor, since downregulation of
miR-122 has been found to correlate with hepatocarcinogenesis (Chang et al. 2004;
Chang and Taylor 2008; Coulouarn et al. 2009; Kutay et al. 2006).
Detection and Management of HCC

Currently, surgical resection and liver transplantation are the best choices to treat
HCC. However, the five-year survival rate in people with resection and/or liver transplantation is less than 30% (Roncalli et al. 2007). Chemoembolization (TACE), ethanol
(Percutaneous), cryotherapy, and radiofrequency thermoablation are being used as
alternatives to resection (Block et al. 2008; Marrero 2006). Recurrences are common
and recovery from such interventions can be compromised. Medically, there have
been even fewer satisfactory options. The recent approval of sorafenib for the advanced
HCC treatment has been received with great anticipation. However, its efficacy is
extremely limited, increasing overall survival by only a few months (Llovet 2007).
The poor prognosis and therapeutic outcomes of HCC is thought mainly as the
result of failure to diagnose the cancer at its early and treatable stage. Hence, early
522
T.M. Block et al.
detection of the cancer is generally considered to be critical to improve outcome

(Hoofnagle 2004; Marrero 2006; Seki et al. 2000; Sherman 2005). Since the
prominent etiology of HCC is chronic hepatitis B followed by hepatitis C (Di Bisceglie
et al. 1998, 2003), there is a clear and identifiable risk population. This allows for a
realistic and focused cancer screening (Block et al. 2007). Moreover, since patients
with cirrhosis (with or without HBV or HCV infection) are at significantly increased
(between 10- and 100-fold) risk of developing HCC, this subpopulation receives the
closest attention (Marrero 2006).
Current methods for HCC detection are either unreliable or impractical. Liver
biopsy is still considered to be the definitive standard to assess the degree of liver
damage in people with chronic hepatitis, although it is usually to be avoided where HCC
is suspected (Block et al. 2007; Lok and McMahon 2001). However, monitoring by
noninvasive methods is currently performed by physical assessment, ultrasound
imaging of the liver, and analysis of serum with a panel of markers including liver
function tests and platelet counts (Lok and McMahon 2001; Lok et al. 2001).
Advanced imaging is costly and even ultrasound detection is expensive and
operator dependent. It also usually requires at least a 12 cm tumor mass to be
present. Unfortunately, most scans are performed when the HCC is at a late stage
and prognosis is very poor (Brechot 1987; Hoofnagle and Di Bisceglie 1997). Since
early surgical intervention is the best hope for patient survival (Block et al. 2003;
Lok and McMahon 2001; Lok et al. 2001), accurate detection of early stage HCC is
necessary to identify the need for intervention. For smaller lesion, 12 cm in diameter,
i.e., those that are the target of HCC screening, radiology is less sensitive, but similarly
biopsy is less accurate as well. This is because of difficult inaccurate needle placement, and because pathological interpretation in these small lesion is controversial
and difficult. In this situation, a diagnostic biomarker would be very helpful.
Liver function lab values (serum tests) are used to assess liver damage but are of
dubious value in diagnosis and detection of HCC (Bruix et al. 2001). Many of the
constituents of the liver function test panels used to evaluate disease status (i.e., degree
of liver damage), such as alanine aminotransferase (ALT) levels (Imbert-Bismut
et al. 2001; Myers et al. 2003; Poynard et al. 2002), vary throughout the course of
chronic hepatitis and are of limited use in early detection of HCC (Sherman 2001).
Since there is a correlation between elevated levels of alpha fetoprotein (AFP)
and the occurrence of HCC, determination of AFP levels is often included as a
serum marker of disease (Aoyagi et al. 1998; Buamah et al. 1984; Lok and McMahon
2001; Lok et al. 2001; McMahon et al. 2000). AFP, a 72,000 Da liver derived
glycoprotein with a function that may be analogous to albumin, has been used
for detection of HCC since 1968 (Alpert et al. 1968). AFP as a sole indicator of
HCC is of limited value, often being elevated in the absence of serious disease
or not elevated when cancer is present or at an early stage (Sherman 2001).
Nevertheless, even the limited correlation between AFP and HCC underscores the
potential of serum as a source of biomarkers of liver disease. The assay for fucosylated
AFP (AFP-L3), as used in the clinical setting, is a bit complicated and requires
specialized equipment. However, its commercialization by Wako Diagnostics proves
that these matters can be overcome. Since the sensitivity of AFP-L3 in detecting
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523
Chronic HBV or HCV

?Viral proteins
Cytokines (necro-inflammation)/ Ligands
(extracellular)
Cytokine receptors/wnt receptor
PI3-K-Activation
Akt activation
mTOR
E-cadherin/b-catenin
PTEN
?Viral proteins
b-catenin
(intra-cellular)
p53
ROS
Anti-apoptosis
Invasion
Metastasis
pRB, p16, myc
Fig. 21.6 Effect of chronic hepatitis virus and other insults to the hepatocytes on pathways and
protooncogenes, whose dysregulation is associated with HCC. Illustration of some of the enzymes,
pathways, polypeptides, and protooncogene products, whose functions have been reported to be
disrupted (activated or inactivated) in either HBV or HCV associated HCC. ROS (reactive oxygen
species). Shaded oval, nucleus. The listing of pathways and polypeptides is not complete, but is
intended to represent some of the more prominent targets. Based on Choudhari et al. (2007)
Tumor Mass Size
Larger Tumor Masses
Less Differentiated
TGFb, Wnt Activated
MYC, AKT Activated
Smaller Tumor Masses
Retains Hepatocyte Phenotype
CTNNB1 Variable
AFP Positive
Poorer Survivor
Better Survivor
Fig. 21.7 Model that uses molecular and biomarker information to subclassify HCC. Based on
model proposed by Hoshida et al. (2009)
524
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early stage HCC is only approximately 50%, although promising, more work or
combinations with additional biomarkers are needed.
There have been significant efforts to discover and use new serum markers for
the early detection of HCC (Wright et al. 2007). For example, des-gamma-carboxy
prothrombin (DCP) has received a great deal of attention as a biomarker of HCC, so
has glypican, serum glycan, and several other circulating biomarkers as reviewed in
(Block et al. 2008). However, none of these prospects has held up as superior or
offering advantages to current methods in multicentered, diverse, and large blinded
studies (Block et al. 2008; Marrero et al. 2009).
Serum markers of HCC, as well as the molecular analysis of the tumors, have
been used to predict outcome, and this is becoming more and more fashionable. For
an example of how molecular classification has been proposed for use in clinical
outcome prediction and care, see Figs. 21.6 and 21.7.
Future Challenges
HCC is a growing problem in the world. Although with clear etiology and in-depth
understanding of HBV and HCV virology, the molecular pathogenesis of HCC
remains largely elusive. Clinically, there are also unmet needs for the early detection
and therapeutic intervention of HCC. Therefore, many challenges and opportunities
exist in this field. For examples, development of novel antiviral drugs or other
therapeutic agents that cure chronic HBV and HCV infections will ultimately
prevent HCC development. In addition, detailed understanding of the genetic (or
epigenetic) lesions of HCC, signal transduction pathways involved in HCC development, hostvirus interaction, antiviral immune response and liver inflammation,
cell of origin, and evolution of hepatocarcinogenesis will provide a roadmap for
the development of HCC therapeutics. Needless to say, successful treatment of
HCC relies on the discovery of biomarkers to identify early-stage HCC as well as
those at greatest risk of developing HCC.
Acknowledgments The authors would like to thank Ms. Erica Litschi for help in manuscript and
figure preparation. The authors acknowledge support from National Cancer Institute and National
Institute of Allergy and Infectious Diseases, and the Hepatitis B Foundation and The Commonwealth
of Pennsylvania.
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Chapter 22
Hepadnaviruses and Hepatocellular Carcinoma

William S. Mason
Introduction
Hepadnaviruses are a family of small, enveloped, DNA viruses that productively
infect hepatocytes, the major cell type of the liver. The prototype virus of this family
is hepatitis B virus (HBV) (Blumberg et al. 1967), which infects humans and higher
primates. Closely related viruses are found in the Woolly Monkey (Lanford et al.
1998), woodchuck (Summers et al. 1978b) and Beechey ground squirrel (Marion
et al. 1980). More distantly related viruses are found in ducks, geese, herons, storks,
and cranes (Guo et al. 2005; Mason et al. 1980; Prassolov et al. 2003; Pult et al. 2001;
Sprengel et al. 1988; Wang et al. 1980). These two groups of viruses are assigned to
the genus orthohepadnavirus and avihepadnavirus, respectively. All hepadnaviruses
are able to cause transient infections, with recovery and immunity to re-infection, as
well as chronic, life-long infections. Chronic infections by the orthohepadnaviruses
can cause hepatocellular carcinoma (HCC), while avihepadnavirus infections do not.
The lifetime risk of HCC is ~25% in humans chronically infected with HBV, higher
in Beechey ground squirrels chronically infected with ground squirrel hepatitis virus
(GSHV), and virtually 100% in woodchucks chronically infected with woodchuck
hepatitis virus (WHV). A vaccine to prevent primary HBV infection and associated
HCC has been available for almost 30 years (Blumberg and London 1982; McAuliffe
et al. 1980); the vaccine is not useful for therapy of chronic infections.
It was hoped for some time that studies with the woodchuck and ground squirrel
models would reveal how chronic infection with HBV leads to human liver cancer.
This has not, so far, been the case. HCC in woodchucks is often associated with
integration of viral DNA into host DNA, typically near to N-myc (Bruni et al. 1999,
2006; Fourel et al. 1990, 1994) and, much less often, C-myc (Wei et al. 1992). Overexpression of these genes then occurs, probably through the action of the WHV
W.S. Mason (*)

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
e-mail: ws_mason@fccc.edu
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transcriptional enhancer (Flajolet et al. 1998; Ueda et al. 1996) or, in some cases, to
a more profound effect of integration on chromosome structure in the vicinity of the
gene, due to disruption of nuclear matrix attachment regions (Bruni et al. 2004;
Shera et al. 2001). The fact that all transformed cells in a tumor contain the same
integrant, and that the nearby oncogene is typically over-expressed, implies that the
integration of viral DNA was required for emergence of the woodchuck tumor, was
probably causative, and perhaps sufficient (Renard et al. 2000).
In contrast, amplification of the C-myc gene, unassociated with integration of
GSHV DNA, is found in 2550% of ground squirrel HCCs (Hansen et al. 1993;
Transy et al. 1992, 1994). Integrations near C-myc and N-myc were not found with
GSHV. Considering that both hosts are rodents/ground squirrels and are infected by
closely related viruses, this dichotomy is unexpected. It suggests that there might
not be a simple relationship between HBV DNA integration and HCC in humans.
In fact, although exceptions have been found in which integration has activated
host genes which are probably important in development of the HCC, including erbA, retinoic acid receptor beta, and cyclin A (Dejean et al. 1986; Murakami et al. 2005;
Paterlini-Brechot et al. 2003; Wang et al. 1990), studies of human HCCs have failed,
in most cases, to reveal a clear role for the sites of integration of HBV DNA (Murakami
et al. 2005; Paterlini-Brechot et al. 2003). This was particularly disappointing since
virtually all human HCCs contain integrated DNA, an observation which indicated
that these tumors resulted from clonal expansion of cells that took place at some time
after the integration event (Brechot et al. 1980; Shafritz and Kew 1981).
While activation of a host oncogene by nearby insertion of an HBV enhancer or
promoter sequence does not so far explain the production and clonal expansion of
malignant cells to produce most human HCCs, the idea that integration of HBV
DNA plays a major role has persisted. For instance, as mentioned above, the possibility that integration activates gene expression by altering chromosome structure
(Bruni et al. 2004; Shera et al. 2001) has been proposed. Another hypothesis, which
has received more attention, but again without conclusive results, is that one or more
normal or aberrant viral gene products, expressed from the integrated viral DNA,
are oncogenic. As discussed below, there is substantial indirect support for this
notion, but there remain many difficulties in connecting experimental observations
in model systems with human HCC.
Another pathway to human HCC, which has received a great deal of attention,
but without definitive conclusions, is persistent injury to the liver caused by the host
immune response. It is clear that the immune response to infected hepatocytes,
mediated by antiviral cytotoxic T lymphocytes, is a feature of HBV chronic infections. This response, along with cell death, activates macrophages, leading to free
radical generation by these cells, as well as in cells targeted by antiviral cytokines
such as TNFa, which is secreted by activated macrophages. This appears to lead to
increased hepatocyte levels of hydroxyguanosine, an oxidized version of guanosine
capable of base pairing with adenosine (Hagen et al. 1994) to create mutations in
host DNA. In addition, persistent killing causes tissue scarring, which leads to fibrosis
and, ultimately, cirrhosis. Cirrhosis severely disrupts the arrangement of hepatocytes
and blood flow through the liver, and may be, along with HBV infection and free
radical generation, an important risk factor for HCC.
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533
Finally, persistent liver regeneration, following genotoxic events effecting

hepatocyte DNA (e.g., free radical formation and DNA mutation), will increase the
risk of HCC (Marongiu et al. 2008). That is, mutation of hepatocyte DNA provides
an essential event that initiates the progression to HCC, but would be ineffective
without the promoting event of liver regeneration (Maeda et al. 2005).
Unfortunately, the importance of these and other factors in the progression to
HCC in HBV carriers remains unresolved. HBV is a serious public health concern
because so many people worldwide are chronically infected with the virus. The
WHO estimates there are ~350 million HBV carriers worldwide and that 60 million
of these may die prematurely from HCC and/or cirrhosis (Lavanchy 2004). Resolving
the association between chronic infections and HCC is particularly difficult because
infections are very prolonged. Chronic HBV infections typically begin at birth
(neonatal transmission) or in the first year of life. As a result, the duration of an infection is generally the same as a patients age. HCC may appear at any age (Chang
et al. 2009), but the incidence peaks after the age of 30 (Beasley 1982; Beasley et al.
1981; Ganem and Prince 2004; Liaw 2009; Yim and Lok 2006). Intervention and
study of younger patients that have not developed HCC is problematic because
HBV infection in these individuals is generally asymptomatic (Lok and McMahon
2007) until significant fibrosis and cirrhosis have developed, by which time the
progression to HCC may also be well advanced.
In the following sections, possible events in the progression to HCC are discussed.
The focus of this review is the role of HBV, and the host response to this virus, in
the progression to HCC. For additional information on HCC, the reader is referred
to recent articles on the role of environment carcinogens (Groopman et al. 2008;
Wild and Gong 2010), the molecular changes that distinguish HCCs from surrounding
liver (Connolly et al. 2008; Lee et al. 2004; Neuveut et al. 2010; Thorgeirsson and
Grisham 2002; Zhang et al. 2009), the occurrence and role of cancer stem cells
(Rountree et al. 2009; Sun et al. 2008; Yamashita et al. 2010), and a relatively new
area of investigation, possible HBV genotype differences in the risk of HCC (Yuen
et al. 2009). The reader should also be aware of the existence of hepatitis delta virus
(HDV), a subviral satellite of HBV that requires HBV envelope proteins in order to
spread. HDV is a viroid-like agent with an RNA genome that can super-infect or
co-infect HBV carriers. HDV infection of HBV carriers increases the severity of
chronic liver disease and the risk of HCC (Tamura et al. 1993; Verme et al. 1991).
The Biology of Chronic HBV Infection

Although the course of chronic infection can vary widely, a generalized picture has
emerged from clinical studies. These studies indicate that infections may pass
through several stages, including an early, immuno-tolerant stage lasting 2030
years or more, an immune clearance stage, an inactive carrier stage, and a reactivation stage (Liaw 2009; Yim and Lok 2006) (Fig. 22.1). It should be kept in mind that
these changes may occur without the patients awareness, as advanced cirrhosis and
534
W.S. Mason
HCC incidence
Cirrhosis and HCC
(may occur at any age, incidence increases in middle age)
Reactivation phase
(Virus titers and inflammation increase)
Inactive phase (little or no HBV replication or inflammation)
Immune-clearance phase
(Virus titer declines
with bouts of acute inflammation)
Immuno-tolerant phase
Age (years): 0
10
20
30
40
50
60
70
Fig. 22.1 Stages of chronic hepatitis B following perinatal infection. Chronic hepatitis B is now
considered to have four phases, an immunotolerant phase in which there is little or no inflammation or fibrosis, an immune-clearance phase in which there may be one or more bouts of hepatitis
associated with a decline in viremia as the immune response attempts to clear the infection, an
inactive carrier phase in which virus titers are low and there is little or no inflammation, and in
some cases, a reactivation phase, in which virus titers and the immune response elevate, with
increased liver inflammation and damage. As noted, these phases may not always be present or, if
they are, may not be apparent to a carrier that is not actively followed in a clinical setting. HCC and
cirrhosis may occur at any age, but generally show dramatic increases after the age of 30 (Figure
adapted from Beasley 1982; Liaw 2009; Wang et al. 2010; Yim and Lok 2006). Chronic HBV
infection initiated in adult life probably lacks an immunotolerant phase (Liaw 2009)
HCC often produce the first outward symptoms of chronic liver disease which are
apparent to a carrier (Evans et al. 1998). Thus, the different stages of infection are
largely defined from clinical monitoring and experience, rather than from populationbased studies (Evans et al. 1998). In addition, the concept of an immuno-tolerant
phase is based on the absence of overt signs of liver damage, including significant
fibrosis and cirrhosis of the liver and elevated levels of liver enzymes in the blood.
Despite its name, ongoing liver damage, which is not apparent by these assays, is
probably a feature of the immuno-tolerant phase of infection.
The liver is made up of a variety of cell types, of which the major is the hepatocyte,
which constitutes ~70% of the cell population of the healthy liver. Human hepatocytes
are arranged primarily in 1-cell thick plates surrounded by fenestrated sinusoidal
endothelial cells. Blood flowing through the sinusoids, across the surface of the
endothelial cell layer, exchanges non-cellular components with hepatocytes. This occurs
through the fenestrations in the endothelial cells, as well as by directional transport
across the endothelial cell layer. Fixed tissue macrophages (Kupffer cells), also part
of the sinusoidal lining, are found in association with the endothelial cell layer,
where they play a major role in response to a variety of microbial infections. Ito
cells (hepatic stellate cells), which can produce collagen in response to liver injury,
are found between the endothelial cell layer and hepatocytes, and are responsible
for development of fibrosis and cirrhosis in response to chronic liver injury (e.g.,
caused by chronic HBV infections).
HBV infection results from exposure to blood or tissues of HBV-infected
patients that leads to entry of virus into the blood stream. From there it is carried
to hepatocytes, the only well-established site of HBV infection and reproduction
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535
in humans. It remains unclear if HBV accesses hepatocytes directly by passing

through the fenestrations in the endothelial layer, or is taken up first by the
endothelial lining cells and from there passed to hepatocytes (Breiner et al. 2001).
Once a hepatocyte(s) is infected, HBV spreads through the liver with a doubling
time of about 4 days (Wieland et al. 2004b), based on studies with chimpanzees,
which are HBV susceptible. Since the human liver contains ~5 1011 hepatocytes,
infection of the entire liver starting from a single infected hepatocyte could take
as long as 4 months (Asabe et al. 2009). Remarkably, in many cases, and in all
those infections which become chronic, infection of the entire hepatocyte population actually occurs. It is inferred that in these cases the HBV infection does not
elicit a significant innate immune response and that the adaptive response is slow
to develop (Wieland et al. 2004a). Even with transient infections that involve, at
their peak, the entire hepatocyte population, adaptive responses may not be apparent until days or weeks after the entire population is infected (Asabe et al. 2009;
Kajino et al. 1994; Summers et al. 2003; Wieland et al. 2004a, b). If this adaptive
response is strong, the infection will clear, typically over the course of a few
weeks (Wieland et al. 2004b). If not, the infection will become chronic. Chronic
liver disease then results from a persistent adaptive immune response that results
in an accelerated rate of hepatocyte killing and compensatory regeneration, as
compared to the uninfected liver. The normal rate of hepatocyte death and replacement is probably around 0.01% per day, but can be 10 to >100-fold higher in
chronic infections (e.g., Mason et al. 1998; Nowak et al. 1996). The extent of
hepatocyte turnover varies in proportion to the degree of hepatic inflammation
and, as a result, can be very different at different times during the course of a
chronic infection.
The belief that the immune response plays an important role in the outcome of
chronic HBV infections came from early studies in animal models, which suggested
that productive hepadnavirus infections are not cytopathic (e.g., Halpern et al. 1983;
Jilbert et al. 1988). These early observations have been supported by a variety of later
observations, including the persistence of infected hepatocytes during antiviral therapy with inhibitors of viral DNA synthesis (e.g., Werle-Lapostolle et al. 2004; Zhu
et al. 2001). These infected hepatocytes should have quickly vanished if the infection
was cytocidal, but they generally persist for months or years after therapy begins.
A separate line of evidence that the immune response plays an essential role in chronic
HBV infections, which may lead to HCC, comes from a study of HBV transgenic
mice. Immune system reconstitution with syngeneic, non-transgenic lymphocytes
reactive to HBV envelope proteins led to chronic liver disease and HCC (Nakamoto
et al. 1998). However, while this shows that a host immune response to products of
an HBV transgene can lead to HCC, the actual role of the antiviral immune response
to a natural HBV infection in the progression to HCC has remained elusive.
Thus, the following generalized view of the immune response to HBV infections
has emerged. Typically, especially in cases that become chronic or that cause clinically apparent transient infections, virus spreads to the entire hepatocyte population.
Hepatocyte infection, per se, is both productive and non-cytopathic. The adaptive
immune response, particularly mediated by CT8(+) T lymphocytes (Asabe et al. 2009;
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W.S. Mason
Thimme et al. 2003; Wieland et al. 2004b), then activates, causing an acute hepatitis.
This response generally abates after several weeks, whether or not the infection has
cleared. HBV clearance presumably depends upon the strength of the immune
response. The residual immune response that persists in chronic carriers is the cause
of liver disease, cirrhosis, and HCC (Liaw 2009; Lok and McMahon 2007;
Rehermann et al. 1996b; Yim and Lok 2006). Interestingly, even if the infection is
resolved, a residual immune response persists, and presumably serves, along with
antiviral antibodies, to prevent future infections, as well as reactivation of the original
infection (Rehermann et al. 1996a). Reactivation may occur from very small
amounts of residual virus that persist in a patient who has recovered from a transient
infection (Hoofnagle 2009; Mulrooney-Cousins and Michalak 2007; Reaiche et al.
2010; Rehermann et al. 1996a; Yotsuyanagi et al. 1997); the mechanism of persistence of these residual infections is unclear. Reactivation is usually associated with,
and presumably the result of, immuno-suppression.
Origin of Hepatocellular Carcinomas

The progression to HCC and the nature of the cells that ultimately undergo neoplastic transformation are still a matter of debate. Some investigators have suggested
that HCC in HBV carriers arise from hepatocyte progenitor/oval cells (Rogler 1991;
Roskams 2006), while others have felt that HCCs in HBV carriers may arise from
differentiated hepatocytes (Thorgeirsson and Grisham 2002). This remains a difficult problem to resolve. Hepatocytes constitute a self-renewing population and are
clearly able to divide to compensate for the death of other hepatocytes. However, it
is also clear that hepatocyte progenitor cells, although rare, do exist, and are able to
expand and differentiate to replace dying hepatocytes during acute and chronic liver
injury, particularly when the injury is caused by agents which prevent mature hepatocytes from fulfilling this role (Evarts et al. 1987; Hsia et al. 1992; Libbrecht et al.
2000; Oertel et al. 2008), but also to some extent during chronic hepatitis B. The
strongest argument that HCCs in adults arise from mature hepatocytes comes from
the fact that HCCs almost always contain integrated HBV DNA and that hepatocytes are the only liver cell, in human liver, that have been unambiguously demonstrated to be infected by HBV. One early study detected high level viral envelope
protein accumulation in the putative hepatocyte progenitor cell population (Hsia
et al. 1994) and this study is often cited to support the conclusion that HBV associated HCC is of progenitor cell origin. We are unaware of any subsequent study that
has re-visited these early results. Interestingly, while neonatal infection does not
typically lead to childhood HCC, exceptions do occur (Chang et al. 2009; Chen
2009). It has been suggested that many HCCs in HBV-infected children have a progenitor cell origin, while those in adults, an hepatocyte origin (Ward et al. 2010).
More work is needed to test this idea.
In brief, it is not clear how the issue of HCC origin can be resolved from deductive
experiments, since HCC cells share many properties with hepatocytes as well as
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with hepatocyte progenitor cells. As noted above, with some caveats, the best
evidence for a hepatocyte origin is the presence of integrated HBV DNA in the
tumor cells.
Hepadnavirus Replication
HBV is a small DNA virus with a genome size of ~3.2 kbp (Fig. 22.2). The virus
encodes only seven proteins. Among these are five structural proteins, including a
nucleocapsid protein subunit (core protein), three envelope proteins, and a reverse
transcriptase, and two nonstructural proteins, known as hepatitis B virus e-antigen
(HBeAg) and X (HBx). HBeAg is a glycosylated, C-terminally truncated, secreted
version of the nucleocapsid subunit which is thought to help the virus evade the host
immune response, at least early in infection (Chen et al. 2005). HBeAg-negative
HBV Genome
~3200 bp
(+)
(-)
Fig. 22.2 The rcDNA genome of HBV. The HBV genome is a relaxed circular DNA with a
complete minus strand, incomplete plus strand, and a short cohesive overlap between the 5 ends
of the two DNA strands. A protein, the viral reverse transcriptase (Pol), which is also the primer for
minus strand synthesis, remains covalently bound to the DNA during virus maturation (filled circle).
The RNA primer of 2nd strand (plus strand) synthesis remains attached to the 5 end of the plus
strand (thin line). All viral open reading frames (ORFs) are in the same direction, though out of
frame where they overlap. Each viral protein has its own mRNA, transcribed from promoters
upstream of the respective coding region, except for Pol, which is translated from the mRNA (the
pregenome) for the core protein (see text; Seeger et al. 2006, for details). The pregenome, which is
also the template for reverse transcription of minus strand DNA, is illustrated for comparison to
the structure of the rcDNA viral genome
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W.S. Mason
HBV strains often arise late in chronic infection, suggesting that this immune evasion
role of HBeAg is eventually lost. HBx is required for virus replication (Keasler
et al. 2009; Xu et al. 2002; Zhang et al. 2001; Zoulim et al. 1994) and is able to
activate transcription of both HBV (Spandau and Lee 1988; Twu and Schloemer
1987) and host mRNAs (Aufiero and Schneider 1990; Avantaggiati et al. 1993;
Balsano et al. 1991; Mahe et al. 1991; Twu et al. 1993). The exact role of this gene
remains obscure. Finally, although the reverse transcriptase is a virus structural
protein that is packaged in nascent viral nucleocapsids, cytoplasmic (Yao et al.
2000) and secreted (Cao et al. 2009) forms of the polymerase have also been
described. Their functional significance has not yet been established.
The genome of HBV is a relaxed circular DNA (rcDNA), which is held in a
circular conformation by a short cohesive overlap between the 5 ends of the two
DNA strands (Fig. 22.2). One strand is always complete within the virus, while the
other is always incomplete, so that the genome is actually only partially double
stranded. The complete strand is the template for viral RNA synthesis and thus is of
negative polarity, while the incomplete strand is of plus polarity. When the virus
infects a hepatocyte (Fig. 22.3), the plus strand is completed and the ends of each
strand are ultimately ligated to form a covalently closed circular DNA (cccDNA),
which is found in the nucleus of infected cells. cccDNA is associated with histones
(Levrero et al. 2009; Newbold et al. 1995) to form the transcriptional template of
the virus, from which all viral mRNAs needed for virus replication are ultimately
transcribed. cccDNA does not replicate but, instead, is produced, from rcDNA
synthesized in the cytoplasm of infected hepatocytes (Tuttleman et al. 1986a, b).
Among the various viral RNAs, the second largest is the pregenome, with a
terminal redundancy of ~120 nts (Fig. 22.2); a larger mRNA, the mRNA for HBeAg,
is only a few nts longer than the pregenome. The pregenome is the template for
translation of both the viral nucleocapsid protein (core protein) and the viral DNA
polymerase (reverse transcriptase), as well as the template for reverse transcription
to synthesize new viral DNA. Because the open reading frame (ORF) for the polymerase is downstream (and overlapping with) that of the core protein on the pregenome mRNA, core protein is the major translation product. Polymerase, which is in
a different translation reading frame than core, is translated following ribosomal
shunting (Sen et al. 2004) to the polymerase ORF, which is thought to be a rare
event as compared to translation initiation at the upstream core AUG. When the
polymerase is translated, it often binds to a stem-loop structure, epsilon, located in
the 5 copy of the terminal redundancy of its own mRNA (Junker-Niepmann et al.
1990). This polymerase/pregenome complex is then packaged, along with host
chaperones (Hu and Seeger 1996; Hu et al. 1997), into icosahedral nucleocapsid
shells composed of 240 or 180 copies of the viral core protein (Bringas 1997;
Crowther et al. 1994; Wynne et al. 1999; Zlotnick et al. 1997). Reverse transcription
of the pregenome takes place within the nucleocapsids (Summers and Mason 1982)
to form new viral rcDNA (Fig. 22.3). Once the plus strand is partially complete, the
nucleocapsids interact with the viral envelope proteins and bud into the endoplasmic reticulum (ER) to form virions (Wei et al. 1996), which are ultimately transported and secreted from the infected hepatocytes into the blood stream.
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Endothelial Cells
Virus
rcDNA genome
90%
Virus
DSL DNA genome
10%
Integration into
host DNA
cccDNA
host DNA
Transcription of
cccDNA
Packaging and reverse transcription

of the pregenome
Hepatocyte
Fig. 22.3 HBV life cycle in hepatocytes. Blood enters the liver and passes through sinusoidal
spaces lined with hepatic endothelial cells. HBV present in the blood infects hepatocytes, probably
after passing through fenestrations in the endothelial cells. The virus envelope is removed and
nucleocapsids transported to the nucleus, where rcDNA is released and converted to cccDNA.
cccDNA serves at the template for six viral mRNAs, including the pregenome, the template for
reverse transcription. The pregenome enters the cytoplasm and is translated into core (nucleocapsid
subunit) and pol proteins. When pol is made, it binds to the 5 end of its own mRNA and the complex is packaged into nucleocapsids, where viral DNA synthesis takes place to form new rcDNA
and DSL DNA. Early in infection, newly made viral DNA is transported to the nucleus to amplify
the cccDNA copy number to about ~550 per cell. Further cccDNA synthesis is then blocked and
newly made DNA is assembled into viruses by interaction of nucleocapsids with viral envelope
proteins, budding into the ER, and export into the blood stream. Virus with linear DNA can initiate
the infection pathway, but may not be able to complete it, since genetic information is lost during
cccDNA formation via illegitimate recombination between the ends of the DSL DNA. DSL DNA
also has a propensity to integrate into host DNA, again by illegitimate recombination
Nucleocapsids can also migrate to the nucleus (Rabe et al. 2009; Tuttleman et al.
1986a) to form more cccDNA (Fig. 22.3). In addition, nucleocapsids lacking viral
DNA (Burrell et al. 1982) typically accumulate in the nucleus in HBV-infected
cells, a phenomenon not found with other hepadnaviruses. HBV core protein was
reported a number of years ago to associate with, and hypothesized to regulate,
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W.S. Mason
Pol-ORF
PreCore/Core-ORF
Pregenome
DSL-DNA
X-ORF
Envelope ORF
PreS1 PreS2
S
S
M
L
PolyA
(+)
()
Fig. 22.4 The structure of double-strand, linear virus DNA (DSL DNA). Reverse transcription of
the minus strand always begins near the 3 end of the pregenome in the terminal redundancy, and
continues to its 5 end, to create a full length minus strand with a short, ~9 base, terminal repeat.
Normally, plus strand synthesis initiates near the 5 end of the minus strand, creating the cohesive
overlap that holds the two strands in a circular conformation. About 10% of the time, plus strand
synthesis begins, instead, at the 3 end of the minus strand, from an RNA primer, to create a double-stranded linear DNA (DSL DNA) (c.f., Fig. 22.2). DSL DNA is the most common substrate for
integration of HBV into host DNA (see text for discussion)
the transcription of HBV cccDNA (Bock et al. 2001). Support for this hypothesis, or
for core protein regulation of host gene transcription, has not yet been established.
rcDNA is the primary product of HBV DNA replication. However, as the result of
an occasional replication error, about 10% of new viral DNA is linear and double
stranded (Staprans et al. 1991), beginning at the 5 end of pregenome RNA (Fig. 22.4).
Double stranded Linear genomes (DSL DNA) that enter the nucleus can form
cccDNA via a process of illegitimate recombination between the free ends (Yang
et al. 1996b; Yang and Summers 1998). This cccDNA is typically defective as a result
of sequence loss during recombination. More interestingly, it can also undergo illegitimate recombination to integrate into host DNA (Bill and Summers 2004; Gong
et al. 1999; Yaginuma et al. 1987; Yang and Summers 1999). A second form of linear DNA also seems to serve, less frequently, as a substrate for cccDNA formation
via recombination and for integration into host DNA. This DNA is probably formed
by strand displacement synthesis through the cohesive overlap of rcDNA (Mason
et al. 2010; Yang et al. 1996b). Because the promoters for the pregenome and for the
PreCore mRNA are located downstream of their respective genes in the smaller double-stranded linear DNA (Fig. 22.4), only the envelope and X proteins could be
expressed from this smaller DNA after integration into host DNA. All viral proteins
might be expressed after integration of the larger linear HBV DNA.
Although most HCCs contain integrated HBV DNA, expression of core protein
has been found in only about 15% of HCCs, and then, only in a minority of cells in
the tumor (Hsu et al. 1989). Envelope protein is detected in about 30% of HCCs
and, in a larger fraction of tumor cells than typical of core (Hsu et al. 1989). HBx
expression has been observed in ~2050% of HCCs (Seo et al. 1997; Su et al. 1998)
but, like core, only in a small fraction of cells in a tumor (Su et al. 1998). As discussed
later, envelope and/or X proteins expressed from integrated DNA have been hypothesized, based on studies in model systems, to have a role in HCC through their
proposed ability to either initiate, promote, or fully transform hepatocytes. However,
their expression may also make these hepatocytes targets of the antiviral immune
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541
response, so that expression might decline in tumors that were, at least hypothetically,
initiated by these viral gene products (Hsu et al. 1989). This might explain why, not
all tumors, or tumor cells, express these viral proteins.
Changes in the Virus and in the Liver During

Chronic HBV Infection
At the peak of transient infections and early in chronic infections, virtually every
hepatocyte is infected, and virus titers in the blood stream are typically between 109
and 1010 per ml. Moreover, productive infection by HBV appears to be non-cytopathic
and the hepatocyte population to be self-renewing. These two points might lead to
the inference that chronic infections are associated with high titer production of
HBV throughout their course. Surprisingly, rather than remaining high, virus titers
actually decline over time, often by several orders of magnitude (Evans et al. 1998).
The decline in virus titers appears to be associated with a decline in the fraction of
infected hepatocytes (Burrell et al. 1984; Gowans et al. 1981; Mason et al. 2008,
2009; Volz et al. 2007) and in the amount of virus replication per infected hepatocyte (Volz et al. 2007). These observations suggest that there is a change over time
in the ability of the hepatocyte population to support HBV replication.
Dramatic changes in the virus population may also occur over time, with the
emergence of mutant virus, often with mutations that would make them replication
defective, as predominant forms of circulating HBV (e.g., Gunther et al. 1999;
Marinos et al. 1996; Preikschat et al. 2002; Yuan et al. 1998, 1999). These and other
mutant strains of HBV presumably survive in the hepatocyte population because of
complementation with wild type virus or because a lost virus function is no longer
required for virus survival. For instance, if horizontal, cell-to-cell spread of virus
were no longer required once a chronic infection were established, virus envelope
determinants essential for infectivity might no longer be essential. The fact that the
hepatocyte population is largely self-renewing means that death of an infected
hepatocyte is often compensated by division of another, also infected, hepatocyte,
and presumably no extra-cellular spread of virus is required in this situation to
maintain the infected cell number.
The host immune response is undoubtedly a factor in the changing virus population,
since deletions of immunodominant epitopes have been documented (Chen et al.
2006; Gunther et al. 1998; Ji et al. 2009; Lee et al. 1996). Perhaps the most well
described change in the virus population is the loss of HBeAg, which occurs in
many patients due to replacement of the predominant wild type strain of HBV by
viruses that are no longer able to make HBeAg. This may be due either to a decline
in transcription of the HBeAg mRNA, as a result of mutations in the promoter for
the PreCore mRNA, or to stop codon or frame shift mutations in the signal sequence
for this secreted protein (Brunetto et al. 1999; Okamoto et al. 1994; Parekh et al.
2003; Sato et al. 1995; Yu and Mertz 1996). HBeAg is thought to induce immune
tolerance to the core protein early in infection, particularly perinatal infection from
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W.S. Mason
infected mothers to their newborn, but to lose this function later, so that its elimination
is now actually beneficial for the survival of HBV-infected hepatocytes (Frelin et al.
2009; Milich et al. 1993). In brief, immune selection for less immunogenic variants
of HBV is probably a major factor in the emergence of mutant viruses. The possibility
of vertical transmission during mitosis may facilitate virus survival even if there is
a loss of a function essential for production of infectious virus. Thus, it is possible
that virus evolution during the course of a chronic infection occurs to a significant
extent at the cellular level, with selective survival of hepatocyte lineages that have
evolved, within themselves, less immunogenic strains of HBV.
Surprisingly, it seems that the hepatocyte population also changes over time,
declining in its ability to support HBV infections. This is strongly suggested by the
reduction in the fraction of infected hepatocytes as a patient ages. It is assumed, as
noted, that 100% of hepatocytes are infected early in an infection, at least for the
first few years. However, this fraction can decline to 30% or less. Since the patients
often remain viremic, the decline seems to reflect resistance of these hepatocytes to
HBV infection. The origin of these hepatocytes and a molecular basis for their resistance to HBV is unknown.
However, precedents for their emergence come from studies of chronic liver injury
with a genetic basis (e.g., as reviewed by Alison et al. 2009; Marongiu et al. 2008).
It has been found in a number of different studies that any source of chronic injury and
death that affects all hepatocytes, or at least the vast majority, leads to repopulation of
the liver by rare hepatocytes that are resistant to the toxicity. For instance, Chisari and
colleagues studied transgenic mice that over-expressed the HBV large envelope protein,
L, in hepatocytes (Chisari et al. 1989). Over-expression of L leads to its accumulation
in the hepatocyte ER, in the form of 22 nm diameter rod-like structures. This accumulation leads to ER proliferation, producing so-called ground-glass hepatocytes (based
on their appearance in stained tissue sections) and, ultimately, leads to hepatocyte
death. The livers of mice that had the highest levels of L expression, and of hepatocyte
injury, were eventually re-populated by hepatocytes that did not transcribe L mRNA
and/or had acquired deletions in the transgene (Chisari et al. 1989; Crawford et al.
2006). Repopulation in this and other models of hepatocyte injury merely requires a
differential survival advantage for a minor subpopulation of cells that can proliferate
to become the predominant hepatocytes in the liver (Alison et al. 2009; Marongiu
et al. 2008; Mason et al. 2008). It has been argued that the resistant cells, at least in the
L over-expression model, may actually be derived from hepatocyte progenitor cells
(Crawford et al. 2006); this may not be a fundamentally distinct issue if these cells
then mature to hepatocytes, which have the capacity for self-renewal. Likewise,
hepatocytes with a primary resistance to HBV would have a survival advantage in an
HBV carrier since they would not be targets of the antiviral immune response, and
could clonally expand to repopulate the liver of HBV carriers.
The basis for HBV resistance could be at a variety of different levels. For
instance, virus-free hepatocytes may be a subset that is especially sensitive to
inhibition of HBV infection by antiviral cytokines. Also, it is not directly known if
HBV-free hepatocytes constitute a fixed or fluctuating population that is responding
to local conditions in hepatic lobules. The population would appear to be stable,
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543
since HBV titers tend to return to baseline levels after withdrawal of antiviral therapy (Dienstag et al. 1995), rather than to the high titers (>109 virions per ml) characteristic of new infections.
A second possible source of resistance to infection is expression of envelope
proteins from viral DNA that has randomly integrated into host DNA. cccDNA
synthesis has been shown, with duck hepatitis B virus (DHBV), to be negatively
regulated by viral envelope proteins (Lenhoff et al. 1998; Summers et al. 1990). In
brief, according to this model, envelope proteins, if sufficiently abundant, lead to
incorporation of all newly made rcDNA into virus, so that little is left to form
cccDNA. With less cccDNA in the cell, there is presumably less viral mRNA synthesis
and envelope protein production, and more opportunity for newly made viral DNA
to be transported to the nucleus, rather than exported as virus particles. In theory,
production of envelope proteins from integrated DNA could shut down this negative
feedback loop and permanently suppress cccDNA synthesis. These envelope proteins
might also induce super-infection resistance, so that cells freed of cccDNA are not
re-infected. It should be noted, however, that attempts to show that envelope proteins
negatively regulate HBV cccDNA formation, as shown with DHBV, have been difficult
primarily because cell culture systems for HBV do not support significant cccDNA
copy number amplification (Ling and Harrison 1997). In addition, it is unclear if
virus-free hepatocytes, most appropriately defined by a lack of DNA replication
intermediates and cccDNA (Fig. 22.3), typically express HBV envelope proteins
from integrated DNA.
The major difference between these two possible modes of resistance to HBV
infection, or at least productive infection, is their origin. The first involves selection
of rare, mutant, or epigenetically altered hepatocytes that are intrinsically resistant
to HBV. HBV infection plays only an indirect role in their emergence, by establishing
an environment that is toxic to normal HBV-susceptible hepatocytes. The second
source of resistance is a direct result of HBV infection and DNA integration, and
presumably, could be initiated in any hepatocyte which was infected by HBV.
Studies with woodchuck hepatitis virus have revealed that integration occurs in at
least ~0.1% of hepatocytes during a transient infection (Summers et al. 2003).
Higher levels of integrated viral DNA have been found during chronic infections in
human, chimpanzees, and woodchucks (Brechot et al. 1981a, b; Mason et al. 2005,
2009, 2010; Shafritz et al. 1981).
Experiments to distinguish these two explanations for HBV-free hepatocytes in
chronically infected patients, or to define alternative explanations, have not yet been
reported. An additional major point, mentioned above, is that the virus population and
hepatocyte population may both evolve during a chronic infection as a result of immune
selection against viral antigens, leading to clonal expansion not only of virus resistant
hepatocytes but also of hepatocytes that are infected with less immunogenic strains of
HBV (Frelin et al. 2009; Mason et al. 2008). Thus, whatever its cause, hepatocyte
repopulation, if it occurs to a significant level as suggested, should also lead to a significant genetic narrowing of the hepatocytes population (Mason et al. 2009), a known
HCC risk factor (Marongiu et al. 2008). In addition, as discussed in the next section,
expression of viral proteins has been proposed to have a more direct role in HCC.
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W.S. Mason
Hepatocyte Evolution and Liver Cancer in Patients

with Chronic HBV Infection
Introduction
The notion that HBV encodes oncogenes is widely cited and may actually be correct,
but this notion is almost entirely dependent upon interpretation of data collected
using model systems. The obvious problem is that HBV infection does not transform hepatocytes; rather, decades of chronic infection are often needed to produce
a cell capable of unregulated growth to form a tumor (Ganem and Prince 2004).
Therefore, no HBV protein is able to transform normal hepatocytes, and any oncogenic
ability, if it exists, is dependent upon other slowly evolving changes in the hepatocyte and/or virus population, presumably extremely rare, since only one or a few
independent tumors will arise in a patients lifetime. Indeed, while HBx and expression of aberrant envelope proteins have been reported to activate expression of a
wide variety of cellular genes (Koike 2009; Lupberger and Hildt 2007), changes in
host gene expression, prior to the adaptive immune response, were not detected in a
serial biopsy study of transiently infected chimpanzees (Wieland et al. 2004a).
We are unaware of any studies showing that in vivo HBV infection, by itself, modifies
expression of host genes in infected hepatocytes. Short-term studies in liver cell
lines, typically derived from non-HBV related HCCs, may reveal what a viral protein
is capable of doing, without proving that it actually does this in infected hepatocytes, or revealing what the consequences might be.
Studies in transgenic mice, where it is possible to target viral gene expression to
hepatocytes, would seem to be more biologically relevant. Even here, however,
there are significant problems of interpretation. For instance, HBx expression
reportedly leads to HCC in some but not all strains of HBx transgenic mice (Kim
et al. 1991; Madden and Slagle 2001; Slagle et al. 1996). Interestingly, in those mice
that did not develop HCC due to HBx expression, HBx was still found to facilitate
liver carcinogenesis. For instance, diethylnitrosamine (DEN)-induced HCC was
facilitated by the presence of an HBx transgene in mice in which HBx, alone, did
not cause HCC (Slagle et al. 1996). HCC was also facilitated by the introduction of
an HBx transgene in C-myc transgenic mice. These latter findings support the idea
that HBx is pro-carcinogenic, even if not fully oncogenic. In contrast, later studies
using mice transgenic for the entire viral genome revealed an increased sensitivity
to DEN-mediated HCC that was not dependent upon HBx expression (Zheng et al.
2007). There is no explanation for these different outcomes, except that other viral
proteins (e.g., envelope) are also pro-carcinogenic. Nor, for that matter, is it clear
that mediators of rodent HCC will behave similarly in human HBV carriers. As noted
earlier, the two rodent models of chronic HBV infection, ground squirrels and
woodchucks, display different profiles of HCC induction from each other and from
humans. In summary, it remains possible that HBV causes HCC simply by stimulating
a chronic immune response to the hepatocyte population, enhancing cell injury,
mutagenesis, death, and regeneration over a very long time period. Alternatively,
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HCC may be a result of this prolonged period of injury, death, and regeneration
working in combination with viral proteins that directly alter cellular metabolism
and, as a result, play a more direct role in cellular transformation.
Hepatocyte Regeneration and HCC

As mentioned above, studies in mouse models and in humans with metabolic diseases
of the liver (e.g., hereditary tyrosinemia) have suggested that progression to HCC is
paralleled in almost every case by death of most of the hepatocytes that are initially
present, and their replacement by clonal expansion of the remainder (see Alison
et al. 2009; Marongiu et al. 2008, for review). This occurs because the agent which
causes HCC, whether endogenous (i.e., hereditary) or exogenous (e.g., a chemical),
also has a major toxic effect on the hepatocyte population. As a result, most hepatocytes die and clonal expansion of rare hepatocytes with resistant phenotypes,
some of which are pre-neoplastic and progress to HCC (Marongiu et al. 2008), is
necessary for host survival. Thus, as in chemical carcinogenesis, liver regeneration
is necessary to promote carcinogenesis that is initiated by mutation of a hepatocyte
or hepatocyte progenitor cell (Maeda et al. 2005). Of course, this does not explain
HCC at the molecular level, but is at least the starting point of a clearly defined
biological process ending in HCC.
Thus, it seems reasonable to assume that the same hepatocyte repopulation
happens in HBV carriers and leads to, or at least promotes the process leading to
HCC. In particular, the clonal expansion of rare hepatocytes that are unable or have
lost the capacity to support productive HBV infections. As a result, these hepatocytes would not be efficient targets for killing by the hosts antiviral immune
response and, therefore, may have the ability to repopulate the liver at the expense
of HBV susceptible hepatocytes.
Two types of observation support the idea of clonal hepatocyte repopulation, in
HBV carriers, due to killing of productively infected hepatocytes and expansion of
rare hepatocytes that are not productively infected: (1) A decline in the fraction of
hepatocytes that support virus replication, to 30% or less, is a characteristic feature
of HBV infection. This has typically been noted in late stages of infection, reflecting
the clinical focus on patients who are in the immune clearance, inactive carrier, or
reactivation stages of the disease (Burrell et al. 1984; Gowans et al. 1981; Mason
et al. 2008, 2009; Volz et al. 2007), when biopsy or surgical specimens are more
likely to be taken. Support for the histologic observations comes from qPCR assays
of liver biopsies. These assays show that in late stages of infection, there is often
insufficient cccDNA, the viral transcriptional template, for it to be present in all
hepatocytes (Volz et al. 2007). (2) Clonal expansion has been observed in hepatocytes in non-cirrhotic liver of chronically infected woodchucks, chimpanzees, and
humans, to produce large clones of 1,000 to 50,000 or more cells (Mason et al.
2005, 2009, 2010). In these experiments, viral DNA, which can integrate at random
sites in host DNA, was used as a marker of cell lineages (Bill and Summers 2004;
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W.S. Mason
Gong et al. 1996, 1999; Summers et al. 2003; Yang and Summers 1999). Determining
the number of copies of an integrant in a liver fragment by inverse PCR indicates
how much clonal expansion of these hepatocytes occurred following the integration
event (Mason et al. 2005). The fact that these hepatocytes contain integrated DNA
means that they were, prior to expansion, susceptible to HBV uptake.
There are several other reasons to think this repopulation could actually occur,
and progress to HCC. (1) Hepatocytes are, to a large extent, members of a closed
and self-renewing population. The population may therefore evolve under selective
pressure, with emergence of initially rare hepatocytes to become the predominant
cell type (e.g., Alison et al. 2009; Chisari et al. 1989; Marongiu et al. 2008; Ponder
1996); (2) Foci of altered hepatocytes (FAH), which develop during chronic
infection and are considered to be pre-neoplastic, generally do not contain replicating
virus, even when surrounded by productively infected hepatocytes (Abe et al.
1988; Fausto 2004; Govindarajan et al. 1990; Radaeva et al. 2000; Rogler 1991; Sell
et al. 1987; Toshkov et al. 1990; Yang et al. 1993; Yang et al. 1996a; Yeh et al.
2001); (3) HCCs, like FAH, are generally not productively infected. As noted earlier, expression of core and HBx are seen in only a minority fraction of tumors, and
only in a minor fraction of cells within those tumors; (4) It has been reported that up
to 50% of HCCs may occur in non-cirrhotic HBV carriers (Bosch et al. 2005),
though the number may be several fold lower (Beasley 1988). This is consistent
with the notion of extensive hepatocyte repopulation even in non-cirrhotic patients.
Thus, hepatocyte repopulation in HBV carriers to evade the host immune response,
or some other unknown factor, may occur, as a risk factor for HCC which precedes
cirrhosis.
An apparently distinct source of clonal hepatocyte repopulation related, only
indirectly, to immune killing of hepatocytes, is well documented in late-stage HBV
patients with cirrhosis. In particular, 50% or more of cirrhotic nodules are clonal
(Aoki and Robinson 1989; Furuya et al. 1988; Mashal et al. 1993; Paradis et al.
1998; Piao et al. 1997; Robinson et al. 1990; Yeh et al. 2001) and often have a
pre-neoplastic appearance. This clonality presumably reflects a selection for rare
mutant and/or epigenetically altered hepatocytes that are able to survive the disruption of lobular architecture and blood flow that occurs within the nodules. Cirrhosis
has long been known to be an epidemiological risk factor for HCC (Beasley 1988).
The histologic findings suggest that cirrhosis and HCC are not merely coincident
outcomes of chronic HBV infection.
In brief, clonal hepatocyte repopulation, which appears to promote emergence of
HCC in liver diseases of non-viral origin, also takes place in HBV carriers, in cirrhotic
nodules and also in non-cirrhotic livers. Clonal repopulation in cirrhotic livers is
likely a direct consequence of changes in hepatic structure and restrictions in blood
and lymph flow in cirrhotic nodules. Clonal repopulation in non-cirrhotic livers
likely results from immune evasion of the antiviral host response. In both cases, rare
hepatocytes expand clonally, presumably because of genetic or epigenetic changes
which allow them to survive in an otherwise toxic environment. The fact that hepatocytes are self-renewing argues against the necessity of a progenitor cell origin for
these rare cells, although some have argued that hepatocyte regeneration during
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547
chronic liver disease has a major progenitor cell component (Libbrecht et al. 2000).
Whether the changes which allow survival of these hepatocytes are also, in some
cases, responsible for HCC, is unknown, though clearly, in most cases, they are not.
A reasonable hypothesis is that these early, pre-HCC, changes are necessary but not
sufficient for transformation of normal hepatocytes to carcinoma cells. Other factors
required for full transformation may include viral protein expression from integrated
DNA and/or additional mutations in regulatory cell proteins or destabilization of
host chromosomes (Barash et al. 2010; Lee et al. 2009; Pineau et al. 2008).
HBx and HCC

Interest in HBx as an HBV oncogene has been persistent since the detection of this
small ORF in the viral DNA sequence and the discovery of its ability to trans-activate
expression of viral and cellular genes in culture. Indeed, early reports indicated that
HBx could transform immortalized cells in culture (Shirakata et al. 1989) and later
studies with transgenic mouse models implied that HBx could act alone (Kim et al.
1991; Koike et al. 1994), or as a co-factor with C-myc (Shirakata et al. 1989) or
with DEN (Lee et al. 1990; Slagle et al. 1996) to transform mouse liver hepatocytes.
However, extension of these findings to HBV patients has been problematic. HBx
clearly does not cause rapid transformation of hepatocytes, even in the mouse models,
nor is there any evidence that infection per se alters hepatocyte behavior. Moreover,
its putative oncogenic potential is limited. While up to 25% of HBV carriers may
develop HCC, the majority do not.
Understanding the role of HBx in infection and HCC is compounded by the wide
range of activities and mechanisms of action that have been reported. Some selected
examples are illustrated in Table 22.1. What remains unclear is whether the different
reported activities of HBx have a common basis. A recent report suggests that HBx
binds to the DDB1 subunit of the CUL4-DDB1 ubiquitin ligase and blocks binding
of receptors molecules to the DDB1, the adaptor subunit of the ligase (Li et al.
2010). The effect would, in theory, be to alter expression of pathways that are
regulated through this E3 ubiquitin ligase. These newer binding results are consistent with early reports that HBx inhibits DNA repair by binding to DDB1, which is
also a component of the DNA repair machinery. It is difficult, however, to reconcile
binding to DDB1 with all the diverse effects of HBx expression, since binding to
DDB1 is apparently not required for its function as a transcription transactivator
(Wentz et al. 2000). Moreover, although in some cases controversial (Madden and
Slagle 2001), other binding partners, including the tumor suppressor P53 (Elmore
et al. 1997; Feitelson and Duan 1997; Greenblatt et al. 1997), and mitochondrial
HSP60 (Tanaka et al. 2004; Zhang et al. 2005a), have been reported in the literature.
In brief, it has not been apparent how to prove that HBx has a direct role in human
HCC. The major problem is that no studies to date show that HBx is needed for
maintenance of the transformed state. Thus, detection of HBx expression in some
HCCs does not prove any expression is essential; in fact, as noted earlier, the majority
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W.S. Mason
Table 22.1 Some reported activities of HBxa

Activates viral gene expression
Colgrove et al. (1989) and Spandau and Lee (1988)
Activates host gene expression
Aufiero and Schneider (1990), Bock et al. (2008), Cohen
et al. (2010), Cross et al. (1993), Twu and Schloemer
(1987), Wang et al. (1998), Yoo et al. (1996), Yu et al.
(2005), and Zhang et al. (2005b, c)
Modifies/activates host transcription Barnabas et al. (1997), Kong et al. (2000, 2003), Lucito
factors
and Schneider (1992), Maguire et al. (1991), Rui et al.
(2006), Seto et al. (1990) and Shamay et al. (2002)
Binds to RNA polymerase
Dorjsuren et al. (1998), Haviv et al. (1998), Lin et al.
complex
(1997) and Qadri et al. (1995)
Modifies/inhibits E3 ubiquiton
Li et al. (2010)
ligase by binding to DDB1
subunit
Causes HCC in mice
Kim et al. (1991)
Collaborates with DEN and
Becker et al. (1998), Madden et al. (2001), Sitterlin et al.
C-myc to cause HCC in mice
(2000), Slagle et al. (1996) and Zhu et al. (2004)
Inhibits nucleotide
Becker et al. (1998), Capovilla and Arbuthnot (2003),
excision repair
Groisman et al. (1999), Jaitovich-Groisman et al.
(2001), Ji et al. (2009), Mathonnet et al. (2004) and
Qadri et al. (1996)
Anti-apoptotic
Elmore et al. (1997), Pan et al. (2001) and Wang et al.
(1995)
Binds to and inactivates/inhibits
Elmore et al. (1997), Feitelson and Duan (1997),
tumor suppressor P53; represses
Greenblatt et al. (1997) and Lee and Rho (2000)
P53 transcription
Induces cellular proliferation
Madden et al. (2001)
Transforms immortalized mouse
Shirakata et al. (1989)
cells
Disrupts intercellular and matrix
Lara-Pezzi et al. (2001a, b)
adhesion
Promotes fibrosis/cirrhosis via
Feitelson et al. (2009) and Martin-Vilchez et al. (2008)
paracrine activation of hepatic
stellate cells
Induces polyploidy
Rakotomalala et al. (2008) and Studach et al. (2009)
Induces chromosome instability
Kim et al. (2008) and Martin-Lluesma et al. (2008)
Shifts TGF-beta signaling to
Murata et al. (2009)
pro-oncogenic pathway
Pro-apoptotic
Bergametti et al. (1999), Liang et al. (2007), Schuster
et al. (2000), Shirakata and Koike (2003), Su and
Schneider (1997), Tanaka et al. (2004), and Terradillos
et al. (1998, 2002)
Induces cell cycle arrest
Bouchard et al. (2001a), Cheng et al. (2009), Friedrich
et al. (2005), Gearhart and Bouchard (2010), Lee et al.
(2002), Park et al. (2000) and Sirma et al. (1999)
Binds to mitochondria and induces
Bouchard et al. (2001b), Chen and Siddiqui (2007),
calcium release, increases
Clippinger and Bouchard (2008), Henkler et al.
(2001), Li et al. (2008), Lim et al. (2010), Rahmani
reactive oxygen species,
et al. (2000), Shirakata and Koike (2003), Tanaka et al.
activates NF-kB and Stat-3,
(2004), Waris et al. (2001) and Zhang et al. (2005a)
induces cell death
a
A sampling of publications illustrating some of the major activities and functions ascribed to HBx
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of tumors do not express detectable levels of HBx, and even those that do, express
it in only a minority of the tumor cells. Future analyses of putative HCC stem cells
may help to resolve this issue.
Perhaps the most consistent finding, in model systems, is that HBx inhibits
nucleotide excision repair (Table 22.1), although possible disagreements exist as to
the molecular mechanism and the target. Binding to DDB1 would suggest inhibition
of global genome nucleotide excision (NER) repair, in which DDB1 participates in
the recognition of distortions in the DNA helix. Based on these data, HBx would not
be expected to inhibit transcription-coupled NER, which generally occurs when
RNA polymerase II is stalled due to DNA damage. However, HBx has also been
reported to bind to downstream components that are common to both transcriptioncoupled and global NER, suggesting that both pathways are inhibited. While many
reported functions of HBx (inhibition of apoptosis; late G1 inhibition of cell cycle
progression) have some obvious function [e.g., survival of infected hepatocytes;
prevention of passage through the cell cycle, which reduces virus replication (Jansen
et al. 1993)], the advantage to the virus of inhibiting NER remains to be determined
and perhaps is only coincident with some other consequence of binding to the DDB1
component of the ubiquitin ligase.
Based on these findings, a simple model is that HBx initiates carcinogenesis by
facilitating accumulation of DNA damage via reactive nitrogen and oxygen species
(Gehrke et al. 2004) and that the immune response promotes carcinogenesis by
accelerating hepatocyte regeneration (Maeda et al. 2005). Other reported functions
of HBx may also increase the risk of HCC (see Table 22.1), though inhibition of
NER would seem the most obvious direct effect.
It should be noted that, despite the still limited amount of data, the claim has
often been made that HBx is expressed in HCCs. This claim would be consistent
with the hypothesis that HBx has a role in maintaining the transformed state of
tumor cells. Moreover, the promoter and most of the HBx gene, except for the last
few amino acids, is present in the linear HBV DNA which is the putative precursor
to most integrations (Fig. 22.3). Therefore, the presence and expression of a carboxy
terminal truncated HBx from many integrated DNAs would not be surprising.
A problem in assuming that HBx has a role in maintaining cellular transformation
is that wild type HBx has been found by several groups to induce cell cycle arrest in
model systems (Bouchard et al. 2001b; Cheng et al. 2009; Gearhart and Bouchard
2010; Lee et al. 2002; Park et al. 2000; Sirma et al. 1999). A resolution to this difficulty may exist in the reports that a carboxy terminal truncated X ORF, found in at
least some HCCs, yields an HBx that has lost the ability to induce cell cycle arrest,
and has an enhanced ability to transform cells in culture (Ma et al. 2008; Tu et al.
2001). This raises the possibility that HBx has at least two roles in HCC, inhibition
of NER leading to accumulation of host mutations early in infection (i.e., initiation)
and direct promotion of pre-neoplastic/neoplastic cells late in infection. Again, it
needs to be noted that truncated HBx, whatever its role in HCC, is not a typical
oncogene because generation of a partially truncated HBx gene is an expected
feature of viral DNA integration, but very few HCCs ever arise. More importantly,
expression of HBx in most HCCs is not, as noted, well documented, and the idea
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W.S. Mason
that HBx, or a truncated HBx, maintains the transformed state, contradicts at least
some studies looking at HBx expression in tumors (Su et al. 1998). Therefore, like
inhibition of NER, the data on other activities of HBX support the conclusion that
it is not required once a cell is cancerous, but may be involved in initiation and/or
promotion of carcinogenesis.
Finally, a significant effect of HBx expression could, in theory, be entirely indirect.
The wild type HBV X protein expressed by replicating HBV has been claimed to be
pro-apoptotic and to induce cell cycle arrest in late G1 (Bouchard et al. 2001a;
Cheng et al. 2009; Gearhart and Bouchard 2010; Lee et al. 2002; Park et al. 2000;
Sirma et al. 1999). This inhibition is readily overcome in vivo, at least partially, to
the extent that infected hepatocytes do divide to replace dying hepatocytes during
the course of a chronic infection (Wolf and Michalopoulos, 1992). It remains possible,
however, that hepatocytes that do not express HBx (e.g., because they are unable to
express HBV) have a greater probability than infected hepatocytes of dividing to
replace dying hepatocytes. This, like selective immune killing of infected hepatocytes,
should contribute to clonal hepatocyte repopulation, an HCC risk factor (Marongiu
et al. 2008), although one inversely related to the functional activity of HBx.
Viral Envelope Proteins and HCC

The three envelope proteins of HBV, encoded by a single ORF, define a nested set
of proteins (Figs. 22.2 and 22.4). The smallest, S, is at the C terminus. Addition of
55 more amino acids to S creates M. The additional sequences in M are referred to
as PreS2. M is not required for assembly of infections virus (Bruss and Ganem
1991; Fernholz et al. 1993; Santantonio et al. 1992). Addition of another ~120
amino acids to M defines L, with the extra sequences referred to as PreS1. These
three proteins combine to form the viral envelope, with S the most, and L, the least
abundant component. During synthesis of L at the ER, the PreS1 domain of only
about 50% of the L protein molecules is translated into the ER. Those PreS1
sequences in the ER (and ultimately, on the outer surface of virions), are involved in
virus recognition of target hepatocytes, while those on the cytoplasmic face appear
to recruit virus nucleocapsids for budding into the ER to form virions (Bruss and
Vieluf 1995; Ostapchuk et al. 1994).
Interestingly, HBV synthesizes much more S, M, and L envelope protein than
are needed to produce progeny virus. The excess is assembled and secreted from
hepatocytes, mostly as 22 nm diameter spheres and, to a smaller extent, rods. The
titer of these surface antigen (HBsAg) particles in sera is typically at least 100
1,000 fold greater than virus titers. The reason for the over-production of envelope
proteins is unclear.
Analysis of spheres and rods purified from sera revealed that the spheres are made
up primarily of S and, to a lesser extent, M and perhaps a small amount of L, while
rods contain S, M, and L (Heermann et al. 1984). As noted earlier, over-expression
of the large envelope protein, L, leads to accumulation of 22 nm rods in the endoplasmic
reticulum (ER) (Chisari et al. 1987; McLachlan et al. 1987; Ou and Rutter 1987).
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551
This is associated with the induction of ER proliferation to give hepatocytes a

ground glass appearance (Gerber et al. 1975). ER proliferation may be a unique
response to L that is only indirectly related to the ER stress response (Foo and Yen
2000), which is also attributed to L overproduction (see below). Ground glass
hepatocytes (GGH) generally accumulate toward the end of chronic liver disease,
rather than early, or during transient infections, and have mostly been studied in
non-tumorous liver samples acquired from patients with HCC.
Two types of GGH have been distinguished, in diseased liver, based on differences in the pattern of intracellular accumulation of viral envelope proteins detected
by immunohistochemistry and light microscopy. One type generally occurs singly
while the other type, in clusters. Those GGH occurring singly are referred to as type
I GGH while those in clusters type II GGH. Su and colleagues have suggested that
type II GGH result from expression of a mutant form of L protein with a deletion in
the PreS2 region (Fan et al. 2000), while type I GGH result from expression of a
mutant form of L with a deletion in PreS1 (Wang et al. 2003, 2006). They have also
suggested that type II GGH are pre-neoplastic. This suggestion is supported by the
fact that type II GGH occur as clusters of cells, which is consistent with (but does
not prove) the notion that the clusters arose by hepatocyte proliferation. Thus, a
major issue is whether envelope proteins, particularly L, are also pro-oncogenic.
In fact, interest in envelope proteins as potential oncogenes started with the early
report that a C-terminal truncated form of the M protein, originally detected in integrated viral DNA from an HCC, could trans-activate host promoters, including
C-myc, C-fos, and C-raf (Caselmann et al. 1997; Hildt et al. 2002; Kekule et al.
1990; Lauer et al. 1992). This activity was not found in M itself and required deletion of the carboxy terminal hydrophobic domain of S, including about 90 amino
acids. The authors suggested the mutant M protein was produced from viral DNAs
that had lost sequences during integration and that the necessary truncation might be
present in at least 25% of HCCs (Lauer et al. 1992). This truncated protein would
not normally be produced during virus replication. However, later studies from this
group suggested that L protein, like truncated M, also functions as a transcriptional
transactivator (Hildt et al. 1996). That is, the transactivation function first ascribed
to truncated M protein may reflect a normal function of L that is sometimes essential during virus replication (Foo and Yen 2000; Hildt et al. 1996).
At high enough levels, expression of L leads to hepatocyte death (Huang and
Chisari 1995). Presumably related to this, L protein expression also leads to an ER
stress response (unfolded protein response), which may lead to apoptosis if not
relieved by some means; e.g., increased production of chaperones involved in
protein folding. Interestingly, cell culture studies have suggested that ER stress also
increases transcription from the HBV M/S promoter, with increased production of
M and S envelope proteins contributing to relief of ER stress. Presumably, more M
and S protein reduced formation of 22 nm rods in favor of virus and 22 nm spherical
particles, which are more readily secreted (Huang et al. 2005; Xu et al. 1997) from
the cell. Thus, at least in cell culture studies, L protein expression appears to induce
an ER stress response that has a feedback role in balancing expression of the three
viral envelop proteins, so that accumulation of potentially toxic levels of L protein
in the ER does not occur.
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W.S. Mason
This raises an obvious question, are GGH always associated with accumulation
of variants forms of L protein that no longer cause ER stress, despite their accumulation to high levels in the ER and the induction of ER proliferation. At present,
there is no answer. With the exception of several recent studies from Su and
colleagues, reviewed in (Hsieh et al. 2007), this issue has not received a great deal
of attention, and it remains unclear if HBV variants in type II GGH are the cause
or only passengers in these focal proliferations. This is a problem because HBV
mutants with deletions in PreS1 and PreS2 are known to accumulate in the liver
during chronic infections (Chen et al. 2006, 2007; Fernholz et al. 1993; Gunther
et al. 1999). Their appearance late in infection is thought to be the result of immune
pressure leading to loss of T and B cell epitopes, which is also true for the PreS2
deletion mutation found in type II GGH (Hsieh et al. 2007). Thus, the connection
between the PreS2 mutation and putative clonal hepatocyte proliferation to form
clusters of type II GGH might be coincidental rather than causative.
One attempt to ascribe a role for the PreS2 deletion mutants of L, in HCC, has
focused on a role in oxidative DNA damage, in the form of 8-hydroxyguanine,
which can lead to insertion of an deoxyadenosine and mutation of host DNA (Hsieh
et al. 2004). In support of this idea, expression of mutant L protein was shown to
modestly upregulate 8-oxoguanine glycosylase, a protein involved in single nucleotide
excision/repair (Hsieh et al. 2004). Enhanced expression of, for instance, vascular
endothelial growth factor-A (VEGF-A), in type II GGH, and in Huh7 cells transfected
with the mutant L, has also been reported (Yang et al. 2009). The mutant L protein
has also been reported to enhance proliferation of Huh7 cells (Yang et al. 2009).
(Huh7 is a cell line derived from a hepatocellular carcinoma (Nakabayashi et al.
1982), probably from an hepatitis C virus carrier). Up-regulation of cyclin A and
induction of anchorage independent growth of HH4 (Tang et al. 2007), a line of
human hepatocytes immortalized by transduction of HPV E6/E7, has also been
claimed (Wang et al. 2005). Both VEGF-A and cyclin A were reported by this group
to be upregulated in type II GGH (Wang et al. 2005; Yang et al. 2009).
A problem with all these studies is uncertainty about the fraction of HCCs that
express either the carboxy terminal-truncated M protein or an L protein with a deletion
in PreS2, or even the wild type envelope proteins. One study, for instance, suggested
that only 30% of HCCs express envelope protein (Hsu et al. 1989). And, as noted
above, it is unclear if expression of the PreS2 deletion mutant of L is always associated with hepatocyte proliferation to form, for instance, type II GGH, or if the GGH
are merely a carrier of a common HBV variant. This might occur if the mutation
eliminated a major T cell epitope, giving the mutant-infected hepatocytes a selective
survival advantage, compared to hepatocytes infected with wt HBV.
In summary, as with HBx, available data do not provide a clear picture of the role
of HBV envelope proteins in carcinogenesis. These proteins are clearly needed to
make virus, and it is not surprising that their expression, particularly of L, may alter
normal host functions, particularly involving the ER, since virus and surface antigen
particles are formed by budding into the ER. On the other hand, there is so far no
data/evidence on changes in host gene expression, in hepatocytes, that occur simply
in response to in vivo infection. Finally, any role of envelope proteins in promoting
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hepatocyte proliferation must be subtle, at least for most of an infection, because

hepatocyte proliferation appears tightly regulated in order to maintain liver size
and function. Mutant viral proteins, even if more oncogenic in model systems than
the wild type, are likely to be under this same restriction, since HBV has a high
mutation rate, but HCC normally does not arise for decades. Thus, as with HBx, any
effect of wild or mutant envelope proteins in cellular transformation might also
depend upon the accumulation of mutations in host DNA that synergize with these
changes in the virus population. These host mutations must be extremely rare, considering the large size of the hepatocyte population (~5 1012 cells) from which they
arise and the long delay between infection and HCC.
Overview
Chronic HBV infection has been known, for almost 40 years, to cause HCC (Beasley
et al. 1981; Myerson et al. 1971; Nayak et al. 1977; Simons et al. 1971; Smith and
Francis 1972; Summers et al. 1978a; Teres et al. 1971; Tong et al. 1971; Velasco
et al. 1971; Vogel et al. 1972), and HBVs mode of replication has been known for
nearly 30 (Summers and Mason 1982). The fact that HBV replicates via reverse
transcription led to the idea that HBV might also cause cancer like a retrovirus,
particularly avian leucosis viruses, which were shown in the early 1980s to transform B cells by insertion of viral DNA in the vicinity of the C-myc gene (Hayward
et al. 1981). This hypothesis was spectacularly successful with the woodchuck
model of HBV. However, results with HBV itself have been disappointing, and a
clear role for HBV DNA integration remains, for most HCCs, elusive. Part of the
problem may be that woodchuck and human hepato-carcinogenesis may have fundamental differences and that human HCC may require many more changes in the
liver, including not just persistent hepatocyte death and regeneration, but oxidative
DNA damage and mutation, insertion of viral DNA into host DNA, ER stress, and
in some cases, expression of proteins from integrated viral DNA. DNA damage
(initiation) and regeneration (promotion) would presumably lead to some HCCs
without any other risk factors being involved. However, a number of additional risk
factors appear to exist, at least hypothetically, including hepatocyte repopulation by
HBV resistant cells, due to persistent immune killing of infected hepatocytes,
cirrhosis, and expression of viral proteins that may not be oncogenes in normal
hepatocytes, but may assume this role in the highly selected hepatocyte population
that emerges during the course of a chronic infection.
Unfortunately, current results do not yet indicate how to chemotherapeutically
treat and destroy HCCs. An additional problem is that new tumors may arise de novo
even after effective surgical removal of an existing tumor. As with chronic hepatitis,
a driving factor in recurrence appears to be the antiviral immune response, with
associated hepatocyte death and regeneration (Budhu et al. 2006; Hoshida et al.
2008). Thus, a major emphasis has been given to cancer prevention. This has been
approached in two ways, vaccination to prevent infection and antiviral therapy to
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W.S. Mason
slow disease progression. Therapy has been successful in slowing or reversing

fibrosis and the progression to HCC (Chien and Liaw 2008; Lai et al. 1998; Liaw
et al. 2004), but for most patients, is not curative, and therefore cannot be withdrawn
without a risk of virus rebound and a bout of immune-mediated acute hepatitis
(Chien and Liaw 2003; Honkoop et al. 2000). In addition, permanent suppression of
HBV will not totally reduce the risk of HCC, which can occur even during the inactive
phase of HBV infection (Chen et al. 2010), when virus productive is virtually
undetectable. An additional and major problem with the most common therapy,
nucleoside analog inhibitors of viral DNA synthesis, is the appearance of drugresistant variants that replace wild type virus as prevalent quasi-species (Locarnini
and Mason 2005). Even with these patients, drug withdrawal can be problematic as
the reemergence of the wild type virus, which generally replicates better than the
drug-resistant strains, can also lead to acute hepatitis (Fung et al. 2005). It is hoped
that these drug therapy issues will be less serious as the standard of treatment
changes from single to multi drug regimens. It is also hoped that a multi-drug regimen
will allow treatment of younger patients, in the immune-tolerant phase of infection,
who are not currently candidates for antiviral therapy by recent guidelines (Lok and
McMahon 2007), in part because they do not have overt liver disease, but equally,
because they do not show a sustained response to monotherapy. Monotherapy generally
shows a sustained response only in patients with active inflammatory disease to
assist in HBV elimination before drug-resistant virus emerges.
In summary, studies over the last 25 years have given a large number of insights
into how HBV gene products might cause HCC, but have not provided clear
evidence that the any viral protein is needed once an HCC has emerged. The
implication is that HBV proteins, like all the other factors that contribute to HCC,
provide only part of a very complex picture of carcinogenesis. Once the tumor has
emerged, disease progression is largely refractory to chemotherapy that targets
the metabolism of tumor cells. The most effective treatments are surgical resection or chemo-ablation of the HCC (e.g., injection of alcohol into the tumor)
(Colombo and Sangiovanni 2002; Teratani et al. 2002), but these procedures are
only efficacious with small HCCs, which are unlikely to be detected unless an
HBV carrier is actively monitored. The best approaches to preventing deaths
from HBV-associated cirrhosis and HCCs are (1) vaccination, (2) effective antiviral
therapies, and (3) ablation of small tumors. Vaccination is clearly the most effective. The expense of post-infection therapies, and the difficulties in identifying
and monitoring carriers make post-infection treatments impractical on a worldwide basis. This may change as better, and less expensive, treatments for HBV
carriers, and more effective treatments for patients with advanced liver disease
and HCC become available.
Acknowledgements I am grateful to Drs. Christoph Seeger and John M. Taylor for a critical
reading of this manuscript.
22
555
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Chapter 23
Hepatitis C Virus and Hepatocellular Carcinoma

Brett Lindenbach
Introduction
Liver cancer is the sixth most common form of cancer and the third most common
cause of cancer death worldwide (Parkin et al. 2005). Hepatocellular carcinoma (HCC)
accounts for a vast majority 84% of all liver cancers, and a large proportion of HCC
cases for instance, up to 70% of cases in Japan are attributable to hepatitis C virus
(HCV) infection (Tanaka et al. 2002; Yoshizawa 2002; Umemura et al. 2009).
HCV is a major cause of acute and chronic liver disease, infecting between 130 and
170 million people (Lavanchy 2009). Once HCV was identified in 1989 as the major
causative agent of non-A, non-B viral hepatitis (Choo et al. 1989), it soon became
apparent that chronic HCV infection is linked with HCC (Bruix et al. 1989; Colombo
et al. 1989). In addition, HCV infection is frequently associated with lymphoproliferative disorders, including non-Hodgkins B-cell lymphomas (Dustin and Rice 2007). In
the ensuing years, much has been learned about the links between HCV and cancer, yet
numerous questions remain. This chapter summarizes our current understanding of
HCV and HCC, with a focus on clinical and molecular virological perspectives.
Clinical Perspective
Epidemiological Link Between HCV and HCC
While the link between HCC and chronic HBV infection was well-known (Chap. 21),
the link between HCC and chronic HCV infection emerged only once diagnostic
B. Lindenbach (*)
Section of Microbial Pathogenesis, Yale University School of Medicine, 354C Boyer Center
for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA
e-mail: brett.lindenbach@yale.edu
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tools for HCV infection became available. Soon thereafter, research teams in Italy
and Spain showed that 6575% of HCC patients had serological evidence of HCV
infection, frequently accompanied by cirrhosis (Bruix et al. 1989; Colombo et al.
1989). These correlations were upheld in other populations, including North America,
Europe, and Japan (Liang et al. 1993; Deuffic et al. 1999; Nagao et al. 2004).
We now know that chronic HCV infection is a major risk factor for developing
HCC (El-Serag 2002). One early study estimated that HCV infection increases the
relative risk for developing HCC by 25-fold when compared to HCV-negative
controls (Resnick and Koff 1993). Similarly, a more recent meta-analysis estimated
that HCV-positive patients have a 17-fold higher relative risk for developing HCC
than HCV-negative controls (Donato et al. 1998).
The development of HCV-related HCC is a slow process, typically occurring
2530 years after infection, and usually after the development of advanced liver
fibrosis and cirrhosis (Castells et al. 1995; Tong et al. 1995). This slow progression,
as well as the fact that many people are unaware of their HCV-infected status, has
made it difficult to quantify the rate of progression to HCC in HCV-infected
individuals. Nevertheless, a published review of the literature identified a few welldesigned and controlled studies that ameliorated such variables and patient selection
biases (El-Serag 2002). Based on this analysis, it is estimated that 335% of HCVpositive individuals develop cirrhosis within 25 years after infection and up to 3%
of HCV-positive individuals develop HCC within 30 years after infection
(El-Serag 2002). It should be noted that numerous studies have shown that once
cirrhosis develops, the annual rate of developing HCC is between 1 and 4%, and has
been reported as high as 7% in Japan (Chiba et al. 1996; Bruno et al. 1997; Gordon
et al. 1998; Serfaty et al. 1998; Degos et al. 2000). Additional risk factors that
contribute to the development of HCC in HCV-positive individuals include coinfection
with HBV, heavy alcohol consumption, older age, race, and male sex (Donato et al.
1997, 1998, 2002; Degos et al. 2000; Freeman et al. 2001).
Antiviral Therapy and HCC

HCV infection was initially treated with interferon alpha, but the rate of sustained
virologic response (SVR; i.e., undetectable serum viral load for 6 months post
treatment) with interferon monotherapy is low, around 1015% (Hoofnagle and di
Bisceglie 1997). As of this writing, the current standard of treatment for chronic
HCV infection is a rigorous 24- or 48-week combined regimen of pegylated interferon alpha and ribavirin (Manns et al. 2001). This combination therapy is expensive
and difficult for patients, but does show a high rate of SVR for some but not all viral
genotypes (described in the section Experimental Systems to Study HCV). In
addition, a series of specifically targeted antiviral therapies for HCV (STAT-C),
which seek to target mechanisms essential for viral replication and should improve
HCV SVR rates, are currently in preclinical and clinical stages of development
(Pereira and Jacobson 2009).
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Since chronic HCV infection contributes to the development of HCC, does

successful treatment of HCV reduce the development of HCC? Answering this question is complicated by the fact that interferon has both antiviral and antitumor activities.
While some studies have suggested that interferon or interferon/ribavirin combination
therapy has preventative effects (reviewed in Liang and Heller 2004), most of these
were retrospective or uncontrolled studies. In one prospective, controlled, and randomized study conducted in Japan, interferon-treated HCV patients had a significantly
lower incidence of HCC than untreated HCV-positive controls, 4% vs. 38% 7 years
of follow-up (Nishiguchi et al. 1995). However, it is notable that this control group
exhibited an unusually high incidence of HCC. Another prospective, controlled, and
randomized study conducted in France found that interferon-treated HCV patients had
a slightly reduced incidence of HCC, 12% vs. 23% over 3 years of follow-up, but this
was not statistically significant (Valla et al. 1999). A prospective, controlled, but
nonrandomized study conducted in Italy showed that interferon treatment had a significant effect on the incidence of HCC, 2.6% vs. 9.8% over 3 years of follow-up
(Mazzella et al. 1996). Interestingly, there was no difference in HCC incidence between
patients who had an SVR vs. nonresponders, which suggested that these latter results
may reflect the antitumor properties of interferon rather than its antiviral effect. More
recently, the large, prospective, controlled, and randomized HALT-C trial failed to
show an effect of long-term, low-dose interferon maintenance therapy on clinical
outcomes, including the incidence of HCC, in patients that did not previously respond
to interferon plus ribavirin (Di Bisceglie et al. 2008; Lok et al. 2009). These results
suggested that successful reduction of viral loads may be necessary to prevent HCC.
Contribution of HCV to the Etiology of HCC

Cancer development is a multistage process that requires multiple cellular checkpoints
to be bypassed. These include continued proliferation, sustained immortality, evasion
of growth inhibition, avoidance of cell death mechanisms, induction of angiogenesis,
altered energy metabolism, evasion of immune surveillance, and acquisition of an
invasive phenotype (Hanahan and Weinberg 2011).
While there is a clear link between chronic HCV infection and HCC, it is not
fully understood how HCV contributes to the etiology of HCC. HCV does not
acutely transform cells, for example, like small DNA tumor viruses (Chaps. 1319).
A prevailing hypothesis is that chronic HCV infection indirectly contributes to the
development of HCC by setting up a long-term inflammatory cascade that leads to
hepatocyte turnover, advanced liver fibrosis (cirrhosis), and generation of DNAdamaging reactive oxygen intermediates (Liang and Heller 2004). While HCV can
induce cirrhosis, and cirrhosis is a risk factor for developing HCC regardless of
HCV infection status, some HCV-infected people develop HCC in the absence of
cirrhosis (De Mitri et al. 1995; Lok et al. 2009). Moreover, there is evidence
that some HCV gene products may directly contribute to cellular transformation
(see the section HCV Tropism). Thus, HCV infection may promote cancer development through both direct and indirect mechanisms.
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Virological Perspective
Experimental Systems to Study HCV
HCV is an enveloped, positive-strand RNA virus, classified as a separate genus in the
family Flaviviridae (Lindenbach et al. 2007). As such, HCV is distantly related to the
classic flaviviruses, such as yellow fever, dengue, and West Nile viruses, as well as
to the ruminant-infecting pestiviruses. Within the hepacivirus genus, HCV exhibits a
large genetic diversity that can be classified into seven major genotypes and numerous
subtypes. It is notable that genotypes 1a and 1b are the most prevalent worldwide and
tend to respond poorly to interferon monotherapy or combination therapy.
HCV has been difficult to study because the virus does not readily grow in cell
culture and because animal models for HCV infection are limited to chimpanzees or
to immunodeficient mice containing human liver grafts. Therefore, many early virological studies examined the structure and function of viral gene products by overexpressing them in various cell systems. The first HCV genetic systems became
available when functional cDNA clones were constructed and shown to give rise to
RNAs that are infectious in chimpanzees (Kolykhalov et al. 1997; Yanagi et al. 1997).
The next major breakthrough came from the construction of selectable, genotype
1a and 1b HCV replicons and genomes that could replicate after transfection into
the human hepatoma cell line Huh-7 (Lohmann et al. 1999; Blight et al. 2000, 2002).
These systems provided genetically tractable tools to dissect the intracellular aspects
of the viral replication cycle in cell culture. However, for reasons that are not entirely
clear, infectious virus was not produced in these early replication systems (Blight
et al. 2002; Pietschmann et al. 2002). More recently, a functional genotype 2a cDNA
clone was shown to replicate and produce high-titer infectious virus in cell culture
(Lindenbach et al. 2005; Wakita et al. 2005; Zhong et al. 2005; Pietschmann
et al. 2006). In addition, infectious genotype 1a and 1b culture systems do now exist
but yield very low titers (Yi et al. 2006; Pietschmann et al. 2009).
HCV Tropism
HCV is hepatotropic, and it is likely that many of the difficulties in culturing this
virus stem from the fact that human hepatocytes are extremely difficult to maintain
in culture (Bhatia et al. 1999; Khetani and Bhatia 2008). It should be emphasized
that the inability to study untransformed, primary hepatocytes in vitro also makes it
difficult to experimentally assess the contribution of HCV infection to cellular
transformation and oncogenesis. Nevertheless, recent advances in the long-term
culture of primary hepatocytes may now allow these questions to be addressed
(Khetani and Bhatia 2008; Ploss et al. 2010; Podevin et al. 2010).
So, if HCV directly contributes to cellular transformation, are transformed HCC
cells infected with HCV in vivo? The answer to this question remains elusive due to
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difficulties in detecting HCV in situ within human liver samples. However, recent
advances in sensitive and specific antigen detection by using two-photon fluorescent
microscopy and semiconducting quantum dots have revealed that HCV infects
720% of hepatocytes, typically within clusters, within frozen liver samples from
chronically infected patients (Liang et al. 2009). In one sample from the study of
Liang et al., HCV was abundantly found in a serial section adjacent to a cancerous
lesion, although it could not be resolved whether the infected cells were malignant
(Liang et al. 2009). These intriguing results beg for further analysis.
In addition to hepatotropism, HCV RNA has been found in extrahepatic compartments, most notably associated with B lymphocytes (Dustin and Rice 2007).
However, definitive evidence for replication in these cells is elusive, as replicating
forms of the viral genome were not detected by using highly sensitive and validated
quantitative methods (Lanford et al. 1995). One likely explanation is that HCV
particles may associate with B cells via interaction with CD81, a known HCV coreceptor, or through interaction of immune complexes with Fc receptors. Despite
these caveats, one group has provided evidence that EpsteinBarr virus-transformed
B cells derived from chronically infected HCV patients contain signs of HCV infection
(Sung et al. 2003). Interestingly, these cells appeared to exhibit a fivefold increased
rate of mutation in the cellular BCL-6, p53, and -catenin proto-oncogenes (Machida
et al. 2004). Intriguing as these results may be, replication of HCV in peripheral
blood mononuclear cells has been difficult to reproduce in other laboratories
(Marukian et al. 2008).
Molecular Biology of HCV

The HCV genome is a monopartite, 9.6-kb coding-sense RNA that encodes a single,
large, open reading frame (Fig. 23.1a). Translation of this genome produces a large
polyprotein that is processed by viral and cellular proteases to produce ten distinct
viral proteins (Fig. 23.1b). The N-terminal region of the genome encodes the
structural (i.e., virion associated) proteins: core, which presumably forms the viral
nucleocapsid, and envelope glycoproteins E1 and E2, which mediate virion binding
and fusion during the initial steps of virus infection. The remainder of the polyprotein
encodes nonstructural (NS) proteins: p7, NS2, NS3, NS4A, NS4B, NS5A, and
NS5B. By definition, NS proteins are not incorporated into infectious virions, but
are expressed within infected cells and coordinate the intracellular aspects of the
viral life cycle.
HCV Proteins and Carcinogenesis

Several HCV proteins have been implicated in cellular transformation by the fact
that they interact with cell cycle regulators or tumor-suppressor proteins, as summarized below. However, most of these studies were performed outside the context
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B. Lindenbach
a
structural
E1
nonstructural
E2
p7 NS2
E1
E2
NS3
4A 4B
NS5A
NS5B
b
lumen
cytoplasm
NS2
NS4A
NS4B
p7
NS3
NS5A
NS5B
Fig. 23.1 HCV genome (a) and polyprotein products (b)
of authentic viral infection, so it is difficult to assess how relevant these observations

are to carcinogenesis in patients.
To better understand the role of HCV proteins in physiologically relevant conditions,
numerous transgenic mice have been constructed. One mouse line that expressed
the entire HCV polyprotein under a liver-specific promoter on the C57BL/6 genetic
background developed liver steatosis (fatty liver disease) and a low but discernible
frequency of liver cancer (Lerat et al. 2002). Interestingly, a parallel transgenic line
expressing the core-p7 region did not develop cancer, suggesting that the viral NS
proteins could contribute to carcinogenesis (Lerat et al. 2002). One caveat to these
mouse studies is that transgene insertion sites can contribute to the phenotype, and
no current study has systematically shown that knockdown or inhibition of HCV
protein expression in transgenic mice reverses the observed phenotype.
The HCV Core Protein

The HCV core protein is synthesized at the endoplasmic reticulum, trafficked to
lipid storage droplets, and incorporated into virus particles (Moradpour et al. 1996;
Miyanari et al. 2007). This pattern of intracellular trafficking may contribute to the
dysregulation of lipid metabolism and the intracellular accumulation of lipids
known as hepatic steatosis (Abid et al. 2005; Jhaveri et al. 2008).
Heterologous overexpression of core protein reportedly affects numerous cellular
pathways that are relevant to cancer. However, conflicting reports abound, which could
reflect differences in experimental systems and/or differential effects of overexpression.
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In some reports, core promotes apoptosis (Zhu et al. 1998; Honda et al. 2000a, b;
Chou et al. 2005) while in other studies core inhibits apoptosis (Ray et al. 1998;
Lu et al. 1999; Sacco et al. 2003). Furthermore, core reportedly inhibits the p53
tumor-suppressor pathway (Ray et al. 1998) while other studies suggest that core
activates this pathway (Lu et al. 1999; Otsuka et al. 2000; Kao et al. 2004; Siavoshian
et al. 2004). Core is also reported to reduce transcription of retinoblastoma (Rb)
tumor-suppressor protein and enhance expression of E2F-1 and Mad2, leading to
inhibited mitotic spindle checkpoint function and polyploidy (Machida et al. 2009).
Core expression can also lead to the accumulation of reactive oxygen species,
induce oxidative stress, and activate antioxidant gene expression (Li et al. 2002;
Okuda et al. 2002; Korenaga et al. 2005). Finally, core can interact with Smad3, a
modulator of transforming growth factor- signaling, and thereby inhibit its antitumor
activities (Cheng et al. 2004; Pavio et al. 2005; Battaglia et al. 2009).
To better understand the role of core in vivo, several core-expressing transgenic
mouse lines have been characterized. One mouse line that expressed high levels of
HCV core protein under a liver-specific promoter in the C57BL/6 genetic background
developed liver steatosis and HCC at a high rate (Moriya et al. 1997, 1998).
However, the expression levels of core in these mice were likely much higher than
those found in HCV infections (McGivern and Lemon 2011). Similarly, another
mouse line constitutively expressing core-E1-E2 in the B6C3F1 genetic background
developed liver steatosis and a variety of hepatic and nonhepatic tumors (Naas
et al. 2005). In contrast, other core or core-E1E2 transgenic mice did not show
increased cancer development (Kawamura et al. 1997; Pasquinelli et al. 1997; Lerat
et al. 2002; Kamegaya et al. 2005), but core and core-E1-E2 transgenes could
sensitize mice to a chemical carcinogen (Kamegaya et al. 2005).
The HCV NS3-4A Enzyme Complex

NS3-4A is a membrane-bound enzyme complex that has serine protease and RNA helicase activities encoded by NS3. NS4A serves as a cofactor for these activities and serves
to anchor the NS3-4A complex to cellular membranes. The NS3-4A serine protease is
responsible for cleaving the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/B junctions during viral polyprotein processing (Steinkhler 2004). In addition, the NS3-4A serine protease downregulates innate antiviral immune activation by cleaving the MAVS/IPS-1/
Cardif and TRIF signal transduction proteins (Li et al. 2005a, b; Meylan et al. 2005).
NS3 has been shown to prevent apoptosis and can interact with and inhibit p53
(Ishido et al. 1998; Kwun et al. 2001; Deng et al. 2006; Tanaka et al. 2006). Mutations
that prevent these activities map to the serine protease domain of NS3 (Deng et al.
2006; Tanaka et al. 2006), although serine protease activity is not required to inhibit
p53 activity (Kwun et al. 2001). However, two contrasting reports suggested that
expression of NS3 or NS4A induces apoptosis independent of serine protease activity
(Prikhodko et al. 2004; Nomura-Takigawa et al. 2006). In addition, NS3-4A can
inhibit cellular response to DNA damage through interaction with Chk2 and ATM
(Ariumi et al. 2008; Lai et al. 2008).
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The HCV NS5A Phosphoprotein

NS5A is a homodimeric, RNA-binding phosphoprotein of unknown function.
However, NS5A is essential for HCV RNA replication and virus particle assembly. In
addition, NS5A has been found to interact with numerous cellular proteins, including
the key regulators of apoptosis, innate immunity, and tumor suppression.
Expression of NS5A in cultured cells has variably been found to inhibit apoptosis
(Gale et al. 1999; Lan et al. 2002), induce oxidative stress (Gong et al. 2001), and
promote anchorage-independent growth (Ghosh et al. 1999). The antiapoptotic
activity of NS5A may be due to interaction with p53 and inhibition of p53-mediated
apoptosis (Majumder et al. 2001; Lan et al. 2002; Qadri et al. 2002). In addition,
NS5A can bind to and activate PI3K, ultimately leading to stabilization of -catenin,
a key regulator of cell growth (Street et al. 2004, 2005). NS5A may also directly
bind and stabilize -catenin (Park et al. 2009; Milward et al. 2010).
Transgenic mice expressing NS5A in the C57BL/6 CBA/J genetic background
showed a high level of liver steatosis and HCC (Wang et al. 2009); however, NS5A
expression was not oncogenic in two other independently derived transgenic mouse
lines with the FVB genetic background (Majumder et al. 2003).
The HCV NS5B RNA Polymerase

NS5B is the RNA-dependent RNA polymerase responsible for replication of the viral
genome. In addition, NS5B can interact with Rb and target it for E6AP-mediated
ubiquitylation and degradation by the proteasome (Munakata et al. 2005, 2007;
McGivern et al. 2009). Amazingly, the region of NS5B that interacts with Rb overlaps
with the active site of the polymerase (Munakata et al. 2005). NS5B mutations that
block Rb interaction were found to cause profound defects in replication but did not
inhibit polymerase activity, suggesting that Rb degradation is important for HCV
RNA replication (McGivern et al. 2009). However, these mutants were not rescued by
Rb knockdown, suggesting that the NS5BRb interaction itself may be important.
Summary and Conclusions

The epidemiological evidence strongly indicates that HCV contributes to the development of HCC. However, HCC usually arises only after decades of HCV infection,
and likely involves both direct and indirect mechanisms. The purely reductionistic
approach has identified candidate HCV genes that could promote direct oncogenic
effects. However, we currently do not yet know how these observations relate to
HCC development in authentic infections. Clearly, the recent advances in HCV and
hepatocyte culture systems will help to clarify some of these outstanding issues.
23
579
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Chapter 24
Human and Animal Retroviruses: HIV-1

Infection Is a Risk Factor for Malignancy
Amy M. Hayes and Kathleen Boris-Lawrie
Comparison of the Distinct Cancer Mechanisms of Simple RNA

Tumor Viruses, Complex Human Transforming Retrovirus,
and Human Immunodeficiency Virus
Retrovirus Can Activate Cellular Proto-oncogenes
At the advent of modern molecular biology, the genetic basis of cancer was discovered
by the study of retroviruses. Study of infectious cancers in birds and mice discovered cellular proto-oncogenes and their transduction and mutation by transforming
retroviruses (Weiss et al. 1982). The hallmark features of cell transformation were
defined, a host of oncogenes were characterized, and the riveting story of retrovirus
insertional mutagenesis, transduction, recombination, mutation, and quasispecies
evolution was revealed by cell and molecular biologists (Chaps. 27 and 28). The
paradigm was revealed that retroviruses may be acutely transforming or slowly
transforming, lack a detectable disease end point, or lead to immunodeficiency of the
infected host, as first observed for the sheep lentivirus, Visna maedi (Fan 1997).
A.M. Hayes K. Boris-Lawrie (*)

Center for Retrovirus Research, Ohio State University, Columbus, OH 43210, USA
Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA
Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA
Department of Veterinary Biosciences, Ohio State University, Columbus, OH 43210, USA
e-mail: boris-lawrie.1@osu.edu
585
586
A.M. Hayes and K. Boris-Lawrie
Viral Oncoproteins Can Cause Transformation Directly

The first human retrovirus, human T-lymphotropic virus type 1 (HTLV-1), was
discovered in 1980 (Poiesz et al. 1981) and ultimately revealed a distinct molecular
mechanism for retrovirus carcinogenesis. Several decades after initial infection with
HTLV-1, a minority of infected individuals present with an aggressive monoclonal
tumor that is refractory to chemotherapy. HTLV-1 induces cell transformation, but
not by activation of cellular proto-oncogene. Instead, the virus-encoded transcriptional
transactivator Tax is an oncoprotein necessary and sufficient to transform cells,
which disrupts cell biology via multiple mechanisms (Chap. 26). The transforming
ability of HTLV-1 Tax is similar in principle to polyomavirus T antigen, adenovirus
E1A, and the human papillomavirus (HPV) E6/E7 oncoproteins, which also dysregulate cell biology by multiple mechanisms (chapters in this volume).
In comparison to the genetically simpler retroviruses that infect avian and murine
species, HTLV-1 possesses a genetically complex genome structure. HTLV-1
encodes the structural and enzymatic genes common to all retroviruses and additional open reading frames for regulatory and accessory proteins. Two small open
reading frames encode the Tax and Rex nucleic acid-binding proteins that transactivate viral gene expression. Alternatively spliced mRNAs encode accessory proteins
that are dispensable in cell culture, but required for viral persistence in patient infections. Human immunodeficiency virus type 1 (HIV-1) shares this genetically complex
structure (Chap. 32). However, HIV-1 does not cause cell transformation nor activate
cellular proto-oncogenes, which may be an adaptation to survival in humans.
Instead, HIV-1 infection is a risk factor for cancers caused by other transforming
viruses, which are less well-adapted to human infection. HIV-1 augments the
pathophysiological drivers of cell transformation and this interface presents a natural
model to decipher the fundamental connection between cellular immunity and
progression to malignancy.
HIV-1-Associated Malignancies Are Prognostic Indicators

of Acquired Immunodeficiency Syndrome
Large cohort studies have documented an increased risk of several cancers in HIV-1infected individuals (Clifford et al. 2005; Engels et al. 2008; Guiguet et al. 2009;
Mbulaiteye et al. 2003). HIV-1 infection is most prominently associated with three
malignancies: Kaposis sarcoma (KS); non-Hodgkins lymphoma (NHL); and
invasive cervical carcinoma (ICC). Each is attributable to coinfection with a slowly
transforming human oncogenic virus. The immune depletion and cytokine signaling
in HIV-1 infection promotes the progression to neoplasia. KS is associated with
human herpesvirus 8 (HHV-8) (Moore and Chang 1995); some NHLs are associated
with EpsteinBarr virus (EBV) (Thorley-Lawson et al. 2004); and ICC is associated with HPV (Lorincz et al. 1987). For an HIV-1-infected individual, presentation
24 Human and Animal Retroviruses: HIV-1 Infection Is a Risk Factor
587
Standard incidence ratio

0
10
15
20
25 200 400 600 800 1000 1200 1400 1600
Kaposis sarcoma
Burkitts lymphoma
Diffuse large B cell lymphoma
Primary central nervous
system lymphoma
Non-Hodgkins
lymphoma
Invasive cervical carcinoma

Oral/pharyngeal cancer
Anal cancer
Lung cancer
Hodgkins lymphoma
AIDS-defining malignancies
Non-AIDS-defining malignancies
Fig. 24.1 HIV-1 infection is associated with an increase in the incidence of multiple types of
cancer. The incidence of selected malignancy is increased in HIV-1-positive patients relative to the
general population. AIDS-defining malignancies (black bar) are prognostic of AIDS and related to
coinfection with transforming oncogenic virus. Non-AIDS-defining malignancies (gray bar) are
associated with environmental factors that include viral coinfection and behavioral risks.
Standardized incidence ratio (SIR) is the cancer incidence in an HIV-1-positive cohort living in the
USA in the period from 1991 to 2002 relative to general population. These SIRs were obtained in
the 5 years after HIV-1 registration by Engels et al. (2008). HAART was introduced in 1996.
HAART status was not tracked herein although two-thirds of the cohort was recruited in the
HAART era
of KS, NHL, or ICC is a prognostic indicator of progression to acquired immunodeficiency syndrome (AIDS). Accordingly, KS, NHL, and ICC are designated
AIDS-defining malignancies.
A cohort study of patients in the first 5 years after HIV-1 registration revealed
that their risk of KS is 1,000-fold greater compared to the general population, which
is largely free of KS [Fig. 24.1, standardized incidence ratio (SIR) 1,300] (Engels
et al. 2008). The risk of NHL is also increased (SIR 7) (Engels et al. 2008). The two
most common forms of NHL in HIV-1-infected individuals are Burkitts lymphoma
(BL) (SIR 15) and diffuse large B-cell lymphoma (DLBL) (SIR 10) (Engels et al.
2008). Primary central nervous system lymphoma (PCNSL) is unusual in the general
population and exhibits an SIR of 250 among individuals with HIV-1 (MacMahon
et al. 1991). The increased incidence of ICC represents progression of cervical
dysplasia attributable to persistence of HPV infection (SIR 3, Fig. 24.1).
Additionally, some non-AIDS-defining cancers exhibit increased incidence
ratios in persons with HIV-1; the SIRs of five neoplasms are provided in Fig. 24.1.
588
Acute HIV-1
infection
Persistent HIV-1
infection
Zone of heightened risk for

AIDS-defining malignancy
Trend in CD4+ counts
~1000- Point of infection

1500
500
300
Burkitts lymphoma
Invasive cervical carcinoma
200
AIDS
100
50
Diffuse large B-cell lymphoma

Kaposis sarcoma
10
Primary central nervous system lymphoma
weeks
years
Time
Fig. 24.2 The risk of AIDS-defining malignancy increases with continuous decline in CD4+ T-cell
count below 500 cells/mL. In the course of infection with HIV-1, CD4+ T-cell counts decrease over
a period of years to the level diagnostic of acquired immunodeficiency (AIDS, <200 cells/mL).
The risk of the AIDS-defining malignancies begins to increase as CD4+ T-cell count declines
below 500 cells/mL (Mbulaiteye et al. 2003; Guiguet et al. 2009). Dashed lines indicate the typical
CD4+ T-cell count at diagnosis of indicated malignancy. Diagnosis of Burkitts lymphoma and
invasive cervical carcinoma is often before the onset of AIDS (Mbulaiteye 2003), whereas diffuse
large B-cell lymphoma (Lim et al. 2005) and Kaposis sarcoma (Gallafent et al. 2005) are commonly diagnosed at CD4+ counts 100 cells/mL and primary central nervous system lymphoma is
observed in profoundly immunocompromised individuals (Hoffmann et al. 2001)
These malignancies are associated with viral and behavioral risk factors. Oral/
pharyngeal and anal cancers are associated with HPV (Melbye and Frisch 1998;
Gillison et al. 2000); hepatocellular carcinoma (HCC) is associated with hepatitis C
virus (HCV) (Saito et al. 1990); and Hodgkins lymphoma (HL) is associated with
EBV infection (Pallesen et al. 1991) that is predominant worldwide. Oral, liver, and
lung cancers are associated with tobacco and alcohol use. The increase in SIR for
these cancers may be due to a direct contribution of HIV-1 infection to cancer progression or due to an increase in risk behaviors in the HIV-1-infected population.
HIV-1-Associated Immunodepletion Heightens

the Risk for Malignancy
A CD4+ T-cell count below 500 cells/mL and sequential declines significantly
increase the risk for the development of AIDS-defining malignancies (Fig. 24.2)
(Mbulaiteye et al. 2003; Jellinger and Paulus 1995; Cingolani et al. 2000).
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Malignancies with the greatest increase in SIR are KS and three NHL subtypes
(Fig. 24.1) (Mbulaiteye et al. 2003). In an American cohort, CD4+ T-cell count did
not significantly increase the risk of ICC or the non-AIDS-defining malignancies
(Mbulaiteye et al. 2003). In a French cohort, presentation of CD4+ T-cell count
below 500 cells/mL was correlated with increased risk of KS and NHL and also HL,
lung cancer, liver cancer, and anal cancer (Guiguet et al. 2009).
Treatment of HIV-1 Infection As a Means to Mitigate

Concurrent Malignancy
HAART Has Changed the Spectrum of HIV-1-Associated
Malignancy
The premise of current antiretroviral therapy is to inhibit two or more viral enzymes
necessary for the hallmark steps in the retrovirus replicative cycle (Broder 2009).
The first reverse transcriptase inhibitor was developed in the 1960s, though its
potential was not recognized at that time. The first inhibitor of HIV-1 protease
became available in 1996; quickly, innovative combination regimens were tested.
Designated highly active antiretroviral therapy (HAART), a typical combination
is a protease inhibitor and two nucleoside reverse transcriptase inhibitors. The principle behind HAART is that the simultaneous application of three drugs decreases
the ease of evolving resistance. A quasispecies resistant to one of the three drugs is
likely to be vulnerable to the other two, and does not experience positive selection.
However, comparative analysis of the doseresponse curve slope for class-specific
anti-HIV drugs has produced a new explanation for the success of HAART. Siliciano
and colleagues determined that the log reduction in single-round infectivity at
clinical drug concentrations is strongly influenced by slope and varies by >8 logs for
anti-HIV drugs (Shen et al. 2008). Observation of a slope >1 was characterized for
nonnucleoside reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors. The results suggest that the efficacy of HAART regiments is attributable to the
superior therapeutic threshold of class-specific anti-HIV-1 drugs.
Since 1996, HAART has been the standard of treatment for HIV-1 in developed
nations, and has dramatically stabilized the immune status of HIV-infected persons.
Large cohort clinical studies to measure the association of AIDS onset, CD4 count,
HAART treatment, and cancer presentation have documented corresponding reductions in the incidence rates of KS and NHL (Engels et al. 2008; Clifford et al. 2005).
However, cohort comparison from 1991 to 1995 or 1996 to 2002 revealed that the
incidence of some non-AIDS-defining cancers has increased. Incidence of nonAIDS-defining cancers, including HCC, oral/pharyngeal cancer, lung cancer, anal
cancer, Hodgkins lymphoma, and nonmelanoma skin tumors, increased from 31 to
58% (Engels et al. 2008). Possible explanations are prolonged survival with exposure to multiple risk factors (including viral coinfections and behavioral factors),
sustained marginal immunosuppression, or both (Engels et al. 2008).
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Combination of HAART with Chemotherapy

Stabilizes Patient Prognosis
Temporary depletion of T lymphocytes is a generic outcome of chemotherapy
(Mackall 2000). Typically, CD4+ lymphocyte levels are more affected by chemotherapy than are CD8+ lymphocyte levels (Mackall et al. 1997) and their recovery to
pretreatment levels can be prolonged to beyond a year after treatment cessation
(Hakim et al. 1997). Especially implicated is cyclophosphamide therapy, which is
part of the standard treatment regimen for NHL (Gershwin et al. 1974).
Chemotherapy-induced depletion of T lymphocytes is of great concern for HIV-1infected patients. However, immune stabilization by HAART has profoundly
improved the prognosis of standard chemotherapy regimens for HIV-1-infected
patients with cancer. A study of 18 patients with NHL (16 DLBL, 1 BL, 1 peripheral
T-cell lymphoma) and 2 with HL examined viral load during HAART plus bleomycin, etoposide, methotrexate, vincristine, prednisolone/cyclophosphamide and
doxorubicin (BEMOP/CA) or infusional cyclophosphamide, doxorubicin, and etoposide (CDE) (Powles et al. 2002). HAART during chemotherapy suppressed an
increase in the viral load and was associated with rapid recovery of CD4+ lymphocytes to baseline in the month after cessation of chemotherapy (Powles et al. 2002).
A subsequent retrospective study compared DLBL and BL survival with HAART
chemotherapy (Lim et al. 2005). Before HAART, patients with NHL had a median
survival time of approximately 68 months (Lim et al. 2005). After HAART,
median survival time for DLBL patients increased to 43 months, although there
was no change in survival for BL patients (Lim et al. 2005). In the case of PCNSL,
HAART addition to chemotherapy dramatically increased survival. Patients without HAART exhibited a median survival of 52 days while patients on HAART had
not reached median survival at 667 days (Skiest and Crosby 2003).
While the risk of NHL has decreased by approximately a factor of 5 with modern
HAART (Carrieri et al. 2003), the potential for developing NHL poses a greater risk
of mortality than the other AIDS-defining malignancies. In a study conducted in
2000 of HIV-1-infected patients, NHL was responsible for 29% of deaths due to
malignancy while KS was responsible for 15% (Bonnet et al. 2004). Primary effusion lymphoma and PCNSL remain consistently fatal diagnoses despite HAART
(Bonnet et al. 2004).
Drug Interactions Demand Management of HAART

and Chemotherapy Regiments
While HAART has facilitated more effective chemotherapy, drug interactions pose
a new challenge to clinical treatment of cancer in AIDS patients. In HAART formulations, protease inhibitors and nonnucleoside reverse transcriptase inhibitors modulate the efficiency of the cytochrome P450 (CYP) pathway. The pathway component
591
CYP34A has broad substrate specificity and is active in the metabolism of multiple
drugs. However, HIV-1 protease inhibitors typically inhibit CYP34A, which can
lead to reduced metabolism for the chemotherapeutic. The protease inhibitor ritonavir has little anti-HIV-1 activity but strongly inhibits CYP34A. Thus, ritonavir is a
useful boosting agent in antiretroviral therapy to enable administration of lower
dosages of drugs metabolized by this enzyme (Merry et al. 1997). However, inhibition of CYP34A by ritonavir can alter metabolism of chemotherapy drugs, which
can result in toxicity when prescribed with anticancer vinca alkaloids (Cingolani
et al. 2010) and docetaxel (Mir et al. 2010). Conversely, the nonnucleoside reverse
transcription inhibitors nevirapine and efavirenz increase CYP34A activity.
Therefore, chemotherapy drugs metabolized by this enzyme may not reach therapeutic levels if these drugs are prescribed simultaneously (Mounier et al. 2009).
Generally, adverse drug interactions can be avoided by adjustment of the HAART
regimen and dosage (Mounier et al. 2009).
Long-Term Control of HIV-1 Is Achievable by Selective Bone

Marrow Reconstitution
HIV-1 enters a susceptible cell by interaction with the CD4 receptor and one of the
two coreceptor molecules, CCR5 or CXCR4 (Choe et al. 1996; Feng et al. 1996).
Homozygous mutation of the CCR5 coreceptor is sufficient to confer resistance to
infection by HIV-1 (Marmor et al. 2001). Recently, bone marrow transplantation was
performed from a donor homozygous for a 32 base pair deletion in the CCR5 gene,
which encodes an inactive receptor (Hutter et al. 2009). The transplant recipient was
an acute myeloid leukemia patient with HIV-1 infection. The treatment has produced
remission of the leukemia and the HIV-1 viral load has been below detectable limits
for over 20 months without further antiviral therapy (Hutter et al. 2009). Although a
minority of the viral quasispecies of this patient had been observed to be CXCR4
tropic, virus load has not rebounded and the patient may experience a durable cure
(Allers et al. 2011). In principle, this clinical outcome validates the significant therapeutic utility of downregulation of coreceptors. It is conceivable that reduced viral
replication and viral load are necessary to permit a robust antiviral response capable
of preventing resurgence of infection. Designated as drug-induced vaccine effect,
studies led by Ruprecht and by Mathes have documented this in primate and feline
models (Ruprecht et al. 1990; Mathes et al. 1992). Alternatively, the rigorous ablation therapy may have completely eliminated any virus reservoir. Current studies are
addressing the source of long-lived reservoirs of HIV-1 persistence that are sufficient
for eventual recovery of detectable HIV-1 viral load. While transplantation therapy
is unlikely to become useful for treatment of concurrent HIV-1 infection and leukemia due to the rarity of appropriate donors, there is interest in developing a similar
therapy involving transduced lymphocytes expressing anti-CCR5 shRNA (Qin et al.
2003). Autologous stem cell transplantation with these transduced lymphocytes
could potentially simultaneously treat HIV-1 and NHL (Gabarre et al. 2000).
592
Viral Coinfection Contributes to Malignancy in HIV-1 Infection

Presentation of Kaposis Sarcoma Led to Discovery of AIDS
HHV-8 is the Kaposis sarcoma-associated herpesvirus (KSHV). In most cases,
HHV-8 causes no overt disease. However, HHV-8 infection can be associated with
KS, NHL subtypes, and Castlemans disease; the particular outcome is related to
cell type infected and expression pattern of virus-encoded interleukin 6 (vIL-6)
(Staskus et al. 1999). Coinfection with HIV-1 and HHV-8 produces a 50% increase
in the 10-year risk of KS (Martin et al. 1998) and an SIR of 1,300 compared to HIV-1uninfected people in the USA (Engels et al. 2008). KS was originally described in
elderly Mediterranean men as a disease with an indolent course (Kaposi 1872). KS
is observed in immunosuppressed organ-transplant recipients (Penn 1979) and in
Africa (Lothe 1960), where HHV-8 infection is widespread by mother-to-child
transmission (Bourboulia et al. 1998). However, in the USA, HHV-8 is primarily
sexually transmitted (Kedes et al. 1996). HHV-8 is not common in other areas of the
world (Ablashi et al. 1999).
Clinical presentation of KS was instrumental in the discovery of AIDS, and
the eventual discovery of HIV-1 as its causative agent. In 1981, eight cases of KS
in homosexual men were ascribed to a sexually transmitted causative agent
(Hymes et al. 1981). In 1982, another unusual appearance of aggressive KS was
attributed to an acquired immunoregulatory defect, and one or more infectious
agents (Friedman-Kien et al. 1982). While the discovery of HIV-1 followed in
1983 (Gallo et al. 1983; Barre-Sinoussi et al. 1983), HHV-8 eluded researchers until
discovery by Moore, Chang, and coworkers in 1994 (Chang et al. 1994). Treatment
of HIV-1 infection with HAART has decreased the risk of developing KS by about
a factor of 5 (Carrieri et al. 2003; Jones et al. 2000).
Synergistic ViralHost Interactions of HIV-1 and HHV-8

Exacerbate the Severity of Kaposis Sarcoma
KS is a complex proliferative mucocutaneous lesion, characterized by hypervascularization, which often first appears on the skin but may affect other mucous
membranes, including the lungs and gastrointestinal tract. KS exhibits the histology
of a benign hyperproliferative lesion, although it is frequently designated a sarcoma
(Brooks 1986). Sarcomas are clonal expansions of transformed cells while KS
typically is polyclonal and multifocal (Gill et al. 1998; Delabesse et al. 1997).
However, later in the disease, lesions may become monoclonal (Rabkin et al. 1995,
1997). KS lesions consist of spindle cells, which are sparse in the initial lesions,
endothelial cells, and infiltrating leukocytes (Flore et al. 1998). As summarized in
Fig. 24.3, HHV-8 infection of cells provides paracrine stimulation that prolongs the
survival of uninfected bystander cells (Flore et al. 1998). While HHV-8 can infect
dendritic cells, monocytes, and endothelial cells, the spindle morphology develops
only in HHV-8-infected endothelial cells (Grossmann et al. 2006).
593
Hyperproliferation
HHV-8-infected spindle cells
Enhances cell proliferation
Free
bFGF
bFGF
Tumor
Induces bFGF
expression
Heparin
sulfate
Competes
with bFGF for
binding sites
Endothelial
cells
IL6 vIL6
Upregulates VEGF expression
VEGF
NFAT1,2 activation
vGPCR
Blood
HIV-1 infected
lymphocytes
secrete IL-6
IL6
HIV-1 infected
lymphocytes
secrete Tat
Tat
Induces leukocyte migration
and invasion of endothelium
IFN
IL-1
TNF
Leukocytes
Fig. 24.3 Multiple points of virushost interface exacerbate Kaposis sarcoma in patients coinfected
with HHV-8 and HIV-1. Kaposis sarcoma lesions are a mixture of HHV-8-infected spindle cells,
endothelial cells, and infiltrating leukocytes. In coinfection, HHV-8 and HIV-1 exhibit synergistic
virushost interactions that result in cellular proliferation and angiogenesis. In HHV-8-infected
spindle cells, expression of interleukin-6 (IL-6) and viral IL-6 (vIL-6) induces expression of vascular
endothelial growth factor (VEGF), which induces proliferation of spindle cells and angiogenesis
of endothelial cells. This upregulation of VEGF is amplified by IL-6 secreted from circulating
HIV-1-infected leukocytes (indicated by linear provirus). Secreted HIV-1 Tat induces leukocytes to
infiltrate endothelium and to release interferon-g (IFN-g), interleukin-1 (IL-1), and tumor necrosis
factor-a (TNF-a). These cytokines induce basic fibroblast growth factor (bFGF) expression in
spindle cells, and Tat enhances release of bFGF from matrix-associated heparin sulfate by competing
for binding sites. Redundant paracrine signaling by VEGF and bFGF contributes to proliferation
of both spindle cells and endothelial cells. HHV-8 viral G-protein-coupled receptor (vGPCR)
cooperates with HIV-1 Tat in posttranscriptional activation of nuclear factor of activated T cells 1
(NFAT-1) and NFAT-2. These transcription factors control the expression of cytokines and growth
factors contributing to spindle cell proliferation. In sum, viral coinfection reinforces local hyperproliferation and hypervascularization, exacerbating Kaposis sarcoma
594
Cytokines, especially IL-6, have a key role in tumorigenesis in KS. HHV-8 alters
the cytokine environment through expression of a virus-encoded cytokine homolog
and manipulates expression of endogenous cytokines. Cells infected by HHV-8
express viral homolog vIL-6 (Nicholas et al. 1997) and vFLIP, which induces IL-6
expression (Grossmann et al. 2006). Additionally, HHV-8 encodes microRNA that
upregulates IL-6 and IL-10 (Nicholas et al. 1997). The virally encoded miR-K12-3
and miR-K12-7 target the mRNA of a negative regulator of IL-6 and IL-10, the
liver-enriched inhibitory protein isoform of CCAAT/enhancer-binding protein (Qin
et al. 2010). Expression of IL-6 and vIL-6 results in proliferation of HHV-8-infected
cells (Miles et al. 1990) and angiogenesis (Cohen et al. 1996), attributable to the
ability of IL-6 to upregulate vascular endothelial growth factor (VEGF). HHV-8
also encodes homologs of macrophage inhibitory protein (MIP): vMIP-I (Nicholas
et al. 1997), vMIP-II (Weber et al. 2001), and vMIP-III (Stine et al. 2000). These
b-chemokine family members aid HHV-8 evasion of cytotoxic T lymphocytes
(CTLs) by shifting the T-cell response from a Th1 type toward a Th2 type.
Increased severity of KS in HIV-1 coinfection is attributable to paracrine stimulation
of proliferation of KS lesion spindle cells and uninfected endothelial cells by
secreted Tat and cytokines (Fig. 24.3). HIV-1 infection of peripheral blood mononuclear cells (PBMCs) leads to expression of the Tat regulatory protein. Within HIV1-infected cells, Tat provides essential transcriptional transactivation of viral gene
expression (Charnay et al. 2009; Coffin et al. 1997) and suppresses microRNAmediated translational repression of HIV-1 (Bennasser et al. 2005; Qian et al. 2009).
Tat secreted from HIV-1-infected cells manipulates cytokine expression by leukocytes (Ott et al. 1998; Izmailova et al. 2003). The activity of Tat on the miRNA
signatures in leukocytes and in circulating plasma microvesicles (Hunter et al. 2008)
and the definition of the biological processes modulated by these miRNA are areas
ripe for investigation (Houzet et al. 2008).
The activity of Tat reinforces at multiple points the pathophysiological interface
of KSHV with the host cell (Fig. 24.3). Secreted Tat induces chemotactic migration
and invasion of the endothelium by leukocytes (Lafrenie et al. 1996) and fosters
growth within the tumor microenvironment. HHV-8-infected spindle cells produce
basic fibroblast growth factor (bFGF), and cytokines released by HIV-1-infected
cells upregulate bFGF further, which bolsters cell proliferation and angiogenesis
(Ensoli et al. 1994). HIV-1 Tat (Rautonen et al. 1994; Zauli et al. 1993) and Nef
(Olivetta et al. 2003) cause increase in serum IL-6 levels (Honda et al. 1990), which
promotes HIV-1 replication (Poli et al. 1990). HHV-8 also upregulates IL-6 in
infected cells and expresses vIL-6, and the further augmentation in IL-6 caused by
HIV-1 coinfection may contribute to greater severity of KS.
Tat itself induces invasion and proliferation of spindle cells and endothelial cells
through multiple paracrine mechanisms (Fig. 24.3). Tat activates cellular integrins,
stimulates VEGF-A receptor fetal liver kinase-1 (Flk-1), activates NFAT-1 and -2,
boosts levels of soluble bFGF, stimulates expression of matrix metalloproteases
(MMPs), and induces lytic replication of HHV-8. These interactions contribute to
angiogenesis and spindle cell proliferation. Tat interacts with cellular integrins
through an arginine, glycine, aspartic acid (RGD) peptide, promoting adherence of
595
HHV-8-infected cells and uninfected endothelial cells (Barillari et al. 1993). Tat
signaling through the VEGF-A receptor Flk-1 enhances spindle cell proliferation
(Ganju et al. 1998) and induces angiogenesis in endothelial cells (Albini et al. 1996).
Furthermore, Tat provides paracrine stimulation of the phosphatidylinositol
3-kinaseRAC-a serine/threonine-protein kinase (PI3K/Akt) pathway that activates
the phosphorylation and nuclear translocation of NFAT-1 and NFAT-2 in HHV-8infected cells, contributing to upregulation of cytokines and growth factors (Pati
et al. 2003).
The activation of NFAT-1 and NFAT-2 by Tat is augmented by the activity of the
viral G-protein-coupled receptor (vGPCR) expressed by HHV-8-infected spindle
cells (Fig. 24.3) (Guo et al. 2004). This receptor is orthologous to the interleukin 8
receptor CXCR2 and signals in the absence of bound ligand. The outcome of this
signaling event is the posttranscriptional activation of transcription factors NF-kB,
NFAT-1, and NFAT-2 in the infected cell (Pati et al. 2003) and activation of hypoxiainducible factor-1a (HIF-1a) resulting in transcriptional activation of VEGF (Sodhi
et al. 2000). The consequences of the redundant upregulation of cytokines and
growth factors, such as VEGF, by HIV-1 and HHV-8 are proliferation and neovascularization of the KS lesion.
Tat provides another redundant activity with HHV-8 by increasing soluble bFGF
in the cellular matrix (Barillari et al. 1999) (Fig. 24.3). Tat competitively binds the
heparin sulfate proteoglycans of the cellular matrix, which increases the concentration of soluble bFGF to induce cell proliferation (Fig. 24.3). Furthermore, bFGF in
spindle and endothelial cells induces the expression of MMPs, and secreted Tat further
stimulates production of MMPs (Toschi et al. 2001; Meade-Tollin et al. 1999). Tat
activates membrane-type-1 MMP, which binds and activates soluble MMP-2, and
inhibits secretion of the membrane-bound tissue inhibitor of metalloproteinase-1
and -2 (Toschi et al. 2001). These activities provide redundant signals for proliferation
of HHV-8-infected cells and angiogenesis in endothelial cells (Barillari et al. 1999).
The paracrine activity of Tat on endothelial cells (Ensoli et al. 1990) induces lytic
replication of HHV-8 (Zeng et al. 2007), attributed to induction of oncostatin M,
hepatocyte growth factor/scatter factor, and interferon-g (Mercader et al. 2000).
In sum, HIV-1 and HHV-8 coinfection results in multiple synergistic virushost
interactions that foster the tumor microenvironment. KS is the malignancy most
responsive to HAART (Carrieri et al. 2003). However, in AIDS patients of the
developed nations, 15% of cancer fatalities remain attributable to KS (Bonnet
et al. 2004).
HHV-8 and HIV-1 Coinfection Is Associated with Presentation

of Rare Lymphoma
NHL in HIV-1 infection is most often associated with coinfection with EBV. However,
a rare DLBL is observed in patients coinfected with HIV-1 and HHV-8: primary
effusion lymphoma (PEL) (Cesarman et al. 1995; Carbone et al. 1996). In some
596
patients, EBV infection may contribute to PEL (Horenstein et al. 1997). Among
patients not infected with HIV-1, PEL makes up only 0.3% of NHL, but among
patients with HIV-1 it makes up 4% of NHL (Gaidano and Carbone 2001). Prognosis
of PEL is poor even in the absence of HIV-1 infection, with a median survival of 6
months (Chen et al. 2007). PEL is refractory to both HAART and chemotherapy.
Similar to the mechanism of KS, IL-6 and other cytokines coordinately stimulate
proliferation of tumor cells and the replication of HIV-1 in PBMC, which promotes
immunodepletion (Honda et al. 1990; Rautonen et al. 1994; Zauli et al. 1993).
HHV-8 and HIV-1 Coinfection Is Associated with Multicentric

Castlemans Disease
Multicentric Castlemans disease (MCD) is not a cancer, but a lymphoproliferative
disorder resulting in the accumulation of cytokine-secreting B cells. Castlemans
disease in the absence of HIV-1 infection is attributed to dysregulation of IL-6 and
presents in unicentric or multicentric forms. In the context of HIV-1 infection, MCD
is associated with HHV-8 infection (Soulier et al. 1995) and is augmented by IL-6
upregulation similarly to KS. This coinfection enhances MCD morbidity and
mortality. MCD is frequently a precursor to HHV-8-positive NHL (Oksenhendler
et al. 2002; Dupin et al. 2000).
Rituximab has become a standard therapy for MCD and depletes proliferating B
cells (Clifford and Demierre 2005). However, rituximab may induce proliferation of
KS lesions by reactivation of HHV-8 (Pantanowitz et al. 2008), an important consideration since 75% of patients with HIV-1-associated MCD develop concurrent KS
(Oksenhendler et al. 1996). Unlike PEL, MCD may respond to treatment with HAART
alone (Lee et al. 2010). In the pre-HAART era, mortality was 7085% (Oksenhendler
et al. 1996). In the post-HAART era, MCD mortality is reduced to about 30% (Mylona
et al. 2008). However, for unknown reasons, the incidence of MCD has risen during
the HAART era, from 2.3 (95% CI 0.024.2) per 10,000 patients years to 8.3 (95% CI
4.612.6) per 10,000 patient years (Powles et al. 2009).
HIV-1 and EBV Coinfection Is Prominent in B-Cell Lymphoma

NHL was classified as an AIDS-defining malignancy in 1987 by the Centers for
Disease Control and Prevention (Centers for Disease Control and Prevention 1987).
NHL is not a discrete disease, but a heterogenous group of over 40 different cancers,
as classified by the World Health Organization International Agency for Research
on Cancer in 2008 (The International Agency for Research on Cancer 2008). The
NHLs occurring in HIV-1 patients overlap with the classifications of the general
population, but are more likely to be high-grade B-cell lymphomas. In persons not
597
infected by HIV-1, most NHLs are EBV negative while most NHLs occurring
with HIV-1 infection are EBV positive. The two most common forms of NHL
among people with HIV-1 are BL and DLBL, both associated with EBV (Engels
et al. 2008). Individuals with HIV-1 also are more likely to develop unusual
lymphomas, such as PCNSL, which is almost universally associated with EBV
(MacMahon et al. 1991). During the clinical progression of HIV-1, lower CD4+
T-cell counts are associated with EBV-associated NHL (Fig. 24.2) (Jellinger and
Paulus 1995; Cingolani et al. 2000).
The majority of adults worldwide are infected with EBV, which is a ubiquitous
B-lymphotropic herpesvirus (human herpesvirus-4) that establishes lifelong infection
(Henle and Henle 1979). Normally, EBV infects nave B cells, induces differentiation to memory B cells, then becomes latent, and escapes immune detection. Upon
differentiation of a memory cell into a plasma cell, EBV replication resumes. EBV
is postulated to contribute to lymphoma when non-nave cells are infected, and
instead of escaping the cell cycle by differentiation into memory B cells continue to
replicate (Thorley-Lawson and Gross 2004).
In vitro infection of B cells by EBV leads to the induction of somatic hypermutation and accumulation of mutations in proto-oncogenes p53 and B-cell lymphoma 6
(Epeldegui et al. 2007). Genomic instability can result from the expression of two
nuclear antigens (EBNA-1 and EBNA-3C) and latent membrane protein-1 (LMP-1)
(Gruhne et al. 2009). EBNA-1 leads to the production of DNA-damaging reactive
oxygen species by upregulating NADPH oxidase 2 (Nox2) while LMP-1 abrogates
the G2 checkpoint (Gruhne et al. 2009). EBNA-3C inactivates the mitotic spindle
checkpoint, potentially leading to aneuploidy (Gruhne et al. 2009). Thus, infection of
non-nave B cells followed by unrestricted replication and accrual of activated oncogenes and aneuploidy contribute to the development of NHLs. The major role of
HIV-1 in the development of all forms of NHL appears to be immune dysfunction.
IL-10 Has a Prominent Role in HIV-1-Related NHL

A second role for HIV-1 in the development of NHL is the dysregulation of IL-10,
in particular in subtypes of B-cell NHL, including BL (Ogden et al. 2005), largecell lymphoma (Marsh et al. 1995), mantle cell lymphoma (Visser et al. 2000),
and chronic lymphocytic leukemia (Fayad et al. 2001). In AIDS-related B-cell
lymphoma, serum IL-10 has been found to be elevated prior to the development
of lymphoma, and is associated with a particular polymorphism in the IL-10
promoter (Breen et al. 2003). The risk for B-cell NHL was shown to be dependent
upon polymorphisms in the IL-10 gene while other cytokines were not implicated
(Wong et al. 2010). HIV-1 infection results in increased IL-10 serum levels
(Ameglio et al. 1994) while treatment with HAART decreases serum IL-10 levels
(Stylianou et al. 1999), suggesting a potential role for HIV-1 dysregulation of
IL-10 in NHL.
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HIV-1 Contributes to NHL by Impairing

Anti-EBV Cytotoxic T Lymphocytes
A longitudinal study of a small sample of 13 individuals with HIV-1 and EBV
coinfection showed a decrease in the population of HIV-1-specific CTL over the
study period while levels of EBV-specific CTL typically remained steady (Kersten
et al. 1997). However, in four out of five patients that developed diffuse large-cell
lymphoma, EBV-specific CTL dropped in conjunction with a rise in EBV viral load
(Kersten et al. 1997). A more recent study found similar trends, but additionally that
in patients who developed NHL CTL gradually lost the ability to produce IFN-g in
response to EBV even as CTL levels remained unchanged, and this loss paralleled
a trend of decreasing CD4+ T-cell count (van Baarle et al. 2001). Others have found
that EBV-specific CTL in people with HIV-1 infection and CD4+ counts below
350/mL have 1.5-fold decreased polyclonality and recognize fewer EBV epitopes
(Legoff et al. 2004). As part of the ongoing immune dysregulation caused by HIV-1
infection, EBV CTL response may be impaired, leading to loss of immune control
of EBV and eventual NHL.
HIV-1 and HPV Coinfection Contributes to Cervical,

Anogenital, and Oropharyngeal Malignancies
The primary cause of ICC is infection with HPV strains HPV-16 or HPV-18 (Lorincz
et al. 1987). HPV infects undifferentiated keratinocytes, and viral replication
begins upon differentiation and ceases when cells exit the cell cycle. The early 6
(E6) and E7 viral oncogenes of HPV induce deregulation of cell growth. E6
promotes ubiquitin-mediated degradation of p53 (Scheffner et al. 1990) while E7
binds retinoblastoma proteins and promotes their ubiquitin-mediated degradation
(Dyson et al. 1989), releasing E2F/DP-1 transcription factors. While the HPV
genome is initially present in nuclei as an episome, integration into the host genome
can occur. Integration typically disrupts the reading frame of E2, which represses
transcription of E6 and E7. Disruption of E2 is associated with a worse prognosis
(Kalantari et al. 1998), usually attributed to loss of control of E6 and E7 expression;
however, cervical intraepithelial neoplasia can develop in the presence of an intact
E2 (Sathish et al. 2004). Another contributing factor is posttranscriptional upregulation of E6 and E7 by the deletion of an AU-rich element in the 3 untranslated region
(Jeon and Lambert 1995). An increase in E6 and E7 mRNA stability engenders a
cell growth advantage (Jeon et al. 1995) and permits increased progression to
neoplasia (Kulmala et al. 2006; Hudelist et al. 2004).
HIV-1 infection increases a womans risk of development of squamous intraepithelial lesions by fivefold compared to sociodemographically matched HIV-1negative women (Ellerbrock et al. 2000). HIV-1-infected women exhibit more
aggressive and treatment-refractory cervical cancer than women free of HIV-1
599
(Maiman et al. 1993). The increased severity of cervical carcinoma in HIV-1 infection
is attributed to impaired immune function. High HIV-1 viral load and low CD4+
T-cell counts are associated with higher frequency and greater severity of HPV
lesions (Davis et al. 2001; Cardillo et al. 2001; Harris et al. 2005). Unlike KS and
NHL, the frequency of occurrence of ICC has decreased only modestly from treatment with HAART (International Collaboration on HIV and Cancer 2000). Without
HAART treatment, premalignant lesions fail to regress and are associated with
greater HIV-1 viral load and lower CD4+ T-cell count (Ahdieh-Grant et al. 2004)
while HAART treatment contributes to regression of premalignant lesions (AhdiehGrant et al. 2004; Heard et al. 2002).
However, HPV infection may persist with HAART even as lesions regress
(Heard et al. 1998; Minkoff et al. 2001). Persistence of HPV infection is associated
with the development of cervical cancer (Rodriguez et al. 2008). The impact of
HIV-1 on the duration of productive infection by HPV is controversial. Some studies
have found delayed clearance of HPV infection (Sun et al. 1997; Moscicki et al.
2004; Ahdieh et al. 2001) while others found little difference in times to clearance of
HPV infection (Koshiol et al. 2006). Besides its role in impairing immune function,
HIV-1 coinfection may contribute directly to HPV replication through stimulation
by Tat. HIV-1-infected cells can be detected within cervical carcinoma lesions
(Vernon et al. 1994) and Tat can induce expression of E6 and E7 in tissue cultures
of HPV-16-infected oral keratinocytes, resulting in cellular proliferation (Kim et al.
2008). In HPV-18-infected HeLa T4 cells, exposure of cells to extracellular Tat
induced transcription of E1 and late 1 (L1) and a rise in L1 capsid protein levels
(Dolei et al. 1999). Therefore, HIV-1 coinfection may promote reactivation of HPV.
The possible modulation of miRNA signatures by HIV-1 gene products and their role
in the reactivation process remains to be determined. Nuovo and colleagues have
identified a strong inverse correlation between microRNA-125b and productive
HPV infection (Nuovo et al. 2010). MicroRNA-125b is abundantly expressed at the
site of infection, the transformation zone epithelia. Marked reduction in miR-125b
was observed in productively infected cells and correlated with expression of the
HPV late gene product, L2. HIV-1 infection induces marked downregulation of
microRNA-125b in lymphocytes (Hayes et al. 2011) and may promote productive
HPV infection by a paracrine signaling mechanism.
The role of HPV infection in other cancers is being increasingly appreciated. Of
greatest note for HIV-1 infection is HPV-associated anal squamous cell carcinoma
(ASCC). HIV-1 infection is associated with a greater risk of ASCC (Fig. 24.2)
(Engels et al. 2008). Similarly to ICC, the impact of HAART on HPV-associated
ASCC is controversial (Bower et al. 2004; Hawes et al. 2002; Wilkin et al. 2004).
Oropharyngeal cancer is associated with HPV infection (Gillison et al. 2000) and
the incidence of oropharyngeal cancer is increased in HIV-1 infection (Fig. 24.2)
(Engels et al. 2008). Environmental factors, such as a higher incidence of smoking
in the HIV-1-positive sample, may contribute to this increase, but severity of disease
has also been found to increase with increasing immunosuppression (Gillison 2009).
Similarly to CIN and ASCC, treatment with HAART did not affect the incidence of
oropharyngeal cancer (Gillison 2009).
600
HIV-1 and HCV Coinfection Leads to More Rapid Progression

of Liver Fibrosis
Persistent HCV infection can result in liver fibrosis and the eventual development of
HCC (Saito et al. 1990). Coinfection by HCV and HIV-1 is a growing concern, since
they share similar transmission routes. HCV is easily spread through sharing needles
for injection drug use, and also may be spread through sexual practices, especially
among those who are already infected with HIV-1 (Tohme and Holmberg 2010).
HIV-1 has a strong influence upon the course of HCV infection, leading to greater
HCV viremia (Beld et al. 1998; Eyster et al. 1994) and more rapid fibrosis (Benhamou
et al. 1999). Another study found fibrosis rate increased with increasing HIV-1 viral
load or with CD4+ T-cell counts of <500 cells/mL (Brau et al. 2006). HAART has
been shown to counteract this more rapid progression of fibrosis, producing similar
fibrosis progression to patients not infected by HIV-1 (Brau et al. 2006). Consistent
with this finding of more rapid fibrosis, patients with HIV-1 and HCV coinfection
develop cirrhosis more rapidly than those without HIV-1, with 15% of patients with
coinfection developing cirrhosis by 10 years after HCV infection compared to 2.6%
in those with only HCV infection (Soto et al. 1997). Furthermore, patients coinfected with HCV and HIV-1 develop HCC earlier than those uninfected by HIV-1,
with patients with coinfection developing HCC at 18 3 years compared to 28 11
years for patients with only HCV (p < 0.05) (Garcia-Samaniego et al. 2001). HCVassociated end-stage liver disease is an increasing cause of mortality in people with
HIV-1 and is a leading non-AIDS cause of death (Salmon-Ceron et al. 2005;
Rosenthal et al. 2003; Macias et al. 2002; Weber 2006).
While HIV-1 coinfection appears to increase the severity of HCV infection, HCV
infection may also impact the clinical progression of HIV-1 infection. Patients infected
with HCV and HIV-1 have a greater risk of progression to AIDS or death, with a hazard
ratio of 1.7 (Greub et al. 2000). The authors also found that in patients coinfected with
HCV and HIV-1 there was a 21% lower likelihood of an increase of at least 50 cells/
mL in CD4+ T-cell counts after 1 year of HAART (Greub et al. 2000). A complicating
factor is the high frequency of intravenous drug use among people with HIV-1 and
HCV coinfection, which also may contribute to adverse effects (Greub et al. 2000).
The advent of HAART has caused a decline in the rate of death in those infected
with HIV-1 by a factor of 10 (Palella et al. 1998; Mocroft et al. 2002). The increase
in life span of those infected with HIV-1 permits chronic conditions to exert their
effect. The outcome in the case of HCV infection is an increase in the rates of
morbidity and mortality due to liver disease. This unintended consequence presents
a new clinical challenge in the management of HIV-1 infection.
Cancer Risk Is Not Elevated in HIV-1 and HTLV-1 Coinfection

Coinfection with HIV-1 and HTLV-1 or HTLV-2 commonly occurs with intravenous
drug use (Casoli et al. 2007). Since adult T-cell leukemia due to HTLV-1 infection
601
develops after decades, prolonged survival of HIV-1-infected patients due to

HAART may reveal increased cancer risk in coinfected individuals, whereas prior
to HAART, coinfected individuals would have succumbed to AIDS. Interestingly,
while patients with HIV-1 and HTLV-1 coinfection have higher CD4+ T-cell counts,
this does not appear to be associated with decreased immunosuppression (Schechter
et al. 1997). However, HTLV-1 coinfection does not result in increased HIV-1 viral
load (Harrison et al. 1997). Moreover, HIV-1 and HTLV-1 coinfection does appear
to be associated with greater HTLV-1-related morbidity, with increased risk of
myelopathy (Harrison and Schechter 1998).
The alternative tropism of HTLV-2 may have a protective effect against HIV-1
progression. HTLV-1 infects CD4+ cells while HTLV-2 infects CD8+ cells and
causes their clonal expansion. HTLV-2 also induces the expression of CCR5-binding
chemokines (Lewis et al. 2000). Competition by these chemokines with HIV-1 virions
for CCR5 binding plus clonal expansion of HIV-suppressing CTL could contribute
to slower HIV-1 infection progression (Casoli et al. 2007).
Perspectives on the Future

In the clinic, increased risk of malignancy is a feature of HIV-1 infection. Screening
of cancer patients for HIV-1 infection is not the current standard of care even though
cancer is an AIDS-defining condition (Chiao et al. 2010). In 2006, the Centers for
Disease Control and Prevention recommended HIV-1 screening as a standard test
offered in all health care settings, given with patient choice to opt out. Presently,
this practice is typically limited to emergency departments and pregnant women.
However, detection of HIV-1 infection has the potential to significantly improve
clinical outcomes in an HIV-1-infected cancer patient. Treatment by HAART in
synch with appropriate management of chemotherapeutic regiments is important to
manage clinical outcome.
In the biomedical research setting, HIV-1 provides a natural model system to
investigate the fundamental connection between the human immune response and
neoplastic progression. The admixture of innate, adaptive, and cell-mediated
responses to HIV-1 during coinfection with EBV, HHV-8/KSHV, or HPVs provides
a vast, interrelated network of intricate interactions that can culminate in perilous
cell growth. Connections are wired between and among cell lineages by cytokine
signaling, paracrine interactions, and secondary effects of immune disbalance. Two
frontiers of ongoing exploration are apparent and their paths are likely to converge.
Progress is underway to characterize the innate response to HIV-1 and other chronic
virus infections, including HCV. Notably, coinfection provides an added dimension
to view the innate response. A less-developed frontier is to define host microRNA
signatures of HIV-1 and other chronic virus infections, to determine their potential
interfaces, and to understand their contribution to neoplastic growth. The convergence of these paths is likely to build new bridges from the bench to the bedside.
602
Acknowledgments We appreciate the assistance of Mr. Tim Vojt on the illustrations, Dr. Kate
Hayes-Ozello for comments on the document, and the support of the National Institutes of Health
for National Cancer Institute RO1CA108882 and P30CA100730 to KBL; P01CA16058 to the
Ohio State University Comprehensive Cancer Center.
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Chapter 25
HTLV-1 and Oncogenesis

Chou-Zen Giam
Introduction
Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell
leukemia/lymphoma (ATL), a malignancy of CD4+ T cells that develops in 35% of
infected individuals over a course of several decades (Taylor and Matsuoka 2005;
Matsuoka and Jeang 2007). It was first isolated in 1980 from Hut102, a T cell line
established from a T-cell lymphoma patient (Poiesz et al. 1980; Gallo 2005). Shortly
after its discovery, HTLV-1 became causally linked to ATL, a leukemia first
described in 1977 in Japan and etiologically associated with a virus (Uchiyama
et al. 1977; Takatsuki 2005). Epidemiological studies indicate that HTLV-1 infection
is endemic in parts of the Caribbean basin, the southern islands of Japan Kyushu
and Okinawa, parts of Africa, South America, and the Pacific islands of Melanesia
and Papua New Guinea (Gessian et al. 1991; Wong Staal and Gallo 1985). To date,
HTLV-1 remains the only retrovirus associated with human malignancy. HTLV-1
infection is highly cell-associated and largely T-cell tropic. The virus preferentially
infects and transforms CD4+ T cells. HTLV-1 is transmitted predominantly via
breast milk, sex, and transfusion of blood products containing HTLV-1-infected
cells. The diseases caused by HTLV-1, namely, adult T-cell leukemia (ATL), HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-1 uveitis,
and other inflammatory skin diseases have their etiologies in the dysregulated proliferation of T-cells and/or ensuing immune dysfunctions. Presently, there is no effective
treatment for HTLV-1-associated diseases (Uozumi 2010; Lezin et al. 2007).
C.-Z. Giam (*)

Department of Microbiology and Immunology, Uniformed Services University
of the Health Sciences, Bethesda, MD 20814, USA
e-mail: cgiam@usuhs.mil
613
C.-Z. Giam
614
HTLV-1 Genome and Replication

HTLV-1 is a complex delta retrovirus that encodes in addition to viral structural
proteins, MA (matrix), CA (capsid), NC (nucleocapsid), RT (reverse transcriptase),
PR (protease), IN (integrase), SU (surface) and TM (transmembrane) glycoproteins,
six accessory proteins: Tax (transactivator encoded by the X region), Rex (regulator
of gene expression encoded by the X region), HBZ (HTLV-1 bZip protein), p12I,
p13II, and p30II. HTLV-1 expresses eight major mRNA species in the sense orientation and 1 mRNA species in the antisense orientation (Fig. 25.1). The unspliced
HTLV-1 mRNA, like that of other retroviruses, serves as the genomic RNA and the
mRNA that encodes Gag (MA-CA-NC) and Gag-Pol (MA-CA-NCRT-IN) polyproteins. The major singly spliced mRNA encodes the envelope (Env) glycoprotein
precursor that is cleaved by a cellular protease to produce the SU and TM proteins.
Tax and Rex are encoded by a doubly spliced mRNA. Tax is a potent activator
of HTLV-1 mRNA transcription, while Rex regulates the transport of the unspliced
and the singly spliced or incompletely spliced viral mRNA to the cytoplasm. In
the early stage of viral replication, cellular transcription factors activate the
synthesis of viral mRNA, which is first doubly spliced to produce Tax/Rex transcript.
Fig. 25.1 Genomic organization of HTLV-1 proviral DNA and viral mRNA trasnscripts. The open
reading frames for Gag, Pro, Pol, Env, Tax, Rex, p12I, p30II, p13II, and HBZ genes are as indicated. The splice donor and acceptor sites in HTLV-1 mRNAs are marked by black and gray triangles.
The nucleotide positions of the splice sites are indicated
25
615
Tax, in turn, augments viral mRNA transcription, while the accumulation of Rex
causes viral mRNA to be diverted to the unspliced and incompletely spliced forms
for the production of viral structural proteins necessary for virus assembly. P30II preferentially sequesters Tax/Rex mRNA in the nucleus, thus downregulating Tax/Rex
expression (Nicot et al. 2004), while HBZ antagonizes the transactivation functions
of Tax to dampen viral mRNA transcription (Gaudray et al. 2002; Lemasson et al.
2007; Basbous et al. 2003). By downregulating Tax expression and Tax activities
respectively, p30II and HBZ are thought to modulate viral replication and possibly
facilitate the establishment of viral latency. The roles p12I and p13II play in viral
life cycle are not fully understood.
HTLV-1 Accessory Proteins and Their Functions

HTLV-1 Tax Functions
Tax is a 40-kDa protein that resides in both the nucleus and cytoplasm and has both
nuclear and cytoplasmic functions.
Tax Activates Viral mRNA Transcription

Tax is a potent activator of HTLV-1 viral mRNA transcription. The mechanism by
which Tax activates viral transcription is depicted in Fig. 25.2. The viral transcriptional enhancer in the U3 region of the long terminal repeats (LTR) consists of three
imperfect 21-bp repeats, each containing a cAMP response element (CRE) core
flanked by 5 G-rich and 3 C-rich sequences. The CREs in the 21-bp repeats are
bound by the cellular basic domain-leucine zipper (bZip) transcription factors
CREB and ATF-1, and possibly other CREB/ATF-like proteins. CREB/ATF-1 and
the 21-bp repeats, in turn, recruit Tax into stable ternary complexes (Armstrong
et al. 1993; Wagner and Green 1993; Yin and Gaynor 1996a; Zhao and Giam 1991;
Suzuki et al. 1993) in which Tax binds the basic domains of CREB/ATF-1 (Adya
et al. 1994; Yin et al. 1995; Baranger et al. 1995; Perini et al. 1995) and makes contacts with the DNA minor groove of the G/C-rich sequences that flank the CRE, thus
achieving the exquisite DNA sequence specificity of Tax-mediated LTR transactivation (Paca Uccaralertkun et al. 1994; Yin and Gaynor 1996b; Lenzmeier et al. 1998;
Tang et al. 1998; Kimzey and Dynan 1998, 1999; Lundblad et al. 1998; Datta
et al. 2000). In the context of the ternary Tax-CREB/ATF-1-21 bp repeat complex,
Tax further recruits transcriptional coactivators, CREB binding protein (CBP)/p300
and, possibly, p300-CBP associated factor (P/CAF) for potent gene activation
(Kwok et al. 1996; Lenzmeier et al. 1998; Harrod et al. 1998, 2000; Bex et al. 1998;
Jiang et al. 1999). Recent studies have shown that transcriptional coactivator, transducers of regulated CREB (TORCs), also participate in the high-order nucleoprotein
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HTLV-I 21-bp TaxREs:
AAGGCCC TGACGTGT CCCCCT
TAGGCTC TGACGTCT CCCCCC
CAGGCGT TGACGACA ACCCCT
CREB/ATF-1
Nucleosome
21bp-repeat
Tax
TORC
Interactions with other
cellular factors?
P/CAF
CBP/p300
Fig. 25.2 Activation of HTLV-1 transcription by Tax. The viral transcriptional enhancer in the U3
region of the long terminal repeats (LTR) consists of three imperfect 21-bp repeats, each containing a cAMP response element (CRE) core flanked by 5 G-rich and 3 C-rich sequences. The CREs
in the 21-bp repeats are bound by the cellular basic domain-leucine zipper (bZip) transcription
factors CREB and ATF-1. CREB/ATF-1 and the 21-bp repeats, in turn, recruit Tax into stable
ternary complexes in which Tax binds the basic domains of CREB/ATF-1 and makes contacts with
the DNA minor groove of the G/C-rich sequences that flank the CRE, thus achieving the exquisite
DNA sequence specificity of Tax-mediated LTR transactivation. In the context of the ternary TaxCREB/ATF-1-21bp repeat complex, Tax further recruits transcriptional coactivators, CREB binding protein (CBP)/p300, TORCs, and p300-CBP associated factor (P/CAF) for potent gene
activation. Recent data indicate that Tax establishes a nucleosome-free region in the LTR
complex assembled on the 21-bp repeats (Siu et al. 2006; Nyborg et al. 2009). This
complex creates a nucleosome-free region in the viral LTR that facilitates mRNA
transcription (Sharma and Nyborg 2008). Whether ATP-dependent chromatin
remodeling factors such as BRG1 is recruited by Tax to the HTLV-1 promoter is a
matter of debate (Zhang et al. 2006; Easley et al. 2010). In the presence of Tax, gene
expression driven by multiple copies of the 21-bp repeat element can increase up to
100-fold or higher.
Tax Activates IKK/NF-kB

HTLV-1-transformed cell lines are known to express in abundance a wide variety of
cytokines and cytokine receptors including IL2 receptor alpha chain. This is largely
because Tax potently and constitutively activates NF-kB. NF-kB/Rel family of transcription factors are controlled by inhibitory I-kB proteins I-kBa, I-kBb, and the
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I-kB-like domains in NF-kB1 and NF-kB2 that sequester NF-kB/Rel in the

cytoplasm as multiprotein complexes (for reviews, see Hayden and Ghosh 2008).
Upon activation by extracellular stimuli such as interleukin-1 (IL-1), tumor necrosis
factor-a (TNF-a), bacterial lipopolysaccharide (LPS), or Tax, I-kBa and I-kBb
become serine phosphorylated by I-kB kinase (IKK). This marks them for polyubiquitination and rapid degradation by proteasome-mediated proteolysis. The degradation of I-kB results in heightened nuclear levels of NF-kB and increased
expression of a plethora of cellular genes under NF-kB regulation, including the
genes of many cytokines and their receptors, adhesion molecules, and immune
modulators (Beg and Baldwin 1993; Liou and Baltimore 1993; Siebenlist et al. 1994).
Dysregulation and/or hyperactivation of the NF-kB/I-kB regulatory pathway as
caused by chromosomal translocation (Neri et al. 1991), oncogene transduction
(Gilmore 1992), viral infection (as in the case of HTLV-I), or targeted gene disruption
(Beg et al. 1995; Klement et al. 1996) leads to cancers of the hematopoietic cells or
chronic inflammatory diseases.
IKK, the Ser/Thr kinase that phosphorylates I-kBa and targets it for lysine-48
(K48) polyubiquitination and proteasome degradation, is the focal point of multiple
signaling pathways that lead to NF-kB activation. The IKK holoenzyme consists of
two catalytic subunits, IKKa and IKKb, together with a regulatory subunit, IKKg/
NF-kB essential modulator (NEMO, referred to as NEMO herein). Tax activates
IKK constitutively (Sun et al. 1994; Good and Sun 1996; Sun and Ballard 1999; Xiao
and Sun 2000; Chu et al. 1998, 1999). This is due primarily to a direct interaction
between Tax and NEMO (Chu et al. 1999; Jin et al. 1999; Xiao and Sun 2000),
which results in constitutive activation of IKKa and IKKb, degradation of all I-kBs, and
activation of both classical and alternative NF-kB pathways (Chu et al. 1999; Jin
et al. 1999; Xiao and Sun 2000). While NEMO is essential for IKK activation, the
mechanism by which NEMO controls IKK activity remains to be fully elucidated.
Both IKKb and NEMO undergo extensive Lys-63 (K63) polyubiquitination
mediated by E3 ubiquitin ligases such as TRAF2 and TRAF6, and a E2 ubiquitinconjugating enzyme, UBC13/UBE2V1 heterodimer (Deng et al. 2000). K63 polyubiquitination is essential for IKK activation. It is thought that K63 polyubiquitin
recruits the TGF-b activated kinase 1 (TAK1) by interacting with TAB2, the ubiquitin receptor subunit of TAK1, to phosphorylate and activate IKK (Leibovitz et al.
1973; Xia et al. 2009). TAK1 has also been reported to interact with Tax and to be
necessary for Tax-mediated NF-kB activation (Wu and Sun 2007). As might be
expected, Tax is extensively modified posttranslationally by phosphorylation, ubiquitination, and sumoylation (Peloponese et al. 2004; Lamsoul et al. 2005). The activating posttranslational modifications of NEMO and IKK, including K63
polyubiquitination of NEMO and monoubiquitination of IKK, are highly induced by
Tax (Carter et al. 2005). Recent data suggest that UBC13/UBE2N associates with
both Tax and NEMO and is critical for their K63 polyubiquitination and Taxmediated IKK activation (Shembade et al. 2007). The importance of K63 polyubiquitination of NEMO in IKK/NF-kB activation has been challenged recently, however
(Tokunaga et al. 2009; Iwai and Tokunaga 2009). Deletion of UBC13 gene in mice
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618
did not affect NF-kB activation significantly (Yamamoto et al. 2006a, b). Furthermore,
head-to-tail linear polyubiquitination of NEMO by the linear ubiquitin-chain assembly complex (LUBAC), which consists of two RING finger proteins HOIL-1L and
HOIP, has been shown to be critical for NF-kB activation via the TNF-a and IL-1b
signaling pathways (Tokunaga et al. 2009). Whether and how Tax interacts with the
E2 and E3 enzymes that carry out either K63 or linear polyubiquitination of NEMO
would be topics of significant interest. Finally, Tax also activates transcription of
serum-response factor-regulated genes by directly interacting with SRF (Fujii et al.
1992). Tax is thought to be the key viral factor responsible for ATL development.
Rex Functions
HTLV-1 Rex is a 27 kDa nuclear phosphoprotein (Younis and Green 2005). It binds
viral RNAs via an RNA element known as the Rex-response element (RxRE) that
resides in the R region of the viral mRNA (Younis and Green 2005). The argininerich region in the NH2-terminus of Rex functions as an RNA-binding motif. Rex
also contains both nuclear localization and nuclear export signals and shuttle
between the nucleus and the cytoplasm. Like HIV Rev, Rex forms homooligomer/
polymer as a part of its interaction with RNA (Younis and Green 2005).
p12I, p13II, and p30II Functions

As indicated in Fig. 25.1, the region between Env and Tax/Rex open reading frames
contains multiple genes in both directions: p12I, p13II, and p30II in the sense
orientation and HBZ in the antisense orientation. p12I, a membrane-associated
protein, appears to play a role in enhancing T cell activation and signaling (Ding
et al. 2003), although contradictory findings have also been described (Fukumoto
et al. 2007). A recent article has reported p12I-mediated downregulation of ICAM-1
and ICAM-2, which is thought to mitigate autologous natural killer cell cytotoxicity
for the infected CD4+ T cells (Banerjee et al. 2007). The p13II is an inner mitochondrial membrane protein with antiproliferation activity (Silic-Benussi et al. 2004).
Its role in HTLV-1 infection and replication is not entirely clear at present. The antiproliferation activity of p13II appears to be related to an interaction with farnesyl
pyrophosphate synthetase and alteration of Ras-mediated apoptosis in T lymphocytes (Hiraragi et al. 2005). P30II is a nuclear and nucleolar protein that functions
as a posttranscriptional modulator of viral replication (Nicot et al. 2004). Data
suggest that p30II retains the doubly spliced Tax/Rex mRNA in the nucleus and
thereby downmodulates viral gene expression by reducing the levels of Tax and Rex
(Nicot et al. 2004). It has also been reported to interact with CBP/p300 and interfere
with LTR transactivation by Tax (Zhang et al. 2001).
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HBZ Functions
As shown in Fig. 25.1, the 3 region of HTLV-1 encodes an mRNA of opposite
polarity to the major HTLV-1 mRNA transcript. This antisense transcript spans the
open region that resides between the env and the tax/rex ORFs, and encodes a basic
domain-leucine zipper protein known as the HTLV-1 b-Zip protein (HBZ) (Gaudray
et al. 2002). The HBZ transcript exists in both unspliced and singly spliced forms,
with the latter representing the dominant species. The spliced HBZ mRNA transcript
does not contain sequences complementary to the Tax/Rex mRNA and, therefore, is
not expected to affect Tax/Rex mRNA by RNA interference (Cavanagh et al. 2006;
Satou et al. 2008). The singly spliced HBZ mRNA encodes a 30 kDa basic domainleucine zipper protein (Gaudray et al. 2002; Clerc et al. 2008). Earlier studies have
indicated that HBZ could downregulate Tax-mediated transactivation of the HTLV-1
LTR by binding CREB/ATF (Gaudray et al. 2002; Lemasson et al. 2007; Basbous
et al. 2003). Interaction between HBZ and CBP/p300 also contributes to downregulation of Tax-mediated LTR transactivation (Clerc et al. 2008). HBZ also interacts
with other bZip proteins including Jun and Jun D (Basbous et al. 2003). Most
recently, HBZ has been shown to selectively inhibit the classical NF-kB pathway by
binding p65 RelA to prevent NF-kB binding to DNA and by facilitating ubiquitinmediated degradation of RelA (Zhao et al. 2009). Most intriguingly, the HBZ mRNA
is widely expressed in ATL cells in contrast to the Tax/Rex mRNA (Saito et al. 2009);
and the HBZ mRNA, but not the HBZ protein, has been reported to stimulate cell
proliferation. This raises interesting questions about HBZs role in ATL maintenance (Satou et al. 2008). The p12I, p13II, and p30II proteins all have been reported
to be critical for HTLV-1 infection in vivo in a rabbit model (Collins et al. 1998;
Hiraragi et al. 2006; Silverman et al. 2004). These earlier results, however, should
be interpreted with caution because mutations introduced into all three open reading
frames could have also altered the HBZ coding sequence.
Cell-Associated Transmission of HTLV-1

HTLV-1 infection is highly cell associated. Cell-to-cell contact is required for the
infection of nave cells. It has been shown that the spread of HTLV-1 occurs through
virological synapses (Igakura et al. 2003) formed between integrin LFA1 of virusproducing cells and intracellular adhesion molecule ICAM1 of target cells (Barnard
et al. 2005; Liu et al. 2006). The ubiquitously expressed glucose transporter 1 (GLUT1)
serves as the receptor for HTLV-1 (Manel et al. 2003). Other cell surface molecules
such as heparin sulfate proteoglycan and neuropilin-1 appear to facilitate viral
attachment and infection as well (Lambert et al. 2009). Curiously, while cells of
many types can be infected by HTLV-1, most are not capable of spreading the infection
further, suggesting that they lack critical cellular factors important for becoming
competent HTLV-1 donors. Recent data have indicated that free HTLV-1 particles
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are assimilated by dendritic cells and transmitted to target cells in biofilm-like

extracellular viral assemblies (Jones et al. 2008; Pais-Correia et al. 2010). While the
T cell tropism of HTLV-1 seems to go against the fact that its receptor is ubiquitously expressed, the dependence of HTLV-1 transmission on dendritic cells, which
interact intimately with CD4+ T cells, may account in part for this tissue tropism.
Natural History of HTLV-1 Infection

Because the levels of cell-free HTLV-1 virus in infected individuals are low, serum
antibody titer (to viral antigens) and proviral load are used to monitor the natural
history of HTLV-1 infection. De novo viral infection of nave cells and clonal expansion of infected cells contribute to the HTLV-1 proviral load in an infected person.
The former requires reverse transcription and synthesis of viral antigens and, thus,
is likely to be accompanied by higher antibody responses; the latter is driven by
increased division/proliferation of latently infected cells and is usually not accompanied by increased viral antigens or an increased antibody response. The proviral
loads in asymptomatic adults with HTLV-1 infection are, in general, stable over the
years and are maintained at around 5,0009,000 copies/105 PBMCs, while those for
HAM/TSP patients are generally higher at around 22,500 copies/105 PBMCs
(Yamano et al. 2002). Studies of ATL patients in Japan by Okayama et al. have
shown that an increase in proviral DNA load frequently preceded the onset of ATL
(Okayama et al. 2004).
Development of ATL
Oligoclonal Expansion of HTLV-1-Infected Cells
Analyses of the clonality of HTLV-1 proviral DNA by inverse PCR have revealed
that during seroconversion the clonality of the infected T cells in a person infected
through spouse is heterogeneous and remains unstable for several years (Tanaka
et al. 2005). By contrast, major clones were common in long-term HTLV-1 carriers.
Longitudinal study of ATL is difficult logistically for the obvious reasons that ATL
develops decades after viral infection and its frequency of occurrence is low.
Progression to ATL is associated with higher proviral load, advanced age, family
history of ATL, and opportunity for HTLV-1 testing (Iwanaga et al. 2010). ATL
cells show monoclonal patterns of HTLV-1 proviral DNA integration and T cell
receptor beta chain gene rearrangement (Maeda et al. 1985). Longitudinal studies of
HTLV-1 carriers have indicated that virus-infected cells initially undergo polyclonal
or oligoclonal expansion (Okayama et al. 2004). Major HTLV-1-infected cell clones
are maintained in carriers for many years. The infected cell clone that contains the
same site of proviral integration as the leukemic cells can be detected 28 years
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prior to the development of ATL. These results implicate two major events critical
to the onset of ATL: (a) the initial oligoclonal expansion of virus-infected T cells
and (b) their subsequent evolution and acquisition of additional malignant potentials
(Okayama et al. 2004).
Tax is thought to promote the initial clonal expansion of specific infected cells.
Its expression and viral transcription, however, are frequently silenced or greatly
attenuated in most ATL cells (60%) due to mutations and/or methylation of the viral
LTR, or other epigenetic changes (Matsuoka et al. 1997; Takeda et al. 2004;
Taniguchi et al. 2005). Thus, the many oncogenic activities of Tax are not absolutely
needed for maintaining malignancy. This contrasts with the oncoproteins of small
DNA viruses such as human papilloma virus E6 and E7 proteins whose constant
expression is needed to drive cancer cell proliferation. It is thought that the oncogenic
activities of Tax are eventually supplanted through acquisition of genetic or epigenetic
changes that activate IKK/NF-kB, the Jak/Stat pathway, and/or the PI3K-Akt
pathway during the evolution of ATL (Tomita et al. 2006; Takemoto et al. 1997;
Fukuda et al. 2005). Importantly, the HTLV-1 HBZ mRNA transcript appears to be
widely expressed in ATL cells (Saito et al. 2009). How HBZ impacts ATL development remains to be fully elucidated. Intriguingly, HBZ mRNA has been reported to
stimulate T cell proliferation recently (Satou et al. 2006). Thus, HBZ may serve as
the viral determinant for ATL maintenance (Fig. 25.3).
Tumor Suppressors and ATL

The role of tumor suppressors in ATL development has been reviewed previously
(Hatta and Koeffler 2002). One of the most prominent features of ATL cells is the
frequent loss of p16INK4a and p15INK4b, especially from acute/lymphomatous ATL
(Yamada et al. 1997; Hatta and Koeffler 2002; Oshiro et al. 2006). Loss of pRb occurs
occasionally (Hatta and Koeffler 2002), and mutations in p53 gene occur in some
(3040%) but not all acute/lymphomatous ATL cases, while genetic alterations in
p18INK4c, p19INK4d, p21Cip1/Waf1, and p27Kip1 are rare (Morosetti et al. 1995; Oshiro et al.
2006). Stabilization and functional inactivation of p53 by Tax has been described
(Takemoto et al. 2000; Pise-Masison et al. 2000, 2001) and may contribute to the
maintenance of wild-type p53 gene in HTLV-1-infected cells. The loss or inactivation
of tumor suppressors correlates with genetic and chromosomal instability in ATL cells.
Genomic Instability in ATL Cells

Unlike cells of other leukemia, ATL cells, especially those of the acute type, are
often aneuploid with complex numerical and structural chromosomal abnormalities.
No single specific chromosomal gain, loss, translocation, deletion, or rearrangement
has been associated with ATL, however. Fujimoto et al. had previously reported
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622
Fig. 25.3 IKK/NF-kB activation by Tax. IKK is the focal point of multiple pathways that lead to
the activation of NF-kB. The core IKK enzyme consists of two highly homologous catalytic subunits a and b of 85 kDa and 87 kDa in sizes, respectively, and a 48-kDa regulatory subunit, NEMO.
Both IKK-a and IKK-b contain NH2-terminal kinase domains followed by leucine zippers (LZ)
and helix-loop-helix (HLH) domains that mediate protein-protein interactions important for IKK
oligomerization and kinase activity. NEMO also contains extensive helical regions and leucine
zipper domains that are involved in posttranslational modifications and protein-protein interactions. It has been proposed that stimulation of cells with proinflammatory cytokines such as TNF-a
leads to the assembly of TNF receptor (TNFR), TRAF2, and RIP1 complex, and the K63 polyubiquitination of NEMO and RIP1 by TRAF2, a E3 ligase, in association with a E2 Ub conjugating
enzyme UBC13/UBE2N. IKK is recruited to the K63 polyubiquitin chain of RIP1 via the ubiquitin-binding domain of NEMO. NEMO, in turn, becomes K63 polyubiquitinated and recruits
TAK1 (TGFb-activated kinase 1) by interacting with its K63 polyubiquitin-binding subunits
(TAB1/TAB2). TAK1 then phosphorylates and activates IKK (a). Recent data, however, indicate
that IKK activation results from linear polyubiquitination of NEMO by a E3 ligase known as the
linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1L and HOIP RING finger
proteins. Linear polyubiquitin recruits TAK1, IKK (by interacting with NEMO), or other IKK
kinases to phosphorylate and activate IKK (b). Activated IKK phosphorylates I-kBa and causing
it to be K48 polyubiquitinated by the SCFbTrCP E3 ubiquitin ligase and targeted for proteasomemediated degradation. In the absence of its inhibitor, NF-kB translocates to the nucleus and activates genes involved in innate immunity, inflammation, and cell survival (c). Tax directly binds
NEMO and activates IKK. The mechanism by which Tax activates IKK via NEMO binding is not
clear at present, but may involve the K63 or linear polyubiquitin assembly systems
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trisomy 3, trisomy 7, a partial deletion of 6q, and abnormalities of 14q11 in ATL

cells (Fujimoto et al. 1999). Oshiro et al. has recently shown distinct chromosomal
alterations in acute ATL lymphoma type versus leukemia type, with frequent gains
at 1q, 2p, 4q, 7p, and 7q, and losses of 10p, 13q, 16q, and 18p in the lymphoma type,
and a gain of 3/3p in the acute type (Oshiro et al. 2006). CARMA1, a critical subunit
of the CARMA1-Bcl10-Malt1 complex that mediates I-kB kinase activation, was
suggested to be a potential target for recurrent 7p22 amplification in the lymphoma
type (Oshiro et al. 2006). Frequent homozygous deletion of 9p21.3 implicates the
loss of tumor suppressors p16INK4a (CDKN2A) and p15INK4b (CDKN2B) as a critical
determinant in ATL evolution (Yamada et al. 1997; Hatta and Koeffler 2002; Oshiro
et al. 2006). Gain of the 3p26-q26 region characteristic of acute ATLL may involve
the phosphatidylinositol 3-kinase catalytic subunit b gene (PIK3CB2) located at
3q22.3 (Oshiro et al. 2006), which has been implicated in T cell proliferation and
the development of the flower cell morphology of ATL ells (Fukuda et al. 2005).
As Tax compromises DNA damage repair, causes DNA damage, and induces
mitotic abnormalities to cause both genetic and chromosomal instabilities, it is
conceivable that Tax is directly responsible for the genetic changes seen in ATL cells.
The roles of these common gains and losses of genetic information during ATL evolution remain to be elucidated. Whether a single multistep pathway initiated by HTLV-1
infection can adequately explain ATL development is also not clear at present.
In Vitro T-Cell Transformation by HTLV-1

Similarities and Differences Between HTLV-1-Transformed Cells
and ATL Cells
Transformation of primary T cells by HTLV-1 in cell culture shows many of the
characteristics of ATL development albeit with important differences especially
with regards to Tax expression, alterations of cyclin-dependent kinase inhibitors,
and tumorigenic potentials in the severe combined immunodeficiency (SCID)
mouse model (Liu et al. 2002; Richard et al. 2001). A comparison between HTLV1-transformed T cells and ATL cells is shown in Table 25.1. Their differences are
likely to be due to the absence of an antiviral immune response and the continual
inclusion of IL-2 in cell culture for in vitro cell transformation. After infection
by HTLV-1 in culture, specific clones of primary T cells can continue to proliferate
indefinitely in an IL-2-dependent manner (Graziano et al. 1987). Over time, the
immortalized T cells acquire the ability to grow without IL-2 and are said to
be transformed (Miyoshi et al. 1981; Graziano et al. 1987; Ratner et al. 2000).
The transition from IL-2-dependent to IL-2-independent growth in vitro is thought
to resemble the progression of chronic ATL, which is characterized by indolent
lymphocytosis, to acute ATL, an aggressive and rapidly fatal disease. In general, the
IL-2-independent (transformed) T cells express Tax constitutively and, as a result,
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Table 25.1 HTLV-1-transformed T cells vs. ATL cells

HTLV-1 transformed T cells
ATL cells
Constitutive Tax expression and IKK/NF-kB
Frequent loss of Tax expression and
activation
NF-kB activated by Tax-independent
cellular mechanisms
Not tumorigenic in SCID mice due to host
Tumorigenic in SCID mice
immune response
HBZ mRNA detectable
HBZ mRNA invariably expressed
Genomic and chromosomal alterations
Damage to DNA repair mechanisms
Frequent loss of p16INK4a, p15INK4b
Downregulation of p27Kip1, inactivation of p21Cip1
Functional inactivation of p53 by Tax
Mutations in p53 gene occur sometimes
Jak3/Stat5 activation
Jak3/Stat5 activation
their IKK/NF-kB pathway is chronically activated. By contrast, most ATL cells are
Tax-negative. The loss of Tax expression from ATL cells may reflect a strong CTL
response against Tax. The ability of Tax to induce cell cycle abnormalities, genomic
instability (GIN), chromosome instability (CIN), and senescence (Majone et al. 1993;
Marriott and Semmes 2005; Kuo and Giam 2006) likely also contribute to a negative
selection against its continuous expression. Despite the strong evidence implicating
Tax in cell immortalization/transformation, outside the context of viral infection,
Tax alone is poorly capable of immortalizing/transforming primary T cells (Bellon
et al. 2010). Whether other viral regulatory proteins, especially HBZ, may synergize
with Tax in cell immortalization/transformation remains to be determined.
Signaling Pathways Involved in HTLV-1 Transformation

in Cell Culture
Earlier studies have found that transition of HTLV-1-immortalized cells to IL-2independence is associated with constitutive activation of the Jak3/Stat5 pathway
(Migone et al. 1995). Altered expression of Src- and Syk-related tyrosine kinases
has been observed in HTLV-1-transformed T cells (Weil et al. 1999). Downregulation
of the cyclin-dependent kinase inhibitor (CKI), p27Kip1, has also been correlated
with cell transformation (Cereseto et al. 1999). All HTLV-1-transformed T cells
express another CKI, p21Cip1/Waf1, in great abundance (Cereseto et al. 1996; Kuo
and Giam 2006). Interestingly, p21Cip1/Waf1 in these cells is localized to the cytoplasm
and therefore functionally inactive (Liu et al. 2008). Downregulation of p27Kip1 and
functional inactivation of p21Cip1/Waf1 appear to be integral parts of T cell transformation
by HTLV-1 (Cereseto et al. 1996; Kuo and Giam 2006; Liu et al. 2008). It is possible
that the altered activities of Src- and Syk-like tyrosine kinases, the activation of the
Jak3/Stat5 pathway, and the impairment to the expression and function of p27Kip1
and p21Cip1/Waf1 are causally connected. Alternatively, they may evolve independently
but act synergistically to transform T cells. Finally, alterations in p27Kip1 and p21Cip1/
Waf1
in HTLV-1-transformed T cells may be functionally equivalent to the loss of
p16INK4a and p15INK4b in ATL cells.
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Aberrant Telomere Maintenance in ATL and Tax-Expressing Cells

Constitutively high telomerase activity in neoplastic cells prevents the replicative
senescence induced by telomere shortening that occurs during successive cell division
cycles. ATL cells display constitutive telomerase activity (Uchida et al. 1999);
however, since tax gene is often transcriptionally silenced in ATL cells, Tax is
unlikely to play any role in the telomerase activity of ATL cells. Tax, however, has
been found to promote hTERT expression in quiescent cells through NF-kB, but
repress hTERT expression in cells stimulated to proliferate (Sinha-Datta et al. 2004;
Gabet et al. 2003). Tax-mediated downregulation of hTERT was suggested to
promote telomere shortening, fusion of chromosome ends, faulty segregation of the
resulting dicentric chromosomes, and genetic instability (Gabet et al. 2003). Finally,
HBZ has been reported to cooperate with with JunD to enhance hTERT expression
(Kuhlmann et al. 2007).
Animal Models of HTLV-1 Infection

Several animal models exist for the study of HTLV-1. Rabbits, nonhuman primates,
and rats can all be infected by HTLV-1 and are useful for investigating virus spread
and host immune responses. HTLV-1-infected rabbits, cynomolgus macaques, and
squirrel monkeys do not show clinical diseases, however (see Zimmerman et al. 2010
for a recent review). The Wistar-King-Aptekman-Hokudai (WKAH) strain of rats
develops spastic paraparesis with degenerative nerve lesions several months after
HTLV-1 infection and has been used as a model for HAM/TSP (Ishiguro et al. 1992).
A chimeric HTLV-1 with its envelope gene substituted by that of the ecotropic
Moloney murine leukemia virus has also been used successfully to infect mice
(Delebecque et al. 2002, 2005). The infected mice developed humoral and cellular
immune responses against the chimeric virus and showed oligoclonal pattern of
proviral integration, but no disease was reported (Delebecque et al. 2005). Finally,
xenograft transplantation of cells from ATL patients or ATL cell lines into SCID
mice has been useful for propagating tumor cells that are difficult to grow in cell
culture (Feuer et al. 1993). The grafts exhibit many of the features of ATL cells in
patients, including PTHrP expression and increased IL-2Ra levels (Feuer et al. 1993;
Kondo et al. 1993; Liu et al. 2002). Interestingly, HTLV-1-transformed T cell lines
derived in cell culture failed to engraft due to NK-cell-mediated lysis, but were
tumorigenic in SCID/bg and NOD/SCID mice that have reduced natural killer
(NK) cell activity (Liu et al. 2002). These results suggest that the lack of HTLV-1
gene expression in ATL cells likely allows them to evade immune surveillance. The
nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gc-null (NOG)
mouse xenograft model has also been used to study HTLV-1 infection and treatments for ATL (Kondo et al. 1993; Dewan et al. 2003, 2006)
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Oncogenic Activities of Tax: IKK/NF-kB Activation, Cell Cycle,

and Transgenic Mice
Tax-Induced NF-k B Activation and Cell Transformation
The oncogenic activity of Tax in cell culture and in transgenic mice is driven principally
by NF-kB (Grossman et al. 1995; Matsumoto et al. 1997; Yamaoka et al. 1996, 1998;
Hasegawa et al. 2006). In fact, NEMO was cloned as a gene whose loss prevented
Tax-induced foci formation/cell transformation of rat fibroblasts (Yamaoka et al. 1998).
The potent activation of NF-kB by Tax is known to upregulate many antiapoptotic
proteins, including Bcl-xL (Tsukahara et al. 1999; Mori et al. 2001a; Nakashima
et al. 2003) c-FLIP, (Krueger et al. 2006; Okamoto et al. 2006), survivin, (Mori
et al. 2001b; Kawakami et al. 2005) HIAP-1, (Ng et al. 2001; Waldele et al. 2006),
and Bcl-2 (Nicot et al. 1997; Akita et al. 2005). An earlier model of ATL development
posits that Tax augments IL-2 and IL-2 receptor expression through NF-kB, thereby
activating an autocrine stimulatory loop that leads to T cell proliferation and leukemia.
Such a model, however, is not supported by further evidence. While IL-2 receptor a
chain expression is upregulated by Tax, other essential IL-2 receptor subunits are not.
Activation of human telomerase (hTERT) expression by Tax through an NF-kBdependent mechanism has been reported recently (Sinha-Datta et al. 2004). As mentioned above, increased hTERT expression in primary T cells as mediated by Tax is
thought to prevent replicative senescence during the early stage of viral infection. Tax
has been shown to inactivate p53 by facilitating the formation of a p65 RelA and p53
complex, which assembles on p53-responsive promoters to block p53-mediated
transcription (Pise-Masison et al. 2000; Jeong et al. 2004).
Tax and Cell Cycle Entry

Earlier studies have focused on demonstrating the functional resemblance of Tax to
the oncoproteins of small DNA tumor viruses. It has been reported that Tax induces
expression of genes encoding D-type cyclins, particularly cyclin D2, and CDK4
(Lemasson et al. 1998; Akagi et al. 1996; Santiago et al. 1999; Iwanaga et al. 2001;
Ohtani et al. 2000) due, in part, to the potent NF-kB activation by Tax (Iwanaga
et al. 2001). Tax also accelerates cell cycle progression through G1 apparently by
binding to and stabilizing the enzymatic complexes formed by cyclin D family
members and CDK4 and CDK6 (Neuveut et al. 1998; Schmitt et al. 1998; Haller
et al. 2000; Li et al. 2003; Haller et al. 2002). Furthermore, Tax has also been
demonstrated to increase E2F production (Kehn et al. 2005; Ohtani et al. 2000).
Notably, Tax has been reported to reduce expression of p18INK4C and p19INK4D
(Iwanaga et al. 2001) and inactivates p16INK4 (Low et al. 1997; Suzuki et al. 1996)
and p15INK4B via direct binding (Suzuki et al. 1999). Some of these proposed activities
of Tax clearly impact on G1S transition.
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Tax and PDZ Domain-Containing Proteins

Soon after the discovery of HTLV-1, a highly related human retrovirus known as
HTLV-2 was isolated (Kalyanaraman et al. 1982). In contrast to HTLV-1, HTLV-2
is not associated with human malignancies. The HTLV-2 Tax is structurally and
functionally similar to the HTLV-1 Tax, but lacks 22 amino acid residues in the
carboxyl terminus. This domain in HTLV-1 Tax has been shown to constitute a PDZ
domain-binding motif and interact with cellular PDZ domain-containing proteins
including tumor suppressor hDlg (Ishioka et al. 2006), MAGI-3 (Ohashi et al. 2004),
scaffolding protein hScrib (Arpin-Andre and Mesnard 2007), and the IL-16 precursor
protein (Wilson et al. 2003). These interactions augment the transforming activity
of HTLV-1 Tax in rat fibroblasts (Hirata et al. 2004) and possibly increase the pathogenicity of HTLV-1.
Tax in Transgenic Mice

Transgenic mice expressing tax develop various tumors depending on the promoters
used for Tax expression. Tax expressed by the HTLV-1 LTR caused neurofibroma, a
tumor of mesenchymal tissue, after a long time (Nerenberg et al. 1987). Interestingly,
one group of LTR-tax mice developed thymic atrophy and died soon after birth,
consistent with the notion that Tax expression is detrimental to T cells (Nerenberg
et al. 1987). Large granular lymphocytic leukemia has been found in mice transgenic
for tax expressed from the T-cell specific granzyme B promoter (Grossman et al.
1995). More recently, tax under the control of the Lck proximal promoter has been
found to induce large-cell lymphomas and leukemia with clinical, pathological, and
immunological features characteristic of acute ATLL (Hasegawa et al. 2006). Here,
again, the lymphoma/leukemia developed after prolonged latency periods. The Lck
promoter-Tax transgenic mice were immunocompromised and prone to opportunistic
infections (Hasegawa et al. 2006).
Oncogenic Activities of Tax: Tax and Genetic/Chromosomal

Instabilities
Tax is known to bring about both genetic instability (GIN) and chromosome instability
(CIN) by causing defects in DNA damage repair and inappropriate chromosome
segregation. Such genetic alterations are thought to increase the rate of mutations and
chromosome aneuploidy that in turn facilitate leukemia development. While there is
general agreement on the role of Tax in causing GIN and CIN, the exact mechanism(s)
by which they are effected has (have) not been fully resolved. It is unclear whether
the seemingly complex GIN and CIN caused by Tax share a common underlying
mechanism and what role GIN and CIN may play in HTLV-1 replication.
C.-Z. Giam
628
Tax and Defect in DNA Damage Repair

With respect to GIN, Tax has been shown to downregulate the expression of DNA
pol b, an enzyme involved in base-excision repair (Jeang et al. 1990). Tax also
induces proliferating cell nuclear antigen (PCNA) expression, which likely contributes to continual DNA replication upon DNA damage and defect in nucleotide
excision repair (Lemoine et al. 2000). Tax has also been reported to disrupt the
ataxia telangiectasia-mutated (ATM) and ATR pathways by interacting with Chk1
and Chk2 (Park et al. 2004; Park et al. 2006; Gupta et al. 2007; Durkin et al. 2008).
It was found to colocalize with and sequester Chk2 and DNA-PK to hamper cellular
response to DNA damage (Gupta et al. 2007; Durkin et al. 2008), while the TaxChk1 interaction is thought to inhibit Chk1 activity, block phosphorylation-dependent
degradation of Cdc25A, and prevent G(2) arrest in response to gamma-irradiation
(Park et al. 2004). Tax was also found to reduce the association between MDC1 and
DNA repair foci, which could potentially compromise cellular mechanism for DNA
damage repair (Chandhasin et al. 2008). Insofar as there is consensus that Tax
induces DNA damage, the multitude of models to explain its mechanisms of action
suggests that additional work is needed to resolve the matter.
Tax and Double-Stranded DNA Breaks

The association between Tax and micronuclei formation was first reported by
Majones et al. (1993). Micronuclei consist of extranuclear chromosome fragments or
whole chromosomes that form as a result of chromosome breaks (clastogenic events)
or chromosome lagging during cell division. Tax-induced micronuclei formation is
associated with increased free DNA 3 ends that are consistent with the increased
occurrence of clastogenic DNA double-stranded breaks (DSBs) (Majone and Jeang
2000). In this vein, Lemoine and Marriott had used the PALA [N-(phosphonoacetyl)l-aspartate]-resistance assay, which measures increased copy number of the CAD
(Carbamyl phosphate synthetase/Aspartate transcarbamylase/Dihydro-orotase) gene
to demonstrate that Tax increase the rate of gene amplification (Lemoine and Marriott
2002), presumably caused by DNA recombination following DSBs. Finally, Ku80deficient cells are refractory to while loss of DNA-PKcs exacerbates Tax-induced
micronuclei formation (Majone et al. 2005). Whether Tax directly induces DSBs,
inhibits the repair of DSBs, or both has not been fully delineated.
Tax and Chromosome Instability

Centrosomes, the microtubule organizing centers (MTOCs), are responsible for
bipolar mitotic spindle formation and proper segregation of chromosomes during
mitosis. Centrosome duplication occurs once in every cell division cycle (centrosome
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629
duplication licensing) and is tightly regulated. Centrosome amplification can lead

to multipolar spindle formation and unequal chromosome segregation. Tax has been
shown to cause centrosome amplification or fragmentation. This has been hypothesized as a contributing factor to the development of aneuploidy in ATL cells.
The underlying mechanism for Tax-mediated centrosomal abnormality is not
entirely clear. Interaction between Tax and RanBP1 that results in a disruption of
Ran-RanBP1 regulation of centriole cohesion has been proposed (Peloponese et al.
2005). A role of NF-kB in Tax-mediated centrosomal amplification has also been
suggested (Peloponese et al. 2005). Finally, Tax1BP2, an extensive coiled-coil protein
identified in yeast 2-hybrid screen as a Tax-binding partner, has been shown to
inhibit centrosome duplication (Ching et al. 2006). Tax1BP2 knockdown or putative
inactivation through interaction with Tax, leads to centrosome hyperamplification.
Another model for Tax-induced CIN posits that a direct interaction between Tax and
HsMAD1, a critical component of the spindle checkpoint, causes spindle assembly
checkpoint defect (Jin et al. 1998), which allows mitosis to proceed even though
proper attachment of sister chromatids to the mitotic spindle is impaired, thus causing
uneven distribution of chromosomes and aneuploidy. Tax-expressing HTLV-1transformed T cells arrest in metaphase after treatment with microtubule-disrupting
agent, nocodazole, suggesting that the spindle checkpoint defect caused by Tax is
likely to be subtle (Liu et al. 2005; Bellon et al. 2010).
Tax Induces Cellular Senescence

Tax-Induced Senescence is a Precancerous Condition
Despite the mitogenic and antiapoptotic activities of Tax outlined above, Tax is difficult
to express in cultured cell lines (Liang et al. 2002). Few ATL cells consistently
express Tax (Taniguchi et al. 2005), and cell transformation by HTLV-1 and especially by Tax in cell culture is inefficient (Graziano et al. 1987). Paradoxically, Tax
expression also leads to drastic upregulation of p21Cip1/Waf1 and p27Kip1, and p53-/pRbindependent cellular senescence (Kuo and Giam 2006). The cellular senescence
induced by Tax is most likely as a cellular protective measure against the potentially
oncogenic activities of Tax such as GIN and CIN. Thus, impairment to this mechanism through downregulation of p27Kip1 and cytoplasmic mislocalization of p21Cip1/
Waf1
renders Tax expression permissible in HTLV-1-transformed cells and allows
NF-kB to remain constitutively activated by Tax to promote cell survival and proliferation. The cellular senescence induced by Tax is not a result of overexpression of
Tax. HTLV-1-infected lymphoid (SupT1) and nonlymphoid (HeLa) cells become
senescent (Liu et al. 2008). Whether primary T cells infected by HTLV-1 also undergo
senescence remains to be determined. It is worth noting that atypical lymphocytes
that are binucleated or contain cleaved/cerebriform nuclei are readily seen in the
blood smears of HTLV-I infected individuals (Taguchi and Miyoshi 1983; Kinoshita
et al. 1985; Shimoyama 1991; Sacher et al. 1999). Such cells resemble senescent
C.-Z. Giam
630
HeLa cells transduced by the Tax gene via a lentiviral vector (Kuo and Giam 2006).
The said pathological findings are consistent with the notion that development of
cellular senescence may reflect a precancerous condition due to oncogene activation
or tumor suppressor inactivation by Tax (Adams 2009). Whether cellular senescence
plays an active role in HTLV-1 replication is not clear. Senescent cells are known to
secret chemoattractants for macrophages and NK cells (Adams 2009), which are
responsible for their eventual elimination from affected tissues. Whether senescent
HTLV-1-infected cells may use this mechanism for virus transmission and spread,
especially to macrophages, can only be speculated upon at this point.
Upregulation of p21Cip1/Waf1 and p27Kip1 by Tax

The p27Kip1 protein half-life in an actively dividing cell is short, but becomes greatly
lengthened in the presence of Tax (Kuo and Giam 2006; Zhang et al. 2009). This
appears to be a result of the unscheduled activation of the anaphase promoting
complex/cyclosome (APC/C) by Tax (Kuo and Giam 2006), which leads to the
premature polyubiquitylation and degradation of Skp2 and inactivation of SCFSkp2,
the E3 ligase that mediates the destruction of p27Kip1. By constrast, the drastic rise
in p21Cip1/Waf1 induced by Tax is due to p53-independent p21Cip1/Waf1 promoter activation (Chowdhury et al. 2003; Zhang et al. 2009) and p21Cip1/Waf1 mRNA stabilization
(Zhang et al. 2009). The massive surge in p21Cip1/Waf1 and p27Kip1 in turn leads to p53and pRb-independent cellular senescence (Kuo and Giam 2006). Cell cycle arrest of
CD34+ hematopoietic progenitor cells transduced by the tax gene has also been
observed (Tripp et al. 2003, 2005), presumably mediated by the same mechanism.
Because p21Cip1/Waf1 can serve as a chaperone for G1 cyclin D-CDK4/6 complex
without inhibiting its kinase activity, it was hypothesized that p21Cip1/Waf1 may thereby
facilitate cell-cycle transition of HTLV-1-infected cells (Kehn et al. 2004). However,
in light of results showing that Tax causes cellular senescence, the increase in p21Cip1/
Waf1
brought on by Tax is most likely for senescence induction.
Data showing that Tax causes mitotic aberrations and senescence support a model
of ATL development in which the p21Cip1/Waf1- and p27Kip1-mediated senescence
program has to be inactivated by cellular epigenetic or genetic changes to accommodate Tax expression and cell proliferation concurrently (Kuo and Giam 2006; Liu
et al. 2008). Alternatively, the silencing of Tax and HTLV-1 expression by 5 LTR
methylation (Taniguchi et al. 2005), or attenuation of Tax expression and activities
by viral factors such as p30II and HBZ may allow virus-infected cells to continue to
proliferate (Mesnard et al. 2006; Satou et al. 2006) and evolve into leukemia.
HBZ and HTLV-1 Leukemogenesis

That HBZ antagonizes the activities of Tax at the levels of LTR transactivation and
NF-kB activation immediately raises the possibility that it may attenuate or silence
viral mRNA transcription and mitigate the various biological effects of Tax including
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senescence. It is conceivable that during HTLV-1 infection, if Tax expression is

dominant over HBZ, then robust viral replication ensues and the senescence checkpoint
becomes activated. Infected cells in this state likely reach a dead end. By contrast,
when HBZ expression is favored, then Tax expression and activities together with
HTLV-1 replication would be moderated and viral latency may ensue. In this vein,
HBZ knockdown has been found recently to correlate with a reduction in T cell
proliferation after HTLV-1 infection (Arnold et al. 2008).
HBZ expression is determined by the abundance of the transcription factors that
activate its promoter and by the sites of HTLV-1 proviral integration. SP1 binding
sites in a TATA-less promoter located in the 3 LTR has been shown to mediate HBZ
expression (Yoshida et al. 2008). Recent data have also indicated that Tax upregulates the expression of HBZ and that this upregulation is influenced by the HTLV-1
integration sites (Landry et al. 2009). Proviral DNA integrated near a strong cellular
promoter that directs transcription in the opposite polarity of the major viral mRNA
transcript is expected to express HBZ preferentially. Those infected cells that
express HBZ in higher abundance are likely to continue to divide and evolve without
triggering the senescence checkpoint because Tax expression and activities are
downregulated. The reduction or silencing of viral replication in turn facilitates
evasion from immune detection. Since ATL can only emerge from HTLV-1-infected
cells that are capable of continuous cell division, and HBZ is likely important for
lowering the Tax level and delaying or preventing the onset of cellular senescence
induced by Tax, it is then logical that HBZ mRNA transcript is widely expressed in
ATL cells. Finally, the ability of HBZ mRNA to stimulate cell proliferation suggests
that it may actually be responsible for leukemia maintenance (Satou et al. 2006).
The role of Tax in ATL development should not be underestimated, however.
The ability of Tax to activate NF-kB remains a key factor in the proliferation, survival,
and immortalization of HTLV-1-infected cells. The new adjustments to the role of Tax
in leukemogenesis would be that (1) because of Taxs ability to induce senescence, its
expression needs to be moderated by HBZ, and perhaps p30II, to allow infected cells
to proliferate and expand; and (2) the full oncogenic potential of Tax can only be realized when the senescence checkpoint is inactivated. The latter would favor elevated
levels of Tax expression and increased NF-kB activation, and at the same time, exacerbate Tax-related genetic and chromosomal instabilities. In this sense, the eventual
silencing of Tax expression would stabilize such preleukemic or leukemic cells genetically and prevent their killing by cytotoxic T lymphocytes (Fig. 25.4).
Conclusions and Perspectives

The major obstacle to the study of HTLV-1 replication cycle remains the low infectivity of viral particles and the rather strict dependence on cell-to-cell contact for
virus infection. This necessitates the coculture of virus-producing cells with target
cells for only a modicum of virus transmission to occur. This technical difficulty is
further compounded by the inability of the infected cells to spread the infection.
Indeed, the low infectivity of HTLV-1 has made it difficult to study key events of
C.-Z. Giam
632
Fig. 25.4 A model for HTLV-1 Leukemogenesis. HTLV-1 is transmitted by cell-to-cell contact.
The expression levels of Tax and HBZ modulate the outcomes of infection. Robust viral replication
stimulated by Tax is accompanied by cellular senescence. HBZ moderates transactivation by Tax,
thereby downregulates viral replication and Tax expression to allow oligoclonal expansion of
infected T cells. Cytotoxic T lymphocyte (CTL) killing can control virus replication in asymptomatic carriers and select for cells that carry latent proviral DNA. HTLV-1-infected cells develop
chromosomal instability. Loss of p16INK4a, p15INK4b, and other tumor suppressors and constitutive
Jak/Stat activation may contribute to the inactivation of the senescence checkpoint to allow persistent Tax expression and NF-kB activation. Loss of Tax expression is favored because Tax is a
primary CTL target and has a propensity to induce genomic instability and cellular senescence.
Inactivation of the senescence checkpoint can facilitate potent NF-kB activation by Tax at the early
stage of leukemogenesis and aid the development of Tax-independent NF-kB activation later.
The mitogenic activity of HBZ mRNA may help sustain the ATL tumor phenotype
viral life cycle such as the mechanisms of viral entry, assembly, and release, and the
roles of viral regulatory proteins and their effects on infected cells during viral
replication. Although recent data suggest that cell-free HTLV-1 particles can be
transmitted to T cells via plasmocytoid dendritic cells (Jones et al. 2008), the efficiency
of virus transmission remains quite modest. Thus, while T cells can be immortalized
and transformed by HTLV-1 in cell culture, the simple question of what fraction of
infected cells becomes so remains unanswered because of the low infectivity of HTLV-1
and the difficulty of tracking infected cells. Existing evidence suggests that immortalization and transformation of T cells by HTLV-1 is a rare occurrence and the notion
that HTLV-1 infection invariably leads to cell proliferation requires critical reevaluation
with better assay systems. Direct transfection of infectious HTLV-1 proviral DNA
clones into cultured cells has been used to analyze the functions of viral accessory
genes, but the in vivo roles of many accessory genes remain elusive.
The existing animal models for HTLV-1 infection do not fully reflect the natural
course of viral infection and disease development in human. Given that ATL develops
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633
over a course of several decades and only in a small fraction of infected persons, it
would be unrealistic to expect an animal model to efficiently recapitulate all features
of HTLV-1 pathogenesis. However, armed with the knowledge that inactivation of
the senescence checkpoint or activation of the Jak/Stat and/or PI3K pathways can
facilitate cell transformation and leukemogenesis, it may be possible to target these
regulatory mechanisms in animal models to accelerate the progression to disease
after HTLV-1 infection.
The absence of Tax expression and the frequent presence of HBZ mRNA transcript
in ATL cells now compel a reexamination of the roles of each gene, especially Tax,
in HTLV-1 life cycle and leukemogenesis. While Tax expression is clearly not
essential for the neoplastic phenotype of ATL cells in vivo, whether HBZ expression
is necessary is not clear at present. Thus, the question remains whether Tax and
HBZ are important respectively for the initiation and maintenance of ATL, or alternatively, both proteins may act complementarily at the beginning of viral infection
up to the precancerous stage of ATL. The induction of cellular senescence by Tax is
consistent with the difficulty in expressing it stably in cultured cell lines, and the
loss of its expression from ATL cells. By moderating the level of expression of Tax
and its activities, HBZ may delay or prevent Tax-induced senescence to allow
HTLV-1-infected cells to proliferate and undergo oligoclonal expansion. Ultimately,
the senescence mechanism triggered by Tax would have to be inactivated to allow
the oncogenic potentials of Tax, such as potent NF-kB activation and induction of
GIN and CIN, to become significant. In this sense, Tax may be considered as an
opportunistic oncogene, and its leukemogenic potential requires specific collaborating
cellular changes that prevent senescence induction. In vivo, Tax-specific cytotoxic
T lymphocyte killing and Tax-induced cell cycle abnormalities eventually select for
the loss of Tax expression from ATL cells. Since constitutive NF-kB activity is
necessary for leukemic cell survival and proliferation, Tax-independent IKK/NF-kB
activation likely evolves at a later stage of ATL development (see Fig. 25.4 for a
depiction of this model). By contrast, since in vitro transformation of T cells by
HTLV-1 takes place in the absence of host immune response, the loss of Tax and
viral expression is not selected, and as such Tax expression and Tax-mediated IKK/
NF-kB activation is retained. This provides an explanation for the frequent inactivation
of the senescence program in HTLV-1-transformed T cells through the downregulation
of p27Kip1 and the functional inactivation of p21Cip1/Waf1. While alterations of p27Kip1
gene are relatively rare in ATL cases (Morosetti et al. 1995), p15INK4b and p16INK4a
deletions are frequent occurrences (Hatta et al. 1995). Whether the latter loss can
functionally substitute for the downregulation of p27Kip1 and inactivation of p21Cip1/
Waf1
is unclear.
Recent progress on the HTLV-1 accessory genes, Tax and HBZ, has begun to
shed light on how their interactions may regulate the proliferation and expansion
of HTLV-1-infected cells and how HBZ may collude with Tax to cause cell transformation and cancer. It will be important in the future to investigate their functions in the context of viral infection in cell culture, in animal models, and in
infected individuals, and to translate basic science findings into treatments for
HTLV-1-related diseases.
C.-Z. Giam
634
Notes
In a recent paper (Zhi et al. 2011, PLoS Pathogens, 7(4): e1002025.doi:10.1371/
journal.ppat.1002025.), the persistent and potentially oncogenic activation of NF-kB
by Tax was found to be responsible for triggering the cellular senescence response.
Down-regulation of NF-kB activity by HBZ, by contrast, delayed or prevented the
onset of Tax-induced senescence. These results support the model depicted in
Fig. 25.4, showing that HTLV-1 executes two alternative genetic programs wherein
robust HTLV-1 replication and elevated Tax expression drive IKK/NF-kB hyperactivation and trigger senescence. Moderation of Tax-mediated viral replication and
NF-kB activation by HBZ, on the other hand, allows HTLV-1-infected cells to proliferate, persist, and evolve. Importantly, inactivation of the senescence checkpoint
can facilitate persistent NF-kB activation and leukemogenesis.
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Chapter 26
Human T-Cell Leukemia Virus Type 2

(HTLV-2) Biology and Pathogenesis
Rami Doueiri and Patrick L. Green
Discovery of HTLV-2
HTLV-2 was first identified in a T-cell line, termed MoT, derived from splenic tissue
of a patient with a variant of hairy cell leukemia (Saxon et al. 1978). Serological
data demonstrated that HTLV-2 was related to, but distinct from HTLV-1, and subsequent sequence analysis revealed that both viruses share approximately 70%
nucleotide sequence homology (Kalyanaraman et al. 1982). HTLV-2 disease association has been less clear in comparison to HTLV-1, which is associated with adult
T cell lymphoma (ATL) and HTLV-associated myelopathy/tropical spastic paraparesis
(HAM/TSP). Although HTLV-2 was discovered in a hairy cell leukemia patient, the
limited number of individuals shown to harbor HTLV-2 in association with specific
diseases has precluded convincing epidemiologic demonstration of a definitive
etiologic role for HTLV-2 in human disease. However, several cases of HTLV2-associated neurological disease have been documented (Hjelle et al. 1992), and
R. Doueiri
Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus,
OH 43210, USA
P.L. Green (*)
Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road,
Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus,
OH 43210, USA
Departments of Molecular Virology, Immunology and Medical Genetics,
The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA
Comprehensive Cancer Center and Solove Research Institute, The Ohio State University,
1900 Coffey Road, Columbus, OH 43210, USA
e-mail: green.466@osu.edu
647
648
R. Doueiri and P.L. Green
more recent findings from an HTLV outcome study (HOST) provide strong support
that HTLV-Z infection is associated with neurodegenerative disease, lymphocyte
prolifecation, and may be a predisposition factor to cancer development and heart
disease (Murphy et al. 2004; Biswas et al. 2009).
HTLV-2 Virion Structure

HTLV-2 is a member of the deltaretrovirus family and, like all retroviruses, is enveloped
and contains RNA as its genetic material (Fig. 26.1). Detailed description of the
virion is beyond the scope of this chapter, but different components are briefly
discussed. The components of the virion are not directly required for cellular
transformation, but they are needed for viral attachment and entry, viral genome
reverse transcription from RNA to double-stranded DNA, and integration into the
cell chromosome, which eventually can lead to cellular transformation. Moving
from outside to the inside of the virus particle, the virion envelope contains part of
the cellular plasma membrane derived during virus exit or budding from the infected
Fig. 26.1 Schematic representation of the mature HTLV-2 virion highlighting the location of the
structural components. The virion envelope is primarily from the host plasma membrane (formed
on budding) and contains the viral Envelope proteins, surface unit (SU), and transmembrane (TM),
alongside other host-encoded proteins. The inner envelope contains the matrix (MA), which is
required for virion assembly at the inner cell membrane. The capsid (CA), which protects the viral
RNA genome, the nucleocapsid (NC), integrase (IN), the protease (PRO), reverse transcriptase
(RT), and tRNApro are shown
26 Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology and Pathogenesis
649
cell, and two viral envelope proteins encoded by the env gene, the surface unit (SU)
and transmembrane (TM) proteins. Beneath the envelope lies three viral proteins
encoded by gag: the matrix (MA), the capsid (CA), and the nucleocapsid (NC)
(which is associated with the RNA genome). Enzymatic viral proteins packaged in
the virion include reverse transcriptase (RT), integrase (IN), and protease (PRO).
Cellular factors also are found in the virion. Of critical importance is the cellular
tRNA pro, which acts as the viral primer for the initiation of reverse transcription.
Genome Structure
HTLV-2 is referred to as a complex retrovirus. In addition to the usual structural and
enzymatic genes gag, pol, and env, there is a unique region at the 3end of the
genome, not found in replication-competent simple retroviruses, which encodes
regulatory and accessory genes. Historically, this region was termed X, which for
HTLV-2 contains five (IV) open reading frames (ORF) on the sense strand and one
ORF on the antisense strand of the DNA proviral genome.
ORFs III and IV encode the positive regulatory proteins, Rex and Tax, respectively.
Tax acts as a transcriptional activator of the viral promoter located within the 5 long
terminal repeat (LTR). Rex functions posttranscriptionally and is required for the efficient expression of structural and enzymatic proteins and thus viral progeny. ORFs I,
II, V, and the antisense ORF encode accessory proteins p10, p28, p11, and the antisense
protein HTLV-2 (APH-2), respectively. These proteins have been less well characterized to date, but emerging data suggest an important role early in the infectious process
resulting in maintenance of proviral load and infected cell persistence in vivo.
In the HTLV-2 infected cell, three major (high copy number) and five lower copy
number messenger RNA (mRNA) species have been detected (Fig. 26.2). Like all
retroviruses, the full-length (unspliced) RNA is utilized for synthesis of gag and
pol-encoded gene products and also serves as the genomic RNA packaged into progeny
virions. A singly spliced mRNA encodes the env gene products, while a doubly or
completely spliced mRNA species encodes both the tax and rex gene products in partial
overlapping reading frames: the start codons for Rex and the downstream Tax lie in
the second exon, with Tax translation favored due to strong Kozac consensus
sequences. Several lower copy number alternatively spliced mRNA species encode
proteins from ORFs I (p10), II (p28), III (truncated Rex polypeptides p20/22), V (p11),
and the antisense ORF (APH-2).
HTLV-2 Structural and Enzymatic Gene Products of the Virion

Gag Gene
The gag (group-specific antigen) region is translated into a polyprotein precursor
(55 kDa) that subsequently is cleaved by the viral protease (Pro) into three mature
650
Fig. 26.2 Structure and organization of the HTLV-2 genome based on the HTLV-2 MO isolate.
The provirus genome is at the top of the figure. Location of the viral proteins, RxRE and CRS, are
shown beneath the provirus. The structure of the structural, regulatory, and accessory proteins is
shown at the bottom of the figure. (Oroszlan et al. 1984; Shimotohno et al. 1985; Halin et al. 2009)
proteins: 19-kDa matrix (MA), 24-kDa capsid (CA), and 15-kDa nucleocapsid (NC)
proteins (Oroszlan et al. 1984). p19 is posttranslationally modified and contains a
myristic acid at the NH2 terminus (Oroszlan et al. 1984). This modification targets
the 55-kDa Gag precursor polypeptide to the inner surface of the plasma membrane
(Hayakawa et al. 1992), which is required for virion assembly and budding of virus
particles from infected cells.
Protease (Pro) Gene

A reading frame that extends from the 3 end of gag to the 5 part of the pol region
encodes the viral Pro. The synthesis of Pro requires ribosomal frameshifting of Gag
(Nam et al. 1988). The protease activity has been elucidated in vitro, and studies
have shown that Pro auto-catalytically processes itself into a mature form and is
responsible for processing the Gag polyprotein into the mature products (Hatanaka
and Nam 1989).
651
Polymerase (Pol) Gene

Similar to pro, a second frameshift is required to express the pol gene (Nam et al.
1988). HTLV-2 pol encodes a polyprotein containing 864 amino acids (Shimotohno
et al. 1985). The 5end of pol encodes a RNA/DNA-dependent DNA polymerase
termed reverse transcriptase (RT), which requires Mg2+ for optimal function (Rho
et al. 1981). The downstream region of pol encodes the RNase H and integrase (IN).
RNAse H is essential to the reverse transcription process by degrading the RNA of
a RNADNA hybrid; IN is required to integrate the proviral DNA into the cellular
genome.
Envelope (Env) Gene

The env gene of the HTLV-2 produces an approximate 67-kDa glycoprotein
(Lee et al. 1984), which then is cleaved into the 46-kDa surface glycoprotein (SU
or gp46) and the 21-kDa transmembrane protein (TM or p21). The SU component
is required for receptor recognition, whereas TM is required for fusion and entry
into cells. Interestingly, human antibodies to HTLV-1 Env cross react with HTLV-2
Env, which led to the initial discovery of HTLV-2 (Kalyanaraman et al. 1982).
HTLV-2 Regulatory Genes

Tax Gene
HTLV-2 encodes two positive regulators of viral gene expression, Tax and Rex,
which are required for efficient viral replication and cellular transformation. Tax-2 is
a 37-kDa phosphoprotein that shares approximately 78% homology with HTLV-1
Tax (Tax-1). Tax is a trans-acting transcriptional activator and increases the rate of
transcription initiation from the promoter in the 5 LTR of the provirus genome. In
contrast to Tax-1, which is detected primarily in the nucleus, Tax-2 is located predominantly in the cytoplasm (Meertens et al. 2004a, Turci et al. 2006). However,
Tax-2 also can be detected in nuclear bodies with RNA polymerase II, splicing complexes and specific transcription factors (Goh et al. 1985; Slamon et al. 1988; Semmes
and Jeang 1996; Bex et al. 1997). Although Tax-2 and Tax-1 share a number of
activities and associations with nuclear proteins, their distinct difference in localization has been proposed to contribute to the differences between HTLV-1 and HTLV-2
biology. Four functional domains have been described for Tax-2 (Fig. 26.3). The
Tax-2 amino terminus contains an activation domain, a nuclear localization signal
(NLS) that lies within the first 41 residues (Turci et al. 2006), and a zinc binding
652

I319R/L320S
(CREB)
S130A/L131F
(NFB)
Zinc finger
Activation domain
100
Cytoplasmic
localization
200
NES
331
Activation domain
PBM
Fig. 26.3 Domain structure of HTLV-2 Tax. The nuclear localization domain (NLS) is located
within the first 41 amino acids in the N-terminus, in addition to an activation domain and a zinc
finger domain. A cytoplasmic localization signal is found at region 90100, and a leucine-rich
region that contains a nuclear export signal (NES) is located at 189202. A second activation
domain is located between residues 189322. Specific mutations affecting transcriptional activation pathways are indicated. The PDZ binding motif (PBM) domain, which is found in Tax-1 but
not in Tax-2, has been added for illustrative purpose
domain between residues 23 and 49 that plays a role in proteinprotein interactions.

The central region of the polypeptide contains a cytoplasmic localization domain from
residues 90100 (Meertens et al. 2004a) and a leucine-rich nuclear export signal at
residues 189202 that appears to be indispensable for Tax-2 function (Chevalier
et al. 2005). The carboxyl terminal 289322 amino acids contain a second activation
domain. One domain absent in Tax-2, but present at the carboxy terminus of Tax-1,
is a four-amino-acid PDZ binding motif (PBM). The significance of this domain and
other activities of Tax are discussed more detail in below.
Rex Gene
In order to efficiently replicate, retroviruses need to override the nuclear retention
of intron-containing mRNAs. The unspliced mRNA (genomic and gag/pol) and the
singly spliced mRNA (env) are intron-containing viral mRNAs and the default pathway in the cell is to retain them in the nucleus until they are processed or targeted
for degradation. At the molecular level, the most notable role of Rex is to regulate
cytoplasmic levels of viral genomic unspliced and env singly spliced mRNAs, thus
controlling the expression of the structural and enzymatic gene products that are
essential for production of viral progeny (Hidaka et al. 1988; Kusuhara et al. 1999).
Rex binds to the viral mRNAs via a cis-acting RNA Rex-response element (RxRE)
and facilitates the export of these mRNA species from the nucleus to the cytoplasm
(Ballaun et al. 1991; Black et al. 1991a; Bogerd et al. 1991). Previous studies
revealed that HTLV-1 Rex (Rex-1), HTLV-2 Rex (Rex-2), and their RxREs are
structurally similar and functionally interchangeable. The HTLV-2 RxRE is 226
nucleotides in length and is located in the R/U5 region of the LTR. A cis-acting
repressive sequence (CRS) has been identified within the RxRE downstream of the
splice donor site (Black et al. 1991a, b; Yip et al. 1991). Thus, a model consistent
with the experimental data is that the CRS retains the unspliced mRNA in the
nucleus to ensure the availability of sufficient amounts of Rex substrate (Ohta et al.
653
S153A*
S151A*
T164A*
19
RBD/NLS
57
71
81
94
124 132
AD/NES
170
Stability
Multimerization
Fig. 26.4 Domain structure of HTLV-2 Rex. The nuclear localization signal domain (NLS) and
the RNA binding domain are located within the first 19 amino acids in the N-terminus. The activation
domain and the nuclear export signal (NES) are located between positions 8194, flanked by two
multimerization domains between residues 5771 and 124132, respectively. A C-terminal regulatory domain from 144 to 164 that contains key phosphorylation sites (Serine 151, Serine 153, and
Threonine 164) is required for efficient function and nucleocytoplasmic shuttling
1988; Black et al. 1991a, b), then Rex binds to the RxRE, overrides the repressive
effects of the CRS, and exports the mRNA to the cytoplasm (Black et al. 1991a
Younis and Green 2005).
Mutational analyses of Rex-1 and Rex-2 identified important domains for their
biological properties including (1) an arginine-rich sequence located at the N-terminus
that serves both as an RNA binding domain and as a nuclear localization signal (NLS),
(2) a central leucine-rich activation domain encompasses the nuclear export signal
(NES), and (3) a multimerization domain composed of two regions flanking the NES
(Siomi et al. 1988; Rimsky et al. 1989; Bogerd et al. 1991; Hope et al. 1991;
Weichselbraun et al. 1992a, b; Bogerd and Greene 1993; Hammes and Green 1993;
Palmeri and Malim 1996; Narayan et al. 2003) (Fig. 26.4). The Rex NLS interacts with
importin-b, and is transported to the nucleus by a Ran-GTP-dependent mechanism
(Palmeri and Malim 1999). Once Rex is in the nucleus, Ran-GTP binds to importin b
releasing Rex and making it available to bind RxRE-containing mRNAs (Gorlich et al.
1994; Gorlich et al. 1996). Rex binding to its RxRE recruits additional Rex molecules,
although this multimerization is not essential for Rex function (Weichselbraun et al.
1992b; Bogerd et al. 1993; Hataka et al. 1998; Narayan et al. 2003). Once a stable Rex/
RxRE/CRM1 complex is formed, CRM1 interacts directly with Rex and its cargo
mRNA and facilitates nuclear transport through a series of proteinprotein interactions
with the FG repeat-containing nucleoporins. The Rex/RxRE/CRM1 complex then is
recognized by Ran-GTP, which finally leads to the exit through the nuclear pore
(Fornerod et al. 1997; Hataka et al. 1998; Heger et al. 1998; Otero et al. 1998; Askjaer
et al. 1999; Fornerod and Ohno 2002; Younis and Green 2005).
Both Rex-1 and Rex-2 are phosphoproteins, and phosphorylation has been shown
to be critical for their function (Adachi et al. 1990, 1992; Green et al. 1992). In HTLV-2
infected cells, four different species of Rex are detected (20, 22, 24 and 26 Kd).
Studies revealed that proteins p24/p26 are encoded by the tax/rex mRNA, and proteins
p20/p22 are amino truncated forms of Rex-2 and are encoded from two alternatively
spliced mRNAs. The function of p20/p22, which appear to be analogous to the
amino terminal truncated p21 Rex of HTLV-1, is not well understood, but evidence
654
suggests that they might interfere with full-length Rex function and localization
(Ciminale et al. 1995, 1997; Kubota et al. 1996; Li and Green 2007). p24/p26 are
the major Rex-2 protein species detected in HTLV-2 infected cells, which have the
same amino-acid backbone, but differ by a conformational change that is induced
by serine/threonine-specific phosphorylation (Green 1991; Narayan et al. 2001;
Lairmore and Franchini 2007). This phosphorylation-induced conformational
change is unique to Rex-2, as the Rex-1 phosphoprotein is detected as a single
27-kDa protein. Rex-2 p24 is localized primarily in the cytoplasm, whereas the p26
phosphorylated form, which is the active species, localizes predominantly to the
nucleus and nucleolar speckles (Yip et al. 1991; Ciminale et al. 1997). Phosphorylation
of Rex-2 correlates with its binding to RxRE-containing RNA and inhibition of mRNA
splicing (Green et al. 1992; Bakker et al. 1996). Mutational analysis of Rex-2
revealed that the C-terminus contains a stability/inhibitory domain that is positively
regulated through phosphorylation. Key residues (Ser 151, Ser 153, Thr 164) in the
C-terminus appear to govern the switch between the p24 and p26 conformation and
active function, supporting a model in which a phosphorylation continuum of Rex-2
at the C-terminus regulates its biological properties (Narayan et al. 2001, 2003;
Kesic et al. 2009; Xie et al. 2009). Interestingly, Rex-2 can also negatively regulate
levels of mRNA that contain LTR sequences (Watanabe et al. 1996) and thus might
also play a role in viral latency. This negative regulation is observed in T-lymphocytes
but not B-lymphocytes, does not require the RxRE, and therefore is a function
distinct from Rex-2 positive posttranscriptional activity (Watanabe et al. 1996).
In addition to Rex facilitating the export of HTLV-2-specific intron-containing
mRNAs, there is evidence that Rex-2 also can inhibit the splicing of these mRNAs.
Rex-2 binding to the mRNA may dislodge or prevent the binding of splicing factors
(Black et al. 1991b; Bakker et al. 1996), thereby allowing the cellular machinery to
recognize it as a processed mRNA and transport it to the cytoplasm. Interestingly,
phosphorylation of Rex-2 is required for both efficient binding to mRNA and
inhibition of splicing. In addition, there is evidence that Rex may increase the translational efficiency of Rex-responsive mRNAs possibly by enhancing binding to the
translation initiation factor 5A (eIF-5A) (Katahira et al. 1994, 1995). Experimental
data indicate that Rex-2 increases the level of incompletely spliced mRNA seven to
ninefold in the cytoplasm, while Gag production increases 130-fold (Kusuhara et al.
1999). Moreover, the homologous HIV-1 Rev has been shown to facilitate translation (Arrigo and Chen 1991; Dagostino et al. 1992).
HTLV-2 Accessory Genes

ORF I, II, and V
The accessory proteins of HTLV-2 are encoded by several proximal X region ORFs
between env and the 3 LTR. These proteins include p10 encoded by ORF I, p28 by
ORF II, and p11 by ORF V (Ciminale et al. 1995). HTLV-2 p28, with the potential to
655
be expressed from two distinct singly spliced mRNAs (both mRNAs also have the
potential to produce p20/p22 truncated Rex), has been the best characterized and is, at
least in part, functionally homologous to HTLV-1 p30. p28 functions to repress viral
replication posttranscriptionally by retaining tax/rex mRNA in the nucleus (Younis
et al. 2004). By repressing Tax and Rex positive regulatory functions, p28 downmodulates viral gene expression. Furthermore, it was shown that p28 was able to efficiently
retain tax/rex mRNA in the nucleus only when the latter is expressed from a fulllength proviral clone and not from a cDNA, initially suggesting that the 5 untranslated region (UTR) of the target RNA, intron sequences, and/or splicing may play a
role in mediating the inhibition. However, additional studies revealed that if the tax/
rex cDNA was expressed from the HTLV promoter, it was repressed by p28. The component of the HTLV-2 promoter, specifically Tax-2, was identified as the factor
required for efficient recruitment of p28 to the newly transcribed mRNA. This finding
suggested a complex interplay between the transcriptional machinery and the posttranscriptional regulation of tax/rex mRNA by p28, thereby coupling transcription
with posttranscriptional inhibition. Yamamoto et al. investigated the functional significance of p28 in HTLV-2 infection, proliferation, and immortalization of primary
T-cells in culture, and viral survival in an infectious rabbit animal model. They showed
that p28 is dispensable for viral replication and cellular immortalization of primary
T-lymphocytes. However, p28 function was critical for viral survival in vivo
(Yamamoto et al. 2008). Together, the results are consistent with the hypothesis that
p28 repression of Tax and Rex-mediated viral gene expression may facilitate survival
of infected cells by downmodulating overall viral gene expression.
p10 and p11 are expressed from the same doubly spliced mRNA in separate but
overlapping reading frames. Although less is known about the role these two proteins
play in the biology of HTLV-2, p10, like HTLV-1 p12, binds to the free chain of
MHC class I but not to the IL-2R b and g chains (Johnson et al. 2000). p11 binds to
MHC class I heavy chain (Johnson et al. 2001) and thus may facilitate escape from
immune surveillance.
Aph-2 Antisense Gene

The majority of retroviral gene products are encoded by the sense strand of the proviral
genome. However, natural antisense viral transcripts have been recognized in retroviruses including HTLV, human immunodeficiency virus, and feline immunodeficiency
virus (Larocca et al. 1989; Vanhee-Brossollet et al. 1995; Briquet et al. 2001). HTLV-1
encodes HBZ (HTLV-1 b-ZIP factor) (Gaudray et al. 2002; Matsuoka and Green 2009),
and recently, APH-2 has been detected in HTLV-2 infected cells (Halin et al. 2009).
APH-2 is a 183-amino-acid protein encoded by a singly spliced mRNA, and although
APH-2 lacks a bZIP motif similar to HBZ, it can still interact with cyclic adenosine
monophosphate-response element binding protein (CREB) and repress Tax-2 mediated
transcription (Halin et al. 2009). Its role in HTLV-2 biology currently is not known, but
drawing similarities from HTLV-1 HBZ, APH-2 may play a role in infectivity, viral
persistence, and cellular proliferation (Arnold et al. 2006, 2008).
656
Genetic Variability and Mode of Transmission of HTLV-2

The rate of genetic variability in the genome of HTLV-2 isolates is quite low, which
is consistent with its mode of transmission. Infected individuals display relatively
low levels of viral replication and virus propagation occurs primarily by mitotic
clonal expansion of infected cells (Cimarelli et al. 1996). To date, there have been
four HTLV-2 subtypes identified. Initially, analysis of env sequences identified
subtypes a and b (Gessain et al. 1985; Hall et al. 1992). HTLV-2a is found mainly
throughout North America and Europe in intravenous drug users (IDU), while
HTLV-2b is predominantly found in the Amerindian population in North, South,
and Central America (Switzer et al. 1995). Recently, a distinct variant of HTLV-2a
has been identified in the Amazon basin that had a full tax gene similar, but distinct
from HTLV-2b. This isolate was classified as HTLV-2c (Ishak et al. 1995; Eiraku
et al. 1996). A new subtype found in the Efe Bambuti pygmy tribe in the African
Congo has been designated HTLV-2d (Vandamme et al. 1998). Owing to the high
incidence of HTLV-2 in Amerindians, the virus was thought to be of new world
origin, contrary to HTLV-1, which is thought to have arisen from a zoonotic transmission of simian T-lymphotropic virus type 1 (STLV-1) from nonhuman primates
to humans (Koralnik et al. 1994; Crandall 1996; Goubau et al. 1996; Voevodin et al.
1996; Liu et al. 1996; Song et al. 1994). However, the discovery of HTLV-2d along
side STLV-2 in Africa supports an ancient African origin of HTLV-2 as well
(Vandamme et al. 1996; Digilio et al. 1997; Van Brussel et al. 1998).
HTLV-2 is transmitted via infected cellular blood products or intravenous drug
use, mother-to-child and sexual contact (Roucoux and Murphy 2004). However, the
route of transmission varies between different populations. Interestingly, vertical
transmission from mother-to-child occurs mainly through breast-feeding, where the
virus has been isolated from breast milk of infected mothers; children born to
infected mothers have a higher prevalence of seropositivity (Heneine et al. 1992;
Vitek et al. 1995).
HTLV Receptor and Infectivity

The frequency of cell-free virus infection with HTLV is relatively low compared to
cell-associated infection. Efficient infection of cells with HTLV in vitro occurs by
cocultivating target cells with gamma irradiated or mitomycin C-treated virus producer cells. In vivo, HTLV-1 and HTLV-2 display distinct tropism for cellular transformation, transforming primarily CD4+ and CD8+ T-cells, respectively (Miyamoto
et al. 1991; Ijichi et al. 1992; Lal et al. 1995). This in vivo tropism is recapitulated
in tissue culture transformation assays (Robek and Ratner 1999; Wang et al. 2000;
Ye et al. 2003). Cell tropism can be determined at the level of viral entry (receptormediated) as has been reported for poliovirus or HIV, or postentry (integration,
transcription, or translation) as reported for murine leukemia virus (Chatis et al.
1984; Browning et al. 1997; Moore et al. 1997; Pleskoff et al. 1997). HTLV-1 and
657
HTLV-2 efficiently enter CD4+ and CD8+ T-cells as well as B-cells, epithelial cells,
and macrophages. However, the transformation tropism is restricted to CD4+ or
CD8+ T-cells (Macatonia et al. 1992; Koyanagi et al. 1993; Jones et al. 2008), which
suggests that the distinct HTLV-1 and HTLV-2 transformation tropism is not specifically determined by virus entry. Studies utilizing HTLV-1/HTLV-2 recombinant
viruses revealed that the viral envelope is responsible for this preferential cellular
tropism (Ye et al. 2003; Xie and Green 2005). The viral envelope has two glycoproteins
SU and TM. SU binds to the cellular receptor, while TM triggers the fusion of the
viral and cellular membranes, facilitating viral entry. Binding studies have confirmed these findings and have revealed differential binding and entry properties of
HTLV-1 and HTLV-2. HTLV-1 is highly dependent on an initial interaction with
heparan sulfate proteoglycans (HSPG) found abundantly on CD4+ T-lymphocytes
and subsequent GLUT-1 and/or neuropilin-1 (NRP-1) interactions. HTLV-2 binding
and entry appears to be less dependent on HSPGs, which are limited on CD8+
T-cells, and highly dependent on interaction with GLUT-1 (Ghez et al. 2006; Jones
et al. 2006).
HTLV-2 and Associated Diseases

HTLV-2 and Neurologic Abnormality
Although HTLV-2 was isolated from a patient with hairy cell leukemia (Kalyanaraman
et al. 1982) and has the ability to transform T-cells in culture (Ross et al. 1996),
clinical data associating HTLV-2 with malignancy have not been conclusive. On the
contrary, an increased body of evidence correlates HTLV-2 infection with a neurological disease similar to HTLV-1 associated myelopathy/tropic spastic paraperesis
(HAM/TSP) (Hjelle et al. 1992; Harrington Jr et al. 1993; Jacobson et al. 1993;
Sheremata et al. 1993; Lehky et al. 1996; Murphy et al. 1997; Silva et al. 2002).
The disease manifestation develops decades after infection, usually consists of neurologic disability and peripheral neuropathy (Murphy et al. 1997), and has a higher
prevalence among women (Biglione et al. 1993; Harrington Jr et al. 1993; Jacobson
et al. 1993; Sheremata et al. 1993; Black et al. 1996; Lehky et al. 1996; Murphy
et al. 1997; Silva et al. 2002; Biglione et al. 2003; Orland et al. 2003; Araujo
and Hall 2004; Biswas et al. 2009). Other reports of rare HTLV-2-associated
disease include the development of a spinocerebellar syndrome (Castillo et al. 2000)
and predominantly sensory polyneuropathy (PSP) (Zehender et al. 1995). (Fig. 26.5)
A clear link between HTLV-2 and neurological disease has been difficult to conclude
mainly due to the lower prevalence of identified HTLV-2 infection worldwide compared to HTLV-1, thus providing limited epidemiological data on disease association.
Additional difficulties are due to concomitant infections with HIV-1, hepatitis B and C,
and human herpes viruses (Berger et al. 1991; Silva et al. 2002; Araujo and Hall
2004; Biswas et al. 2009; Rosenblatt et al. 1992). Additionally, HTLV-2 neurologic
disorders present with myelopathic and cerebellar features, similar to diseases such
658

HTLV-2 infected
CD8+ T-cell
Rantes
CCRs
HIV-1 infected
CD4+ T-cell
Cell death/viral
spread
Fig. 26.5 List of neurologic signs and symptoms in HTLV-2 infected individuals
as multiple sclerosis and spinocerebellar degeneration (Oger and Dekaban 1995;

Peters et al. 1999; Araujo and Hall 2004). Therefore, a thorough investigation and a
careful interpretation of the symptoms and family history must be carried out before
linking HTLV-2 with a particular disease manifestation.
HTLV-2 and Hematopoiesis

Although the HTLVs infect T-cells in vivo (and to a lesser extent B-cells and monocytes), little has been done to evaluate the effect of the infection, particularly with
HTLV-2, on hematopoiesis. Previously it was noted that HTLV-2 infection causes
elevated lymphocyte counts (Itoyama et al. 1988; Prince et al. 1990, 1994). In the
HTLV Outcome Study (HOST), Bartman et al. reported that HTLV-2 infected
patients have an increase in absolute lymphocyte counts compared to HTLV-1
patients and seronegative individuals (Bartman et al. 2008). The mechanism for this
significant increase is not well understood but several reasons have been postulated
including (1) downstream effects of Tax-2 on T-cell proliferation and antiapoptotic
factors, (2) inhibition of the immunologic responses to respiratory infections
(Murphy et al. 2004; Asquith et al. 2007) and/or (3) an inflammatory response to the
viral infection (Murphy et al. 1997; Oliveira et al. 2009). Interestingly, in infected
individuals, antibodies are raised against all three Gag proteins p15, p24 and p19,
and two envelope proteins gp46 and p21 (Clapham et al. 1983; Hoshino et al. 1983;
Essex et al. 1984; Ratner 1996). Recently, an HTLV-2-specific CTL response against
659
Tax-2 has been identified in infected individuals (Oliveira et al. 2009). The CTL
response was described previously in HTLV-1 infected individuals and was found to
play a pivotal role in controlling proviral load (Jacobson et al. 1990; Parker et al.
1992). In addition, high levels of CTL responses have been described in HIV
infected individuals resulting in suppression of viral load and delayed AIDS
progression (Goulder et al. 1997), as well as in healthy CMV seropositive individuals (Gillespie et al. 2000). The direct implication of a CTL response in HTLV-2
infected individuals is not yet clear but data suggest that it is a factor in limiting the
number of infected cells as measured by proviral load, and might be important in
preventing disease development in infected individuals.
HTLV-2 and HIV Coinfection

In the past decade, there has been an increased incidence worldwide of HTLV coinfection with other viruses, but particularly with HIV-1. The rate of coinfections
between HIV-1 and HTLV-1 is increasing among south Americans and Africans,
while HIV-1 and HTLV-2 is increasing among north American and European intravenous drug users (IDVUs) (Khabbaz et al. 1992; Briggs et al. 1995; Salemi et al.
1995; Hershow et al. 1996; Egan et al. 1999; Soriano et al. 1999; Goedert et al. 2001;
Guimaraes et al. 2001). The fact that HTLV-1 and HIV-1 infect CD4+ T-cells while
HTLV-2 has a preferential tropism for CD8+ T-cells appears to influence HIV-1
pathogenesis (Casoli et al. 2007; Beilke et al. 2004). The outcome of HTLV-2
HIV-1 coinfection suggests that HTLV-2 has a protective role by maintaining CD8+
and CD4+ T-cell counts and lowering HIV-1 replication, resulting in delayed progression to AIDS. In vitro experiments investigating coinfection of peripheral blood
mononuclear cells (PBMCs) with HTLV-2HIV-1 provide valuable information on
the mechanism by which HTLV-2 plays a protective role. Casoli et al. reported that
in HTLV-2HIV-1 coinfection, HTLV-2 expression occurs earlier than HIV-1
(Casoli et al. 2000). In addition, HTLV-2 infected CD8+ T-cells secrete increased
concentrations of three HIV-1 suppressive chemokines including macrophage inflammatory protein 1a (MIP-1a or CCL3), MIP-1b (CCL4), and RANTES (CCL5)
in vitro. Interestingly, the concentration of the chemokines (mainly MIP-1a) was
inversely related to HIV-1 replication, and upon the addition of antichemokine monoclonal antibodies (Mabs), the protective HTLV-2 function was abrogated (Casoli
et al. 2000). MIP-1a, MIP-1b and RANTES are CCR5 ligands, and CCR5 acts as a
coreceptor for non-syncytium-inducing (NSI) macrophage-tropic strains of HIV-1,
which dominate at the early onset of HIV-1 infection (Cocchi et al. 1995; Dragic
et al. 1996; Oravecz et al. 1996; Moriuchi et al. 1998; Zagury et al. 1998). In addition,
the HTLV-2 positive regulator Tax-2 has been shown to induce the expression of
MIP-1b and RANTES (Lewis et al. 2000), but little is known about its effect on
MIP-1a. Interestingly, Tax-1 positively regulates the expression level of MIP-1a
(Baba et al. 1996). Tax-2 also can inhibit HIV-1 replication by interacting with the
major histocompatibility complex class II transcriptional activator (CIITA) (Accolla
660
Fig. 26.6 A model of the interplay between HTLV-2 and HIV-1 in coinfected individuals. HTLV-2
induces several CC chemokines leading to inhibition of HIV-1 replication
et al. 2001; Casoli et al. 2004). CIITA can inhibit Tax-2 transactivation of the HTLV
LTR, and similarly inhibit HIV replication by targeting the HIV positive regulator
Tat (Accolla et al. 2002). HTLV-2 infected cells also secrete additional chemokines
and cytokines that induce a protective Th1 response against invading pathogens
(OGarra 1998); a Th2 response seems to positively correlate with progression of
HIV-1 infection (Clerici and Shearer 1994) (Fig. 26.6).
Although the effect of HTLV-2 on HIV-1 appears more prominent than that of
HIV-1 on HTLV, it has been demonstrated that HIV-1 Tat up-regulates both HTLV-1
and HTLV-2 gene expression in coinfected individuals (Beilke et al. 1998; Roy et al.
2008). The long-term implication of such interactions warrants further investigation
for its importance on AIDS and HTLV-associated disorders. For example, it is
poorly understood whether HTLV-2HIV-1 coinfection can lead to additional
complications, and epidemiological data suggest an increase in the incidence of
neurologic disorders and liver dysfunction in HTLV-2HIV-1 dually infected individuals (Beilke et al. 2004). On the contrary, special consideration should be given
to HTLV-2HIV-1 patients regarding highly active antiretroviral therapy (HAART).
661
HAART can reduce HIV-1 morbidity and mortality but appears to increase HTLV-2
proviral load. In addition, the disorders affecting HAART treated HIV-1/HTLV-2
coinfected individuals are more of an inflammatory nature than of opportunistic
pathogens. This is consistent with a greater incidence of neurological diseases in
HIV-1/HTLV coinfected individuals (Casoli et al. 2007).
Tax Oncoprotein Mechanisms of Action

Tax is a positive regulator of viral replication and displays oncogenic properties
including the transformation of rodent fibroblasts and induction of tumors in
transgenic animals. In addition, several studies using infectious molecular clones of
HTLV-1 and HTLV-2 showed a direct role of Tax in transformation of human
T-lymphocytes, and ample evidence points to a pivotal role of Tax in HTLV pathogenesis (Nerenberg et al. 1987; Tanaka et al. 1990; Grassmann et al. 1992; Yamaoka
et al. 1992; Ross et al. 1996; Robek and Ratner 1999). Tax functions both at the
transcriptional and posttranscriptional levels by modulating viral and cellular proteins
required for viral replication and transformation. The different subtypes of HTLV-2
(AD) encode slightly different Tax proteins. Tax-2B, 2C, and 2D are similar but
not identical and vary slightly in length (356, 356, 344 amino acids), whereas
Tax-2A is shorter and contains 331 amino acids (Feuer and Green 2005). However,
it is not known if the different forms of Tax-2 contribute differently to viral replication, lymphocyte immortalization, and/or persistence.
Tax and Transcription

Tax was first identified as a positive regulator of viral transcription. Interestingly,
Tax is capable of transactivating several cellular genes important for cell proliferation and eventually transformation. Tax is expressed early after infection, and
transactivates viral gene expression by recruiting activating transcription factor
(ATF) and cyclic-AMP response element binding (CREB) proteins to the cyclicAMP response element (CRE) present at the viral promoter located at the 5LTR.
Tax recruits these coactivators of transcription to its response element (TRE)
located in the U3 region of the 5LTR (Zhao and Giam 1992; Wagner and Green
1993; Adya et al. 1994; Anderson and Dynan 1994; Yin et al. 1995; Bantignies
et al. 1996). Recently, Tosi et al. have demonstrated that Tax-2 has the ability to
recruit CBP/p300 to the viral LTR, while Tax-1 recruits CBP/p300-associated factor
(PCAF), histone acetyl transferases (HATS) and CBP/p300 (Tosi et al. 2006). This
difference might, in part, explain the differential profile of genes activated by Tax-1
compared to Tax-2. A mutational analysis was carried out to better understand the
contribution of the different domains of Tax-2 to its transcriptional activity. Tax-2
662
mutations that disrupted the zinc-finger domain and that affected the CREB/ATF
domain failed to transactivate the HTLV-2 LTR (and HTLV-1 LTR) (Ross et al.
1997). However, it was found that HTLV-2-mediated T-cell transformation is
dependent on Tax-2 activation of both NFkB and CREB/ATF, which then induces
IL-2 independent T-cell transformation (Feuer and Green 2005; Ross et al. 1996).
The activation of NFkB is absolutely essential at early stages of cellular transformation, while CREB/ATF activation by Tax is required for sustained cell growth
(Ross et al. 2000). Recently, it has been shown that Tax-2 can interact with CIITA
and NF-Y and this interaction was found to inhibit Tax-2-mediated activation of
the viral LTR. CIITA is the master regulator of the expression of MHC class II
genes that are important for the homeostasis and regulation of the immune system
(Reith et al. 2005). CIITA is a non DNA binding transcriptional integrator recruited
to the promoter by multiple transcription factors including nuclear factor Y (NF-Y)
(Kretsovali et al. 1998; Spilianakis et al. 2000; Fontes et al. 1999a, b). One study
showed that CIITA targets HIV-1 Tat and represses its transactivation of the HIV
LTR (Accolla et al. 2002). Similarly, CIITA inhibits Tax-2 transactivation of the
LTR promoter in HLA-II positive cells by recruiting NF-Y (Casoli et al. 2004; Tosi
et al. 2006). The repression of Tax-2 transcriptional activity is the major, and
probably the exclusive mechanism involved in reducing HTLV-2 replication in
B-cells and T-cells (Tosi et al. 2009).
Tax Posttranscriptional Effects

The transformation properties of Tax are a consequence of the ability of the protein
to deregulate the transcription of genes and signaling pathways involved in cellular
proliferation, cell cycle control and apoptosis (Hall and Fujii 2005). Interestingly,
the Tax-1 carboxy terminus was shown to be responsible for increased transformation efficiency in rodent fibroblasts and increased micronuclei formation (Rousset
et al. 1998; Hall and Fujii 2005). This region contains a PDZ binding motif (PBM),
which is absent in Tax-2. The PDZ domain is a proteinprotein interaction region,
and is important for the interaction of Tax-1 with tumor suppressors such as hDLG,
APC, Dlg-1, Scribbles, or precursor of interleukin-16 (proIL-16), and membraneassociated guanylate kinase (MAGUK) with inverted orientation (MAGI)-3 (Suzuki
et al. 1999; Wu et al. 2000; Adamsky et al. 2003; Wilson et al. 2003; Ohashi et al.
2004; Yao et al. 2004; Ishioka et al. 2006; Okajima et al. 2008). In addition, this
domain was found to contribute to HTLV-induced proliferation and immortalization
of primary T-cells in vitro and viral survival in the rabbit animal model (Tsubata et al.
2005; Xie et al. 2006). A chimeric Tax-2 encoding the last 53 amino acids of Tax-1,
which contains the PBM, resulted in increased transformation of Rat fibroblasts
(Endo et al. 2002), increased micronuclei formation and increased T-cell proliferation in vitro (Xie et al. 2006). Data suggest that the interplay between the PDZ
domain and interacting partners are major players in the differences in pathogenesis
between HTLV-1 and HTLV-2.
663
Tax/NFk B Interaction
NFkB is a family of transcription factors that include RelA, RelB, c-Rel, NFkB1
(p105 catalyzed into p50), and NFkB2 (p100 catalyzed into p52). These transcription
factors play an important role in immune function, innate and adaptive responses,
development, and cell survival (Vallabhapurapu and Karin 2009). NFkb activity is
tightly controlled in T-cells, but is constitutively active following HTLV infection.
In the canonical pathway, NFkb is inhibited by inhibitor of kb (Ikb), which in turn
is under the control of inhibitor of kb kinase (IKK). Tax ubiquitination is crucial for
its interaction with the IKKg subunit (NEMO) and subsequent activation of NFkB
(Shembade et al. 2007). One study reported that once Tax-2 is polysumoylated, it
localizes to the nucleus (similar to Tax-1), thus bringing it in close proximity to
RelA (Turci et al. 2009) and bypassing IKK and Ikb. On the contrary, the noncanonical
pathway involves IKK phosphorylation of p100 in the NFkB2/p100 complex with
subsequent processing into p52. In contrast to Tax-1, Tax-2 does not affect the
noncanonical pathway (Higuchi et al. 2007; Higuchi and Fujii 2009). A region in
Tax-1 located at 225232 and not conserved in Tax-2 is responsible for the Tax1-p52 interaction (Shoji et al. 2009).
Tax-2 and Cell Cycle Dysregulation

Cell cycle is a tightly regulated process, its perturbation is a hallmark of cellular
transformation, and is a common target for viral oncoproteins (Feuer and Green 2005).
The cell cycle is regulated by cyclin-dependent kinases (CDKs) and different D and
E type cyclins. CDKs are under the control of CDK inhibitors (CDKI) such as p16ink
and p21cip1/waf (Akagi et al. 1996; Mori et al. 2002). p21cip1/waf is part of the CDK2/
D1/PCNA complex and is required for G1 and G2 control; however, over-expression
of p21 inhibits G1 and G2 control via p53-dependent and -independent mechanisms
(Macleod et al. 1995). Stably transfected cell lines that produce Tax-2 (or Tax-1)
have been useful in understanding the effect of Tax-2 on cellular proliferation. Tax-2
modulates the cell cycle at several steps. Tax-2 activates the expression of p21cip1/waf,
but at a lower extent than Tax-1 (~2535% less) (de La Fuente et al. 2000; Sieburg
et al. 2004). In addition, Tax-2 has been shown to reduce cellular proliferation kinetics
in Jurkat cells but to a lesser extent than Tax-1 (Sieburg et al. 2004; Feuer and Green
2005). Tax-2 induces the formation of multinucleated cells, suggesting that it might
block progression or completion of mitosis or cytokinesis due to disruption of cell
cycle checkpoints leading to unscheduled S phase entry and accumulation of DNA
damage (Lemoine and Marriott 2001; Marriott et al. 2002).
A great deal of knowledge about Tax comes from over-expression in transfected
cell lines that do not represent the viral target cells in vivo, or are not likely to be
encountered by the virus. Studies in primary hematopoietic CD34+ progenitor cells
(HPC) failed to show arrest of the cell cycle, suppression hematopoiesis, or modulation of p21cip1/waf1 or p27kip1 gene expression via Tax-2 (contrary to Tax-1) (Feuer and
Green 2005).
664
Tax-2 Modulation of Apoptosis

Apoptosis, a form of well controlled programmed cell death, is marked by a defined
sequence of morphological changes. Apoptosis is required during embryonic development and in adult life to maintain proper homeostasis. It has been well documented
that disruption of apoptosis can lead to cancer, neurodegenerative diseases, and immunological diseases. Expression of both Tax-1 and Tax-2 protects cells from apoptosis
by modulating the activity of several pro-apoptotic genes. For example, inhibition of
p53 function is associated with impaired function of Tax-2A. Interestingly, Tax-2B
has been found to prevent apoptosis in infected cells by modulating p53 activity, an
activity shared with Tax-1 (Mahieux et al. 2000; Van et al. 2001). Tax-2B represses
p53 in T-cells, which is correlated with NFkB activation. However, in nonlymphoid
cells, Tax-2B uses CBP binding to inhibit expression of p53, most likely through the
interaction of CBP and the CR2 sequence of Tax-2 (Meertens et al. 2004b).
Tax-2 also has been found to protect Jurkat T-cells as well as murine fibroblasts
from apoptosis following serum deprivation (Saggioro et al. 2001; Sieburg et al. 2004).
In addition, Tax-2 protects the cells from Fas-mediated apoptosis by up-regulating
Bcl-XL expression (Zehender et al. 2001).
HTLV Experimental Models

The development of culture and animal models to study HTLV-2 infection are important
since they have the potential to yield valuable information about requirements for
infectivity, persistence, prevention and treatment of the virus infection (Cockerell et al.
1991). HTLV experimental systems present a great challenge due to the poor replication
of the virus in vitro and the inefficiency of cell-free infection; efficient infection requires
the cocultivation of irradiated cells with PBMC. In addition, HTLV infects a wide
variety of cells including B-cells, T-cells, endothelial cells, glial cells, and monocytes;
but only primary T-cells are susceptible to transformation (Ho et al. 1984; Hoffman
et al. 1984; Akagi et al. 1992; Koyanagi et al. 1993). Transformation of T-cells by
HTLV is defined as IL-2-independent growth. Morphologically, transformed cells are
evident microscopically as refractile cell clusters within 710 weeks of coculture;
however, establishing transformed cells requires months in culture. Miyamoto et al.
demonstrated that rabbit leukocytes can be transformed in vitro by HTLV-2 (Miyamoto
et al. 1990). Rabbits then were assessed for their ability to be infected with HTLV-2.
The in vivo infectivity was evident as early as 2 weeks and persisted until 24 weeks (the
end of the experiment) as measured by anti-HTLV antibody response (against p24 and
env gp46) and demonstration of viral antigens or detectable proviral sequences in tissues
of inoculated rabbits (Cockerell et al. 1991). Interestingly, HTLV-2 was found to be
less infectious, and to replicate less efficiently in the rabbit model than HTLV-1
(Cockerell et al. 1991; Lairmore et al. 1992). In addition, HTLV-2 infection does not
seem to correlate with any clinicopathologic evidence of disease in rabbits (Cockerell
et al. 1991; Yamamoto et al. 2008; Xie et al. 2009).
665
Conclusion
In recent years, there has been increased interest in HTLV-2 because it has been
associated with several neurological abnormalities, increased viral spread due to
IVDU and contaminated drug-related paraphernalia, and an effect on AIDS progression in HIV-1HTLV-2 coinfected individuals. Furthermore, comparative studies
between HTLV-1 and HTLV-2 have provided additional insight into the activity and
function of viral regulatory and accessory proteins in the context of the host and
during viral replication. Nevertheless, similarities and differences occur between
HTLV-1 and HTLV-2 that account for the different pathological effects observed. It
is becoming apparent that these differences cannot be attributed to any one single
viral or host protein, but likely involves a contribution of Env (tropism), Tax, and
accessory gene products and their differential interaction with host cellular factors.
Despite advances made in understanding the molecular basis of viral infection, little
is known about HTLV-2 pathogenesis, and research is warranted to better understand
(1) the causes of the long latency period of HTLV-2 infection, (2) the reasons why
HTLV-2 is associated with few cases of neurodegenerative diseases but no malignancies (unlike HTLV-1), (3) the effect of the viral infection on the immune system
both short-term and long-term, and (4) the role of the host (in particular the restriction
factors) in controlling HTLV-2 infection.
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Chapter 27
Mechanisms of Oncogenesis by Avian

and Murine Retroviruses
Karen Beemon and Naomi Rosenberg
Introduction
The oncogenic retroviruses (formerly called RNA tumor viruses) have played an
important role in cancer research since their discovery in chickens 100 years ago.
The discovery of retroviral oncogenes established the central paradigm that cancer is
a genetic disease. Since retroviral oncogenes are captured host genes, study of animal
retroviruses is very relevant to understanding mechanisms of human cancer initiation
and maintenance. Investigations of tumors induced by oncogene-containing retroviruses first demonstrated that altered or overexpressed cellular genes can provide a
dominant signal initiating oncogenesis. The obligate integration step that characterizes retroviral replication allows these viruses to alter the expression of cellular genes
by insertional mutagenesis. Our knowledge of the ways in which cellular genes can
contribute to cancer is based on fundamental studies of retroviruses. Furthermore,
many genes that participate in human tumor development were first isolated as
retroviral genes or as sites of retroviral integration. Thus, studies directed at understanding viral mechanisms of oncogenesis revealed insights into more general cellular
mechanisms of tumor induction. Novel ways to disrupt normal cell function and to
regulate gene expression have all emerged from the study of these viruses.
Several different mechanisms of oncogenesis have been associated with different
classes of retroviruses (Table 27.1). First, the oncogene-containing retroviruses,
such as the avian Rous sarcoma virus (RSV), the murine sarcoma viruses (MSV),
K. Beemon (*)
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
e-mail: KLB@JHU.edu
N. Rosenberg
Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine,
Boston, MA 02111, USA
677
678
K. Beemon and N. Rosenberg
Table 27.1 Mechanisms of retroviral oncogenesis

Mechanism and representative viruses
Oncogene(s)
Oncogene capture
Rous sarcoma virus
v-src
MC29
v-myc
Fujinami sarcoma virus
v-fps
Avian myeloblastosis virus
v-myb
Reticuloendotheliosis virus-T
v-rel
Abelson murine leukemia virus
v-abl
Moloney murine sarcoma virus
v-mos
Ha/Ki murine sarcoma virus
v-ras
Feline sarcoma viruses
v-fes, v-fms, v-fgr
Simian sarcoma virus
v-sis
Walleye dermal sarcoma virus
rv-cyclin
Insertional mutagenesis
Avian leukosis virus
Murine leukemia virus
Feline leukemia virus
Mouse mammary tumor virus
myc, myb, erbB, TERT, bic, and others

myc, myb, and others
myc, myb, and others
wnt-1, fgf, and others
microRNA induction
Avian leukosis virus
Reticuloendotheliosis virus-T
Radiation leukemia virus
Friend murine leukemia virus
SL3-3 murine leukemia virus
miR-155
miR-155
miR-106-363 cluster
miR-17-92 cluster
miR-17-92, miR-106-363 clusters
Env signaling
Friend spleen focus-forming virus
Jaagsiekte sheep retrovirus
Avian hemangioma virus
altered env product

altered env product
altered env product
Accessory genes
Bovine leukemia virus
Human T-cell leukemia virus
tax
tax, HTLV-I basic leucine zipper factor (HBZ)
and Abelson murine leukemia virus, have all captured oncogenes from their hosts.
Second, a large number of viruses lacking oncogenes, including avian leukosis virus
(ALV), murine leukemia virus (MLV), feline leukemia virus (FeLV), and murine
mammary tumor virus (MMTV), activate cellular oncogenes by insertional mutagenesis. A third type of oncogenesis involves activation of cellular microRNAs,
either by insertional mutagenesis (ALV and MLV) or by transcriptional activation
(reticuloendotheliosis virus). A fourth type of viral oncogenesis involves signaling
by the viral env glycoprotein gene and is used by Friend spleen focus forming virus
(SFFV) and Jaagsiekte Sheep Retrovirus (JSRV). Lastly, accessory (nonstructural)
genes of human T-lymphotropic virus (HTLV) and bovine leukemia virus (BLV)
such as tax and HBZ are key to cancer induction by these agents. HTLV is discussed
in a separate chapter in this book. We discuss the first four mechanisms of oncogenesis here.
27
Mechanisms of Oncogenesis by Avian and Murine Retroviruses
679
Types of Oncogenic Retroviruses

The prototypical oncogenic retroviruses were discovered over 100 years ago. RSV
was discovered by Peyton Rous in 1910 (Rous 1910) and shown to be a filterable
agent that causes solid tumors in chickens. Ellerman and Bang discovered ALV in
1908 (Ellerman and Bang 1908) and showed that the virus causes leukemia and
lymphoma in chickens. Subsequent studies in mammals and other hosts led to
discoveries of other tumor-inducing viruses, some of which contained oncogenes in
their genomes and others that did not. The early discoveries of Bittner (Bittner 1942)
and Gross (1951a, b) revealed that retroviruses were associated with mammary
tumors and thymic lymphomas in mice. Subsequent studies revealed that many
animals are infected by oncogenic retroviruses including cats, cows, rats, sheep,
goats, koalas, several primates, and some fish (Rosenberg and Jolicoeur 1997;
Hanger et al. 2000). Retroviral particles have also been found in tumors in vipers,
but their role in the disease process has not been studied thoroughly (Andersen et al.
1979). The isolation of HTLV in 1980 marked the discovery of a retrovirus that
caused malignant disease in humans (Poiesz et al. 1980).
All of these viruses belong to the retrovirus subfamily called Orthoretrovirinae
(Linial et al. 2005), a group that contains six different genera that are named for the
first six letters in the Greek alphabet (e.g., Alpharetrovirus, Betaretrovirus, etc).
The classification scheme is based on genome structure, virion morphology, and
sequence relationships. All six of these genera contain members that have oncogenic
potential. In addition to classification by genera, oncogenic retroviruses are often
divided into two groups based on the mechanisms by which they induce tumors. One
group of viruses contains oncogenes, while the other group does not. Viruses of the
first type were derived from the second type by a process called oncogene capture.
Retroviral oncogenes, called v-onc genes, were derived from cellular sequences
called protooncogenes (c-onc genes) and incorporated into the viruses by recombination during virus integration and replication. Typically, these viruses are replication
defective because one or more genes required for virus replication have been lost as
a consequence of recombination with the c-onc gene. Viruses of this type typically
induce tumors with a relatively short (several week) latent period and require a helper
virus for replication. These viruses also typically transform cells in tissue culture, a
property that advanced studies of oncogenic mechanisms used by these viruses.
The second type of oncogenic retrovirus does not contain genes derived from
cellular sequence and typically induces tumors by integrating into the host genome
and altering the expression of a cellular gene. The majority of these viruses induce
tumors after a much longer latent period than viruses that contain v-onc genes with
tumors arising several months or more after infection. In addition, viruses of this
type do not transform cells in tissue culture. ALV and MLV are prototypes of these
viruses. A second type of virus that lacks an oncogene uses a modified env gene
product to stimulate cell growth. Viruses of this type, such as JSRV and SFFV, are
associated with tumors that arise in a relatively short latent period. They also transform
some cell types in tissue culture. These properties reflect the fact that a virus-encoded
product is responsible for causing altered growth.
680
Fig. 27.1 Oncogene capture and the generation of v-onc-containing viruses. The process by which
oncogenes are thought to be incorporated into retroviruses is illustrated. Each step is described in
more detail in the text. Cellular exons: dark boxes; Cellular regulatory sequences: gray boxes;
RNA: dashed lines with spliced information shown as solid lines
Oncogene Capture and the Generation

of v-onc-Containing Viruses
Oncogene capture is the process by which portions of c-onc genes are incorporated
into retroviruses. Although over 100 different retroviruses have captured v-onc genes
(Rosenberg and Jolicoeur 1997), the process is actually rare. Most of these viruses
arose once and were identified because the animal from which the virus was isolated
was part of a laboratory experiment or in close contact with people. Most commonly,
these latter instances have involved pets, especially cats, and farm animals, especially chickens. The rarity with which v-onc gene capture occurs has made it difficult
to study the process directly. Thus, the precise mechanisms involved have received
limited experimental validation. Indeed, much of the information that has contributed
to the model by which oncogene capture occurs has involved comparison of the viral
and cellular sequences and a general knowledge of retrovirus replication.
The integration of a replication-competent retrovirus into the host genome, an
obligate step in the life cycle of all retroviruses, is the first step in oncogene capture
(Fig. 27.1). Following integration, the viral genome is transcribed using cellular
machinery and although most transcripts terminate in the 3 LTR, a fraction fail to
do so and continue into flanking cellular sequences generating transcripts called
readthrough transcripts. In one instance where this phenomenon has been studied
(ALV), about 15% of the transcripts were shown to be of this type (Herman and
Coffin 1986). In cases where the virus integration has occurred near a c-onc gene,
the read-through transcript can contain these sequences. Because integration is
largely random (Bushman et al. 2005), this event occurs rarely. However, if the
27
681
readthrough transcript can lead to expression of the c-onc gene product, the cell is
likely to be at a selective advantage.
The second step in oncogene capture involves the packaging of the readthrough
transcript in virions that are released from the cell. Retrovirus virions contain two
copies of the virus genome, a feature that is necessary for successful replication
following infection of a cell. Reverse transcription requires both copies to generate
the cDNA copy that goes on to integrate into the genome. The final step in the
process occurs after the virion containing one normal genome and one hybrid transcript infects a new cell. As part of the reverse transcription process, the template
switching between the two RNAs can lead to recombination and incorporation of
the cellular sequences within the viral genome (Fig. 27.1). The general aspects
of this model are consistent with studies done using plasmids that mimic features of
some of the intermediates that are postulated to play a key role in oncogene capture
(Swain and Coffin 1992).
RSV and Oncogene-Containing Retroviruses

The earliest characterized oncogene was the v-src oncogene of RSV (Martin 2001).
The genomic RNA of RSV was found to be about 2 kb larger than that of ALV
(Duesberg and Vogt 1970). Comparisons by RNase T1 fingerprinting between ALV
and RSV genomic RNA showed them to be very closely related. However, RSV had
an additional genomic sequence that was mapped near the 3 poly(A) sequence
(Wang et al. 1975). Hybridization experiments, using a src-specific probe, showed
that uninfected cells had related sequences; these were called c-src (Stehelin et al.
1976). In vitro translation experiments, together with generation of src-specific antibodies, defined the Src protein as a 60 kDa phosphoprotein called pp60src (Beemon
and Hunter 1978; Brugge and Erikson 1977). The c-Src protein had a C-terminal
amino-acid sequence, with repressive activity, that was replaced in the v-Src protein.
Surprisingly, Src was found to be a tyrosine-specific kinase, a novel specificity at
the time (Hunter and Sefton 1980).
To date, approximately 100 different oncogene-containing retroviruses have
been described. These viruses are derived from a variety of different hosts, with the
majority coming from chickens, mice, cats, and rats. All of these animals can be
readily infected with the replication-competent, non-oncogene-containing retroviruses
that serve as the viral parent of these agents. Such infections are a prerequisite for
generation of retroviruses that contain oncogenes.
As noted earlier, most of these viruses are replication defective because the function
of at least one viral gene required for replication has been lost during oncogene
capture. RSV is a notable exception to this generalization and is the only replicationcompetent, v-onc gene-containing virus; it probably arose from more than one
recombination event. The other viruses of this type exist naturally in combination
with a replication-competent virus that provides the proteins required for replication.
Viruses performing this function are typically referred to as helper viruses.
Although helper viruses play a role in the spread of viruses containing v-onc genes,
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Fig. 27.2 Oncogene-containing retroviruses encode viral-onc fusion proteins. The structure of
representative v-onc gene containing retroviruses is illustrated. The first five viruses were derived
from ALV, and the last two were derived from MLV. Sequences derived from the replicationcompetent parent of the viruses are shown as open boxes; v-onc gene sequences are shown as
shaded boxes. The proteins encoded by the v-onc genes are also illustrated but viral proteins that
are expressed by some of the viruses are not shown. The drawing is not precisely to scale
study of helper virus free stocks, prepared using plasmids that provide the proteins
required for replication, has revealed that the helper virus is not required for tumor
induction or for transformation of cells in tissue culture (Green et al. 1987; Andersson
et al. 1979; Dunbar et al. 1991; Fichelson et al. 1995).
The genome structure of viruses that contain v-onc genes varies, but in all cases,
the structure leads to a protein product that is directly capable of stimulating cell
growth (Rosenberg and Jolicoeur 1997). In many instances, the v-onc gene exists as
a fusion with part of a viral gene, particularly the gag gene that normally encodes
virion structural proteins (Fig. 27.2). The product of such genes is typically a fusion
27

Table 27.2 Functions of viral oncogenes
Protein Function
Gene
Growth factor
v-sis
Receptor tyrosine kinase
v-erbB
c-erbB
v-kit
v-fms
Nonreceptor tyrosine kinase
v-src
v-fps
v-abl
v-fgr
v-fes
G protein
v-rasK
v-rasH
Serine/threonine kinase
v-mos
v-raf
v-akt
Adapter protein
v-crk
v-erbA
v-myc
v-myb
v-ets
v-fos
v-rel
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Virus
SSV
AEV
ALV
HZ4-FeSV
SM-FeSV
RSV
FuSV-ASV
Ab-MLV
GR-FeSV
ST-FeSV
Ki-MSV
Ha-MSV
Mo-MSV
3611-MSV
Akt8
CT-10-ASV
AEV
MC29
BAI-AMV
ALV
E26-AMV
FBR-MSV
protein containing sequences from both genes, and the determinants derived from
the viral gene can influence the localization or function of the oncoprotein. Some
v-onc fusions involve the viral env gene. Lastly, some v-onc genes are expressed
without additional sequences derived from viral genes. In a few cases, viruses that
contain two v-onc genes have been isolated. Examples include some isolates of
avian erythroblastosis virus that contain both v-erbA and v-erbB (Engelbreth-Holm
and Rothe-Meyer 1935), MH2, an avian virus that induces several types of tumors
and contains both v-mil (v-raf) and v-myc, (Begg 1927) and E26, an avian virus that
induces myeloblastosis and contains both v-myb and v-ets (Ivanov et al. 1962).
Because the protooncogenes that were captured in these cases are not physically
linked in the genome, these viruses appear to have arisen as a consequence of two
independent capture events. Experimental evidence shows that both oncogenes
contribute to the types of tumors induced by these viruses (Metz and Graf 1991;
Beug and Graf 1989; Hartl et al. 2006).
A survey of the different proteins encoded by retroviruses expressing v-onc genes
reveals examples of molecules in many signaling pathways that modulate cell growth
and differentiation (Table 27.2). For example, a number of oncoproteins, including
Abl, Fes, Fps, and Yes, encode protein intracellular tyrosine kinases. Others, such as
Fms, Fgr, Kit, ErbB, and Met, encode protein tyrosine kinase receptors, while Sis is a
growth factor. Serine/threonine kinases including Akt, Raf, and Mos, the G protein
Ras, and the adaptor protein Crk are expressed by one or more oncogenic retroviruses.
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Fig. 27.3 Activation of cellular oncogenes by insertional mutagenesis. Four different mechanisms
by which cellular protooncogenes can be activated by insertional mutagenesis are illustrated:
I. downstream transcriptional activation; II. enhancer mediated activation; III. read-through transcription; IV. alteration of 3 regulatory elements. Additional details are presented in the text
Transcription factors including Myc, Myb, Fos, Jun, ErbA, and the Cbl E3 ligase are
other types of signaling molecules that are responsible for the oncogenic properties
of rapidly transforming retroviruses.
Activation of Cellular Oncogenes by Insertional Mutagenesis

Many oncogenic retroviruses do not contain oncogenes, and viruses of this type are
common in many species and exist naturally. They are the predominant cause of
retroviral induced tumors outside of laboratory settings (Rosenberg and Jolicoeur
1997). The long latency associated with oncogenesis by these viruses and their close
relationship to endogenous retrovirus elements that exist in the genomes of many
animals contributes to their maintenance in animal populations. The oncogenic properties of these viruses reflect the fact that integration is an obligatory part of the retroviral
life cycle and thus these agents are insertional mutagens that disrupt the DNA structure
at the site of integration (Fig. 27.3). These disruptions can separate exons of cellular
genes, resulting in the production of nonfunctional proteins or proteins with altered
function. They can also separate regulatory elements such as 3 untranslated sequences
that control the stability of cellular mRNAs from coding sequences. As a consequence,
altered expression of genes near integration sites can contribute to oncogenesis.
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A second consequence of integration relates to the structure of the integrated

provirus, which has strong promoter and enhancer sequences within the long terminal
repeats (LTRs) that are located at both ends of the genome. The LTRs also contain
other regulatory sequences such as polyadenylation sequences that are required for
proper expression of viral RNAs. These sequences allow the virus to integrate moreor-less randomly in the host genome and express the viral genes. However, these
sequences can also affect the expression of genes in the vicinity of the integration.
The promoter sequences found in the LTRs substitute for the promoter normally
associated with a cellular gene in the vicinity of the integration. In addition, the
strong enhancer sequences located in both LTRs can stimulate expression of cellular
genes, including some that are located at considerable distance from the integration
site and like all enhancers, can function independent of orientation. Examples of all
of these mechanisms have been found in a wide range of retrovirus-induced tumors.
Promoter Insertion
Study of ALV-induced bursal lymphoma led to the discovery of the promoter insertion
mechanism of retroviral oncogenesis. This tumor arises in the Bursa of Fabricius, an
organ that generates B lymphocytes in birds. The first clues to the mechanism were
the presence of common integration sites in all cells, demonstrating a clonal relationship and indicating that the cells in the tumor arose from a single virus-infected
cell. These early Southern blot analyses suggested that the integration site of the
virus might be related in some fashion to tumor induction (Neel et al. 1981; Payne
et al. 1981). Further study showed that the tumors contained novel fusion mRNAs
with both viral and cellular sequences. Hayward and coworkers (1981) found that
most ALV-induced tumors had sense integrations in the cellular myc locus, an oncogene previously observed in the oncogene-containing MC-29 virus that induces
myelocytomatosis.
In most tumors, the provirus had integrated into the first intron of myc, downstream of a transcriptional pause site. Surprisingly, the 3 LTR drove transcription of
the downstream cellular myc gene by a mechanism called Promoter Insertion
(Hayward et al. 1981). The 5 LTR was not used in these tumors because of mutations downstream of the 5 LTR which somehow inactivated it (Fung et al. 1982;
Goodenow and Hayward 1987). Since the first c-myc exon is noncoding, this resulted
in overexpression of the normal cellular Myc protein, a transcription factor that is
normally expressed only briefly during the cell cycle. This was the first evidence of
tumor initiation by disregulated expression of a normal cellular gene. It was predicted
that nonviral agents might also induce elevated expression of c-onc genes. In fact,
this was rapidly born out when translocations leading to myc overexpression were
discovered in Burkitts lymphoma (Klein 1983; Taub et al. 1982). Myc has also
been activated by insertional mutagenesis by MLV and FeLV in a large number of
mouse and cat tumors (Neil and Stewart 2011).
Retroviruses use promoter insertion to induce tumors involving activation of
other protooncogenes. For example, in certain lines of chickens, the c-erbB gene is
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activated by ALV integration, an event that induces erythroblastosis (Nilsen et al.

1985). In these tumors, transcription initiates in the viral 5 LTR and reads through
the poly(A) site into the downstream c-erbB gene, which encodes an EGF receptor
protein family member. Alternative splicing generates both Gag-ErbB and GagEnv-ErbB products. v-erbB is the oncogene of avian erythroblastosis virus and is an
amino-truncated form of the EGF receptor, lacking the EFG-binding domain.
Remarkably, ALV integrations within the middle of the c-erbB gene result in
generation of a similarly truncated form of the EGF receptor.
Promoter insertion can also be associated with oncogenesis in other animals
(Rosenberg and Jolicoeur 1997). For example, a variety of MLVs use this mechanism
to induce thymic lymphoma, a T-cell tumor that is one of the most common types of
retrovirus-induced tumor in mice and rats. Moloney MLV can activate the protooncogenes gfi-1 (Gilks et al. 1993), lck (Shin and Steffen 1993) and others via this
mechanism. Several CasBr strains of MLV, viruses that induce several hematologic
tumors, have been shown to cause tumors in the same fashion (Bergeron et al. 1993;
Askew et al. 1991). Promoter insertion has also been documented in T cell lymphomas
induced by FeLV in domestic cats (Neil et al. 1984; Forrest et al. 1987).
Insertions Affecting Regulatory Sequences

While the majority of ALV integrations in classical lymphoid leukosis were in myc
intron 1 or upstream of it, integrations were also observed in the 3 untranslated
region (3UTR) (Payne et al. 1982). The 3 UTR insertions most likely supplied a
new poly(A) site, leading to a shorter 3UTR. While it was not clear why this event
was oncogenic at the time, we now know that mRNAs are often downregulated by
interactions of microRNAs or regulatory proteins with the 3UTR. Thus, a shorter
3UTR would likely stabilize the mRNA, leading to overexpression of Myc.
Activating retroviral insertions have also been seen in the 3 UTRs of other oncogenes,
including pim1 and gfi1 (Dabrowska et al. 2009; Selten et al. 1985). Recently, global
shortening of 3UTRs of the majority of mRNAs has been observed in cancer cells
(Mayr and Bartel 2009) and in proliferating cells (Sandberg et al. 2008), thus making
the surprising observation of 30 years ago a more general phenomenon.
A second example of regulatory sequence disruption has been documented in
ALV-induced tumors that arise following infection of 1014 day gestation chick
embryos. In contrast to infection of newly hatched chicks where most ALV infection
leads to bursal lymphoma after 46 months, about 10% of the chicks infected as
embryos die from B-cell lymphomas in 12 months (Kanter et al. 1988; Pizer and
Humphries 1989). These short latent period tumors often involve integration into
the c-myb locus, a site that is not associated with the longer latency tumors that typically arise following neonatal infection. The incidence of short-latency lymphomas
was much higher with a virus called EU8 that was found to have a 42-nt deletion in
the gag gene that was important for the short latency (Jiang et al. 1997; Smith et al.
1997). This deletion occurred in an RNA regulatory sequence called the negative
regulator of splicing (NRS), which inhibits splicing and promotes polyadenylation
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(McNally et al. 1991; OSullivan et al. 2002). A single point mutation in this
element also led to rapid tumorigenesis (Polony et al. 2003). Most myb integrations
were in intron 1, leading to a slightly truncated Myb protein which is highly oncogenic (Jiang et al. 1997). The truncated Myb protein lacks the N-terminal 20 amino
acids, but their function is not known. The rapid-onset lymphomas required infection of embryos at mid-gestation and involved Myb overexpression, suggesting that
the target cells are different than for the later occurring leukemias associated with
myc integrations.
Enhancer-Mediated Insertional Mutagenesis

During the same period when investigators were examining oncogenic mechanisms
in the bursal lymphoma model, other scientists were focused on tumorigenesis in
murine models using a variety of murine viruses including Moloney and Friend
MLV, mouse mammary tumor virus (MMTV), and other viruses. Similar to the situation
with ALV, tumors induced by these viruses were clonal with respect to virus integration. However, the initial analyses were complicated by the fact that the presence of
large numbers of cross-reacting endogenous virus sequences made it difficult to
identify integration patterns. Also, in most cases, no fusion RNAs similar to those
detected in ALV-induced bursal lymphoma were found. Rather, enhancer sequences
in the U3 region of the retrovirus LTRs were found to activate genes in the vicinity
of the integration site.
Early clues to the importance of the enhancer elements in insertional mutagenesis
came from studies in which changes in the enhancer were shown to affect the type
of tumor that was induced. For example, chimeric viruses constructed using F-MLV,
which induces erythroleukemia and M-MLV, which induces T cell lymphoma,
revealed that determinants in the LTR determined the type of tumor that was induced
(Chatis et al. 1983). Repeats within the core sequence of the LTR enhancer or point
mutations in these sequences also influence tumorigenicity by these viruses
(Lenz et al. 1984; Morrison et al. 1995). Another example is variants of MMTV that
induce T cell lymphoma rather than mammary tumors (Ball et al. 1988).
Enhancer insertion involves activation of expression of the promoter of a cellular
gene (or relief of repression) by complexes formed at the enhancer sequences in the
retroviral LTR enhancer. In many cases, the integration site is upstream of the
transcription start site of the cellular gene and in the opposite orientation. Since
retroviral enhancers tend to be stronger than cellular enhancers, enhancer insertion
leads to overexpression of the product of the affected gene, thereby leading to
altered cell growth. In some cases, more than one cellular gene is affected and the
products of all of the genes cooperate in tumor induction (see below).
Enhancer insertion allows for considerable flexibility in the position of the integrated
provirus with respect to the cellular gene that is affected. Indeed, insertions that
affect genes located as much as 50100 kb away have been reported and may affect
higher order chromatin structure and looping. These include insertions into pvt1/
c-myc, evi1/mecom, and c-myb (Bartholomew and Ihle 1991; Hanlon et al. 2003; Lazo
et al. 1990). In some cases the effects of long-range insertions have been reevaluated
688
recently due to the discovery of microRNA gene activation nearer the integration
site (Beck-Engeser et al. 2008). In the future, additional classes of noncoding RNA
may be found to be oncogenic, possibly leading to alternatives for some of the longrange interactions proposed.
Many protooncogenes have been identified as targets of enhancer-mediated
insertion, including some that can also be targeted by other mechanisms of insertional mutagenesis or have been recovered in v-onc gene containing retroviruses.
In addition, many of these genes have subsequently proven to be overexpressed in
human tumors that are not associated with retrovirus infection. Among these are
c-myc, bmi1, runx2, sox4, pim, ets, and many others (Neil and Stewart 2011).
MMTV uses enhancer insertion to activate wnt1, fgf3, and several other genes in
mammary tumorigenesis (Dickson et al. 1984; Fung et al. 1985). In some cases, a
cryptic cellular internal promoter is activated, resulting in a truncated protein as in
the case of integration into jdp2 (Stewart et al. 2007).
Although most of the genes activated by these viruses in tumors have direct
effects on cell signaling and growth, genes with other functions can also be affected.
ALV activates telomerase reverse transcriptase (TERT) by enhancer insertion
(Yang et al. 2007) in a fraction of short-latency B cell lymphomas that arise following
infection of chick embryos. In these tumors, TERT is overexpressed and telomerase
activity is increased. Because the integrations near TERT were clonal, the data
suggest that these insertions occurred early in tumor development and that TERT
activation may play a role in tumor initiation. This pattern is distinct from human
tumors where telomerase activation is thought to be a late event in tumorigenesis
(Shay and Bacchetti 1997). Because some of these tumors also overexpressed c-myb
by a mechanism independent of proviral insertion, upregulation of Myb may also
contribute to oncogenesis (Yang et al. 2007).
Insertional Mutagenesis in the Setting of Gene Therapy

Until recently, examples of oncogenesis by proviral insertion were limited to naturally occurring examples in a few animals and laboratory experiments conducted
using rodents, chickens and cats. In the past decade, an unintended and unfortunate
example of insertional mutagenesis emerged from two gene therapy trials in which
gammaretrovirus-based vectors were used in attempts to correct hematopoietic deficiencies in individuals with X-linked severe combined immunodeficiency (SCID-X1)
(Hacein-Bey-Abina et al. 2003, 2002, 2008; Santilli et al. 2008). SCID-X1 results
from mutations affecting the gene encoding the common g chain, a component of
cytokine receptors that are critical for normal hematopoietic cell development. As a
consequence, individuals affected with the condition are unable to mount normal
immune responses and typically succumb to lethal infections early in life.
In these trials, the study subjects were infused with stem cells that had been
infected with a nonreplicating retrovirus vector that expressed the common g chain.
Hematopoiesis was restored in all of the study subjects that became engrafted with
the stem cells but five of the 20 individuals went on to develop leukemias that arose
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from the transplanted cells. Analyses revealed that the tumors in four of the five
subjects had vector insertions in the vicinity of the LMO2 gene (Nam and Rabbitts
2006; Howe et al. 2008; Hacein-Bey-Abina et al. 2008) that activated expression
probably via an enhancer-mediated mechanism. Insertions involving BMI1 and
CCND2 were also found as were other somatic changes that likely contributed to
tumor development. Although these genes are not prominent among those that are
classically associated with insertional mutagenesis in murine models of tumor
induction, insertions into these genes have been documented and changes in their
expression are associated with oncogenesis (Dave et al. 2009).
A second example of insertional mutagenesis in a gene therapy setting has been
documented in a trial focused on X-linked chronic granulomatous disease (X-CGD)
(Ott et al. 2006). This immunodeficiency stems from a defect in gp91phox, a critical component of the oxidative response that helps to control microbial infection.
A protocol generally similar to that used in the SCID-X1 trial was followed. Two of
the study subjects contained clonally-expanded populations of cells with proviral
insertions in MDS-EVI1, PRDM16, or SETBP1. In this case, the original defect associated with X-CGD was not corrected, perhaps in part because of silencing of vector
expression through methylation. Although leukemia did not develop in these cases,
both subjects developed myelodysplastic syndrome, a preleukemic disorder, associated
with the effects of proviral insertion (Stein et al. 2010). One of them succumbed to
sepsis, and the second was treated with an autologous stem cell transplant.
The experiences from these trials, coupled with the wealth of experience with
models of leukemia induction by conventional murine and avian retroviruses, have
led to renewed attempts to design retrovirus vectors that will not affect expression
of host genes. A variety of approaches are being explored, including the use of
lentivirus-based vectors that have been shown to have different integration patterns
relative to cellular genes (Montini et al. 2006, 2009; Lewinski et al. 2006; Bushman
et al. 2005). Nonetheless, clonal dominance has been noted in a study subject treated
for b-thalassemia by stem cell transplant with a lentivirus vector (Cavazzana-Calvo
et al. 2010). In this instance, a clear clinical benefit from the gene transfer is evident,
and there is no evidence of tumor development or premalignant conditions.
Disruption of Gene Function and Insertion Into Tumor Suppressor Genes

Most retrovirus integrations affect expression of cellular genes by upregulating
genes in the vicinity of the integration site. However a fraction of all integrations
disrupt expression of the gene product. Those that contribute to tumorigenesis likely
disrupt tumor suppressor genes. The rarity with which this happens likely reflects
two features. First, a more limited spectrum of integration sites is likely to block
expression of a gene or gene product than those that can activate a gene. In addition,
for the gene product to be lost both copies of the gene need to be compromised and
retroviral integration into both copies is unlikely. Nonetheless, loss of the second
allele by other mechanisms can occur, and there are multiple examples of this event
in human cancers (Sherr and McCormick 2002; Fletcher and Houlston 2010).
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Fig. 27.4 Activation of micro-RNAs by insertional mutagenesis. Four examples of insertional

mutagenesis events that activate miRNAs are illustrated. Additional details are presented in the text
Despite these limitations, retrovirus integration into tumor suppressor genes has
rarely been observed in tumors induced by ALV or MLV. Nf1, a gene that encodes a
Ras-GAP and is involved in neurofibromatosis in humans, is targeted in myeloid
leukemias induced by MLV in BXH mice (Largaespada et al. 1995). In this instance,
integrations into a large intron led to an mRNA that encodes a truncated and nonfunctional protein. In addition, Trp53, the gene that encodes the p53 tumor suppressor
is frequently targeted in erythroleukemias induced by Friend MLV (Ben-David
et al. 1988). However, even in this instance, inactivation of p53 in these tumors
typically involves deletion and loss of the second allele independent of retroviral
integration at the locus. Despite these examples, other studies using mice lacking a
functional Trp53 reveal modest effects on Moloney MLV tumorigenesis, and other
experiments have suggested that loss of heterozygosity is not a common feature of
MLV-induced tumors (Lander and Fan 1997).
Activation of microRNAs
The discovery over 20 years ago of common ALV integration sites in the noncoding
B-cell integration cluster (bic) in B-cell lymphomas (Clurman and Hayward 1989)
was the first example of retroviral activation of a micro-RNA precursor (Fig. 27.4).
The function of bic was puzzling for a long time, since it did not contain a long open
reading frame but was replete with termination codons in all reading frames. Thus,
it appeared to be an oncogenic noncoding RNA (Tam et al. 1997). Further, it had
extensive secondary structure, which was conserved between chickens, mice and
humans. Overexpression of myc and bic from retroviral vectors in chickens led to
death from tumors in 30 days rather than 60 days with myc alone. While overexpression
of bic alone did not lead to accelerated tumorigenesis over vectors alone, the tumors
that appeared were of a higher grade (Tam et al. 2002). In addition, transgenic mice
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overexpressing bic in B cells led to death from tumors in 6 months (Costinean et al.
2006). The identification of microRNAs led to the observation that the largest bic
hairpin is a precursor to miR-155.
Thus, miR-155 was the first identified onco-miR. Its transcription is also
activated by the rel oncogene in B cell lymphomas induced by avian reticuloendotheliosis virus T (REV-T) and was shown to promote cell survival (Bolisetty et al.
2009). EpsteinBarr virus (EBV) also activates miR-155 and this is needed for
immortalization of B cell tumors (Yin et al. 2008; Linnstaedt et al. 2010).
Furthermore, homologs of miR-155 with common targets are encoded by both
human Kaposis sarcoma-associated herpes virus (KSHV) (Skalsky et al. 2007) and
by an avian herpes virus, Mareks disease virus (Morgan et al. 2008). miR-155 is
also overexpressed in a large variety of human tumors, including B-cell lymphomas,
breast, colon, pancreatic, and lung tumors. When mir-155 was knocked out in mice,
the immune system was severely compromised (Thai et al. 2007). miR-155 is also
induced as an inflammatory response and has been claimed to be at the interface of
cancer and inflammation (OConnell et al. 2007).
A number of other retroviruses integrate into microRNA loci and cause their
overexpression (Fig. 27.4). One example is the Kis2 locus, which contains the miR
106-363 cluster and is targeted and activated by radiation leukemia virus (RadLV)
and MLV SL3-3 in mouse T-cell leukemias (Landais et al. 2007). This microRNA
cluster is also involved in human T-cell leukemias. Both Friend MLV and SL3-3
MLV commonly integrate into and activate the miR 1792 cluster of seven microRNAs in erythroleukemias and T-cell lymphomas, respectively (Cui et al. 2007;
Wang et al. 2006). This microRNA cluster is also amplified in human B cell
lymphomas and other tumors. This microRNA cluster cooperates with myc to
induce more rapid tumors in mice (He et al. 2005).
In fact, some viral integrations far away from protein-coding genes have been
reanalyzed and found to actually affect micro-RNA genes. The pvt1 locus encodes
several microRNAs and is targeted by retroviral integration in mouse and rat T-cell
lymphomas (Beck-Engeser et al. 2008). The pvt1 locus is about 50 kb upstream of
the c-myc oncogene. Myc is also overexpressed in these tumors, possibly due to
indirect effects of the Pvt1 microRNAs.
Retroviral integration readily activates oncogenes, but it only rarely inactivates
tumor suppressor genes (see above). Thus, discovery of onco-miRs led to the idea
that perhaps they were suppressing the expression of tumor suppressor genes (Kent
and Mendell 2006). Conversely, microRNAs that are downregulated in tumors may
target oncogenes. A number of targets of miR-155 have been identified, some of
them possible tumor suppressors, such as tumor protein 53-induced nuclear protein
1 (TP53INP1), which is reduced in pancreatic cancers (Gironella et al. 2007).
Restoration of TP53INP1 decreased cell growth in culture and inhibited tumor
formation in vivo. Similarly, JARID2 (Jumonji), which potentiates retinoblastoma
protein (Jung et al. 2005) and negatively regulates cell growth is a target of miR-155
in chicken and human cells (Bolisetty et al. 2009). Overexpression of miR-155 has
been shown to enhance cell survival (Bolisetty et al. 2009), which may explain its
cooperation with apoptotic oncogenes.
692
Oncogene Cooperativity
Although oncogenic retroviruses have developed a variety of strategies to subvert
cellular growth, even in the case of those that express v-onc genes, activation of
additional oncogenes plays a central role in tumor development. These changes
contribute both to tumor progression and to modulating the differentiation state of
the cells that comprise the tumor. Indeed, like many other discoveries made with
oncogenic murine and avian retroviruses, work with these agents contributed to our
current understanding of tumorigenesis as a multistep process.
Cooperativity between viral oncogenes was first noted as investigators became
aware of retroviruses that had captured more than one oncogene. One focus of these
investigations involved strains of avian erythroblastosis virus (AEV). Some strains
such as AEV-H carry only the v-erbB gene, while others, such as AEV-ES4, have
both v-erbB and v-erbA (Beug and Graf 1989; Hihara et al. 1983). The close relationship of these strains facilitated studies examining the pathogenesis of the two
viruses and revealed that chickens infected with AEV-ES4 develop a more rapid
disease than those infected with AEV-H. In addition, in vitro experiments using
erythroid precursors revealed that expression of ErbA affects expression of genes
that would normally stimulate erythroid development, thereby suppressing differentiation (Hayman and Beug 1992; Metz 1994). This suppression, in cooperation with
the growth stimulatory signals transmitted by ErbB, contributes to the enhanced
oncogenic potential of AEV-ES4 (Fuerstenberg et al. 1992). Similar findings have
been observed when the E-26 strain of AMV was compared to the AMV-BAI strain.
As noted earlier, E-26 expressed both v-myb and v-ets, whereas the BAI strain
expresses only v-myb. In this instance, a broader range of hematopoietic cells can
be transformed by the E-26 strain (Metz and Graf 1991).
Cooperativity between oncogenes is not limited to situations where two v-onc
genes have been captured or to retroviruses that have captured these genes. Analyses
of integration patterns in tumors induced by ALVs and MLVs revealed that most
tumors, despite their clonal nature, contained more than one integration site, raising
the possibility that more than one gene was activated by proviral insertion in these
tumors. For example, chickens with B-cell lymphomas frequently had integrations
in both myc and bic loci (Clurman and Hayward 1989). Since bic encodes the
precursor to microRNA-155 mentioned above, this is an instance of cooperativity
between protein-coding and noncoding genes. There are also many examples of
cooperation between protein-coding genes.
Strong evidence in support of this idea emerged when investigators began to use
mice carrying transgenes that encoded known oncogenes. For example, infection of
mice expressing a c-myc transgene with Moloney MLV revealed that tumors developed
with an accelerated kinetics. Analyses of common proviral insertion sites in the tumors
led to the identification of bmi1 and pim1 as oncogenes that collaborate with c-myc to
induce B-cell lymphomas (Moroy et al. 1991; Berns 1991; van Lohuizen et al. 1991).
Other studies have identified additional gene networks that contribute to oncogenesis. With the advent of genomic technology and high-throughput methods to
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identify viral integration sites, several investigators have conducted large screens of
tumor integration sites in different mouse backgrounds, including studies involving
mice lacking functional tumor suppressor genes such as Trp53, Cdkn2a (Lund et al.
2002; Uren et al. 2008), or combinations of these genes (Kool et al. 2010). These
data have been compiled in the readily accessible Mouse Retrovirus Tagged Cancer
Gene Database (RTCGD), developed by Neal Copeland and collaborators to catalog
retroviral insertion sites in many murine cancers (http://RTCGD.ncifcrf.gov/)
(Akagi et al. 2004), and additional computational tools have been developed to
assess the significance of integration patterns (de Ridder et al. 2006). These advances
are helping to maximize the utility of information obtained in these screens and aid
investigators in relating these patterns to discoveries in human cancer.
Endogenous Murine Retroviruses and Oncogenesis

As noted earlier, integration is an obligate part of the retrovirus life cycle and is
nonreversible. When integration occurs in a germ cell, all progeny of that cell will
carry the integrated provirus. This phenomenon has led to the presence of integrated
retroviruses that are a part of the genetic makeup of many species, including humans
(Jern and Coffin 2008). Although the majority of these elements have become
inactive as a consequence of mutations that have accumulated over the centuries
that they have existed, some animals carry copies that are expressed. In certain
strains of laboratory mice, these viruses play a special role in oncogenesis.
The first clues to the role of endogenous retroviruses (ERV) in tumorigenesis
emerged over 60 years ago in pioneering studies by Gross (Gross 1951a, b) using
the AKR strain of laboratory mice. Almost all of these animals develop thymic
lymphomas by 1 year of age. Long before the complexity of endogenous viruses
was understood, Lilly and colleagues (1975) used classical genetic approaches to
demonstrate that a single locus, now known as Akv1, was required for tumor induction.
This locus corresponds to an endogenous provirus that is expressed from birth in
AKR mice. Additional studies revealed that this virus, called AKR-MLV, undergoes
recombination with other endogenous viruses expressed by the mice (Stoye et al. 1991;
Hartley et al. 1977). The recombination events are central to the development of the
tumors (Fig. 27.5).
The newly formed viruses were initially recognized by virtue of an extended host
range. AKV-MLV infection is limited to rodent cells that express the mCAT1 receptor
(Albritton et al. 1989). However, viruses present in the tumor tissue could infect
rodent cells and cells derived from other sources, including mink lung. Many of the
viruses caused a specific type of cytopathic effect in these cells and were named
MCFs or mink cell focus forming viruses (Hartley et al. 1977). The extended host
range reflected recombination within the env gene that affects the amino terminal
portion of the surface (SU) glycoprotein. A second recombination event alters the
sequences within the U3 region of the LTR, a change that enhances transcription.
Both of these events are important for generating the leukemogenic virus.
694
Fig. 27.5 Endogenous murine retroviruses and oncogenesis. In AKR mice, the endogenous retrovirus, AKR-MLV is expressed from birth. It recombines with other ERVs in the genome to produce
a leukemia virus, which causes thymic lymphomas by 1 year. The recombinant virus has an
expanded host range due to changes in the SU glycoprotein and the LTR
The importance of ERV in tumorigenesis is not unique to AKR mice. Other

strains of laboratory mice, including C58, HRS, and CWD, have a high frequency
of spontaneous tumors and similar recombination events occur in these animals.
Recombinants are also generated in the course of tumor induction by Moloney MLV
and in MMTV-mediated induction of breast tumors. This phenomenon is not
limited to mice; recombinations have been documented in FeLV-induced T cell
tumors in cats (Rosenberg and Jolicoeur 1997).
Friend Virus and env Gene-Mediated Oncogenesis

Although classical oncogenes are involved in tumor induction by most murine and
avian retroviruses, Friend virus uses a distinct mechanism to induce tumors (Cmarik
and Ruscetti 2010). Friend virus stocks contain two retroviruses, a replicationcompetent virus called Friend MLV and a replication-defective component called
spleen focus forming virus (SFFV). The virus induces a rapid erythroid leukemia in
mice, and the SFFV component is responsible for the disease. While these general
features are reminiscent of v-onc gene-containing retroviruses, SFFV does not contain
an oncogene but rather expresses an altered env gene product that contains an internal
deletion and is not processed in the way functional Env gene proteins are processed
(Kabat 1989). Other changes affect the carboxyl terminal sequences of the molecule.
The SFFV-encoded Env protein, called gp55, exerts its leukemogenic effects by
interacting with the erythropoietin receptor (Epo-R) that is expressed by developing
red blood cells (Ferro et al. 1993; Nishigaki et al. 2001; Wang et al. 1993). Erythropoietin
(Epo) typically stimulates these precursors by interacting with its receptor, initiating a
cascade of signaling events that orchestrates the differentiation process. When gp55
interacts with the Epo-R, signaling cascades that are similar to those activated by Epo
are triggered (Fig. 27.6), but in this case, constitutive expression of gp55 results in a
sustained signal that drives cell proliferation (Cmarik and Ruscetti 2010).
The effects of gp55 are not solely mediated via the Epo-R. A second cellular
molecule called Stk also plays a role. This tyrosine kinase is expressed in two
forms: a full length form and a truncated form called sf-Stk (Iwama et al. 1994).
27
695
Fig. 27.6 Friend Virus and env Gene-Mediated Oncogenesis. The Friend SFFV altered env
protein, gp55, interacts with the Epo Receptor and sf-Stk, initiating sustained signaling that drives
proliferation
The latter form, highly expressed on erythroid precursors, also plays a key role in
SFFV-mediated disease. The role of sf-Stk emerged from studies that identified
Mstr1 as the gene responsible for Fv2, a locus associated with resistance to SFFVinduced disease (Persons et al. 1999). Only strains that express the truncated form
are susceptible to the virus, a feature that reflects the ability of gp55 to interact and
stimulate signal transduction via sf-Stk, but not the full-length form (Cmarik and
Ruscetti 2010). Mechanisms similar to the one used by SFFV are likely important
for tumors induced by the avian hemangioma retrovirus (Alian et al. 2000) and the
sheep retrovirus JSRV (see Chap. 30).
Host Factors Influencing Oncogenicity

A variety of host genes have been identified that affect tumorigenesis by oncogenic
retroviruses. The majority of these can influence the process by affecting the outcome
of infection and do not relate directly to the oncogenic effects of the virus. Other genes
influence the growth and differentiation of the host cells that are the targets of the
virus, while others, as described above for Mstr1, affect key signaling molecules that
are important for stimulation of cell growth. Lastly, genes that influence the immune
response to the virus can also play a key role in determining the outcome of infection.
Genes that directly affect aspects of viruscell interaction are highlighted here.
One class of genes that play an important role is encoded by ERVs. Genes in this
class, including Fv4, Rmcf, and Rmcf2, exert their effects by expressing env gene
696
products that block infection by exogenous viruses (Stocking and Kozak 2008).
Some chicken ERVs function in a similar fashion. These effects are mediated by a
phenomenon called super-infection resistance whereby an Env protein expressed
from an ERV prevents the interaction between the virus receptor and another virus.
ERV gag gene products can also influence infection.
A second type of ERV effect is exemplified by the Fv1 locus, which restricts
infection at a stage after reverse transcription but before integration. This restriction,
originally identified as affecting oncogenesis by Friend virus (Rosenberg and
Jolicoeur 1997), affects virus replication and thereby reduces oncogenic potential.
Although the precise way in which the Fv1 product exerts its effects are still not
fully understood, a Gag-related protein similar to those encoded by the ERV-L family
of ERVs is responsible (Best et al. 1996).
Other host genes can affect virus replication and thereby influence the oncogenic
potential of retroviruses. The clearest example illustrating this point is the APOBEC
family of proteins. These molecules are cytidine deaminases that affect replication
during reverse transcription through mutations caused by substitution of A residues
for G residues. The protein also appears to be packaged in virions in at least some
circumstances. Mice lacking the single gene that encodes APOBEC, mA3, have a
higher virus load and develop thymic lymphomas more rapidly than wild-type mice
when challenged with Moloney MLV (Ross 2009).
Host genes that influence development of the cells that are transformed by retroviruses have been most thoroughly studied in the mouse. Examples of genes of this
type include W and Sl, genes that influence the development of erythroid precursors.
The W locus encodes the c-Kit receptor and the Sl locus encodes stem cell factor
(SCF), the ligand for the receptor (Besmer 1991). Signaling mediated by SCF interaction with c-Kit is critical for appropriate development of hematopoietic precursors,
and mutations affecting these pathways influence the frequency of cells that are
susceptible to Friend virus-induced disease. A second host gene that plays a similar
role is FoxN1, a gene that encodes a forkhead family transcription factor and is
responsible for the nude mutation (Nehls et al. 1994). Nude mice do not develop a
normal thymus because FoxN1 has undergone a spontaneous deletion in these
animals and thus these animals lack the cells that give rise to thymic lymphomas.
Acknowledgments We acknowledge the help of Mohan Bolisetty and Sarah Short with the
preparation of the manuscript.
KLB was supported by NIH grants R01CA048746-20 and R01CA124596-04.
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Chapter 28
Rous Sarcoma Virus: Contributions

of a Chicken Virus to Tumor Biology, Human
Cancer Therapeutics, and Retrovirology
Leslie J. Parent
Discovery of A Filterable Agent That Causes

Tumors in Chickens
A century ago, Peyton Rous published a landmark article demonstrating that a spindle
cell tumor could be transferred from one Plymouth Rock chicken to another
(Rous 1910). Rous, a pathologist at Rockefeller Institute for Medical Research, had
been given a hen with a large tumor protruding from its right breast. He removed the
tumor and described its histological appearance as containing irregularly arranged
cells, with frequent mitotic forms as well as multinucleated and giant cells. He
inoculated pieces of the tumor into the peritoneal cavity of the original hen, which
developed a large intraperitoneal tumor and died 35 days later. He also injected bits
of the tumor into other animals and found that transplantation of the tumor was most
successful in young birds and occurred between closely related birds of the same
lineage, but not in more distant relatives or fowl purchased at market.
In 1911, Rous reported that tumors could be transmitted using a small amount of
liquid derived from the filtrate of macerated tumors. He noted that the filterable
agent, or virus, caused tumors with the same histology as the original neoplasms, and
metastatic disease occurred by dissemination through the bloodstream and lymphatics
as well as by direct extension. Rous demonstrated that the agent in the filtrate fulfilled
Kochs postulates, proving that the tumor had an infectious origin (Rous 1911).
Although Ellerman and Bang had reported in 1908 that filtrates could transmit leukemia and lymphoma in chickens, this disease was not recognized as a malignant
process at the time (Vogt 1997; Javier and Butel 2008). Because of these circumstances, Rous sarcoma virus (RSV) became known as the first tumor virus identified,
and it was at the forefront of the emerging fields of tumor virology and cancer biology.
L.J. Parent (*)

Departments of Medicine and Microbiology and Immunology,
Penn State College of Medicine, Hershey, PA 17033, USA
e-mail: lparent@psu.edu
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L.J. Parent
However, it was not until 1966, when Rous was 85 years old, that the Nobel committee
formally recognized the value of his discovery and awarded him the Nobel Prize in
Medicine or Physiology. In his Nobel Lecture, Rous described how his discovery of
RSV was largely ignored for many years because the transmissible nature of the
tumor was thought to be an anomaly unique to chickens (Rous 1972).
In 1910 I described a malignant chicken sarcoma which could be propagated by transplanting
its cells, these multiplying in their new hosts and forming new tumors of the same sort.
In other ways the growth showed itself to be a neoplasm of a classical sort, yet, as reported in
1911, its cells yielded a causative virus Hence the findings with the sarcoma were met with
down-right disbelief, though soon several other, morphologically different, spontaneous
chicken tumors were propagated by transplantation and from each a virus was got causing
growths of its kind. Not until after some 15 years of disputation amongst oncologists were the
findings with chickens deemed valid, and then they were relegated to a category distinct from
that of mammals because from them no viruses could be obtained (Rous 1972).
Although Peyton Rous turned his attention to other areas of research, RSV was
distributed to scientists throughout the world. RSV was actively studied not only for
its relationship to tumorigenesis, but also as a tool to explore the fundamentals of
molecular biology and virushost interactions (Martin 2004). The number and
breadth of seminal discoveries that arose from studying RSV is quite remarkable:
mechanism underlying cellular transformation; viral oncogenes and protooncogenes;
signaling pathways that regulate cell growth; protein tyrosine kinases; retroviruses;
virally encoded enzymes reverse transcriptase (RT), integrase (IN), and protease
(PR); RNA packaging elements; retroviral gene therapy vectors; and host factors
that contribute to the assembly of enveloped viruses. These discoveries have had
far-reaching impacts on human health, leading to the development of tyrosine kinase
inhibitors for cancer therapy and the inhibition of human retrovirus replication by
interfering with the enzymatic activities of RT and PR.
As a testament to the importance of RSV to the study of viral oncogenesis and
cellular biology, two additional Nobel prizes in Medicine or Physiology were subsequently awarded: one to Harold Varmus and Michael Bishop for the discovery of the
Src protooncogene and the other to David Baltimore and Howard Temin for their
independent discoveries of reverse transcription and the RT enzyme. Remarkably,
despite its humble beginnings, to this day RSV remains a powerful tool that continues
to provide novel insights into tumor virology, retrovirus replication, and cellular
transformation. Unfortunately, during his lifetime, Rous could not have imagined how
his work in the early twentieth century would lay the foundation for treating human
cancers, challenge the central dogma of molecular biology, or contribute to controlling
the worldwide AIDS epidemic caused by a distantly related human retrovirus.
Pathogenicity of RSV in Birds and Mammals

RSV readily establishes infection in chickens and other avian species, and it
induces malignancies that ultimately lead to death of the animal. Inoculation
of RSV into chicks younger than 1 month in age leads to the development of
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sarcomas in as little as 23 days. The tumors that arise are fibrosarcomas and
histiocytic sarcomas that involve organs including liver, spleen, and lung (Rous 1911).
Interestingly, although infection of adult birds also results in malignant transformation with formation of sarcomas, these tumors undergo spontaneous regression
in immunocompetent animals (Fine and Sodroski 2000).
Tumor induction by RSV is not strictly limited to birds, although the sarcomas
formed in other species are not highly pathogenic. In rats and mice, injection of
RSV in young animals rarely causes tumors locally, but malignant transformation at
disseminated sites does not occur (Altaner and Temin 1970; Fine and Sodroski 2000;
Maeda et al. 2008). In rodents, tumors at the inoculation site spontaneously undergo
involution over time as the animals grow older. In cultured mammalian cells, cells
do become infected, and integration of the viral genome occurs. These infected cells
are transformed, although in most cases virus replication is abortive and virus particles are not released from tumor cells (Kotler 1971). The defect in virus release is
complemented by fusion of chicken and mammalian cells, suggesting that a chickenspecific host factor required for RSV assembly is missing from mammalian cells
(Coffin 1972). However, the block to budding is not absolute, because virus-like
particles are released from mammalian cells upon expression of the RSV Gag protein
from a nonretroviral promoter (Wills et al. 1989). To date, the avian factor needed
for RSV to be released from mammalian cells following proviral integration has not
been identified.
Rous Sarcoma Virus and the Biology of Transformation

During the 1950s, more than 40 years after Rouss initial discovery, many important
developments set the stage for the major breakthroughs that would follow. Several
groups of scientists reported that RSV-induced tumors were not isolated to avian
species; rats, mice, hamsters, and rabbits also developed sarcomas when infected
with certain strains of RSV (Martin 2004). The development of tumors in mammalian species legitimized the study of RSV and increased interest within the scientific
community. The initial convincing evidence that the tumor-inducing activity associated with RSV was clearly linked to an actual virus was provided by electronmicroscopic examination of pelleted tumor extracts that revealed spherical virus
particles 7075 mm in diameter and enclosed by a double-layered membrane
(Epstein 1956, 1958, Fig. 28.1). To ensure that the virus particles had biological
activity, a sample of the pelleted fraction was divided into two portions, one that was
subjected to microscopic examination and the other used to infect cells, which subsequently become became transformed (Epstein 1958). Around this time, quantitative methods for infectivity were developed that permitted careful investigation of
the biological properties of the virus and the infected tumor cells (Rubin 1955).
A particularly significant advance during this period was the invention of the focus
assay, in which an agar overlay was applied to infected cells to isolate single infected
cells into discrete foci that were all infected by a single virus (Temin and Rubin 1958).
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L.J. Parent
Fig. 28.1 (a) Diagram of immature (left) and mature (right) RSV virions depicting the arrangement of proteins, lipids, and RNA in the virus particles. vRNA viral RNA, CA capsid, MA matrix,
NC nucleocapsid, PR protease, SU surface glycoprotein, TM envelope transmembrane domain,
RT reverse transcriptase, IN integrase. Figure modified from (Garbitt 2004) with the authors
permission. (b) Electron-microscopic images of RSV virus-like particles produced by Gag expression
that demonstrate immature morphology (left) and authentic RSV virions that have been released
from the cell and have undergone maturation with arrangement of the core to form an electrondense center. Images obtained by L.Z. Scheifele and L.J. Parent with assistance from Roland
Meyers, Penn State College of Medicine Imaging Core Facility (Scheifele 2004) and used with
the authors permission
This assay allowed for single virus variants, or genetic mutants, to be cloned and
characterized in detail. The results of experiments based on this assay led Temin to
propose the provirus hypothesis (described below) and contributed directly
to the discovery of Src, the transforming protein encoded by RSV (Vogt 1997;
Martin 2004).
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The biology of transformed cells was also studied using viruses isolated from
foci of tumor cells in culture. RSV was a potent transforming agent and induced
cancer in cultured cells more rapidly than any other virus, with initial changes of
transformed morphology observed within 14 h following infection (Hanafusa 1969).
Under optimal conditions, cellular transformation was produced in 90% of chicken
embryo fibroblasts within 24 h. Transformation was most efficient if cells were
infected within 4 h after subculture, to ensure that cells were actively dividing when
exposed to virus. Incubation of cells with DEAE-dextran also increased the efficiency
of transformation by enhancing virus-cell attachment. Cells transformed by RSV
exhibited either rounded or fusiform shape with increased intracellular vacuoles, an
ability to form colonies in agar suspension, increased growth rate, decreased dependence on serum factors for multiplication, changes in cell surface markers, and
metabolic alterations including glucose utilization (Hanafusa 1969; Martin et al. 1971;
Temin 1971; Bader 1972). Isolation of viral mutants with differing abilities to transform cells led to the hypothesis that the virus encoded a gene product that was
responsible for maintaining the transformed state.
Discovery of a DNA Intermediate Required for RSV Replication

The focus assay developed by Temin and Rubin allowed investigators to study
the effects of single viruses on the properties of uniform populations of infected
tumor cells (Temin and Rubin 1958). In the early 1960s, Temin observed two
distinct phenotypes of tumor cells infected with the Bryan strain of RSV (Temin
1961). The wild-type virus, referred to as morphr, caused rounding of the cells,
in contrast to the mutant strain morphf, which resulted in fusiform cellular morphology. Temin recognized that the morphological differences in the cells were
maintained during long-term passage of the cells or by transfer of the virus to
new cells, suggesting that the viral genetic material had become stably associated with the cell, likely through integration into the cellular DNA (Temin 1960,
1962; Temin and Kassner 1976).
A major breakthrough in understanding the ability of RSV mutants to cause
reproducible, transferrable cellular phenotypes came when Temin observed that
actinomycin D, a DNA-dependent RNA synthesis inhibitor, interfered with virus
production in infected cells (Temin 1963a). Inhibition of DNA synthesis prevented
establishment of RSV infection, but only when cells were treated prior to formation
of the provirus. Furthermore, Temin found that RSV infection was dependent on
new DNA synthesis and cell mitosis, and RSV-infected chicken embryo fibroblasts
contained DNA that was homologous to RSV RNA (Temin 1964a, b). Based on
these experiments, he publically reported his DNA provirus hypothesis, which
postulated that infection of chicken cells by RSV leads to the formation of one or
two copies of a regularly inherited structure with the information for progeny virus
and for cellular morphology (Temin 1976). In other words, the RSV RNA genome
was converted to a DNA intermediate, which was integrated into the cell genome
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L.J. Parent
and used as a template to synthesize RSV RNA. Remarkably, Jan Svobda also
proposed the existence of a provirus around the same time based on independent
studies of rat cells infected with RSV (Svoboda et al. 1963; Temin 1976). However,
data supporting the provirus hypothesis were considered by many scientists in the
field to be too indirect and, therefore, inconclusive.
Temins conviction that a DNA provirus was intrinsic to RSV replication was
unwavering, and he and others continued to perform more rigorous experiments that
were consistent with the theory. Then, in 1969, a key insight was made that greatly
energized the field. In RSV-infected cells, RSV-derived DNA was synthesized in the
absence of protein synthesis, suggesting that the enzyme that carried out viral DNA
synthesis was present in incoming virus particles (Temin and Mizutani 1970; Temin
1976). These experiments prompted the development of an assay to look for RNAdependent DNA polymerase activity in RSV virions (Temin 1976; Weiss 2003).
Purified virus particles were permeabilized with nonionic detergent, incubated with
dideoxynucleotides dATP, dCTP, dGTP and radioactive dTTP in the presence of
magnesium and dithiothreitol, and the incorporation of radioactively labeled TTP
into DNA was measured (Temin and Mizutani 1970). There was a high degree of
TTP incorporation under the standard assay conditions, but DNA synthesis was
abrogated by pretreatment of the virions with ribonuclease A, suggesting that viral
RNA was used as a template for production of DNA.
Simultaneously, David Baltimore performed a similar polymerase assay using
permeabilized Rauscher murine leukemia virus, discovering the same RNAdependent polymerase activity in virions, and concluding that all RNA tumor viruses
shared the same enzyme (Baltimore 1970). Baltimore and Temins back-to-back
manuscripts in Nature were groundbreaking, and the editors of the journal coined
the term reverse transcriptase for this enzyme discovered in RNA tumor viruses
(Baltimore 1995). Soon thereafter, RNA tumor viruses were called retroviruses
based on their reverse transcriptase activity (Dalton et al. 1974). In 1975, only five
years after their discovery, Temin and Baltimore shared the Nobel Prize in Physiology
or Medicine with Renato Dulbecco. The relationship of reverse transcription to the
study of cancer was eloquently described in Temins Nobel Lecture:
The Nobel Prize is an honor not only for me but also for all those who have been working
with avian RNA tumor viruses The genetic information in RNA is transferred to DNA
during the replication of some viruses, including some that cause cancer. This transfer of
information from the messenger molecule, RNA, to the genome molecule, DNA, apparently contradicted the central dogma of molecular biology, formulated in the late 1950s.
This mode of information transfer was first postulated and established for the replication of
Rous sarcoma virus, a strongly transforming avian C-type ribodeoxyvirus In most of this
discussion of virus replication and virus origins, I have not mentioned cancer. In fact, the
absence of such discussion makes an important point: RNA tumor virus replication is not
sufficient for cancer formation by RNA tumor viruses. Strongly transforming RNA tumor
viruses like RSV cause cancer by introducing genes for cancer into cells I do not believe
that infectious viruses cause most human cancers, but I do believe that viruses provide
models of the processes involved in the etiology of human cancer. (Temin 1992).
The original RT assay was refined and used to delineate the molecular steps of the
reverse transcription process in avian retroviruses (Boone and Skalka 1980, 1981).
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Fig. 28.2 Schematic diagram showing the integrated provirus of RSV, with the viral genes gag,
pol, env, and src indicated. The unspliced viral RNA that serves as the viral genome and as the
template for Gag and Gag-Pol fusion protein expression is shown below, in addition to the Gag and
Gag-Pol proteins. The spliced viral RNAs that dictate Env and Src expression are illustrated with
their respective protein products. Nucleotide numbering is based on the Prague C strain of RSV
(Coffin et al. 1997). LTR long terminal repeat, U3 unique 3 region, R repeat, U5 unique 5 region,
DR1 direct repeat 1, DR2 direct repeat 2, PBS primer binding site, PPT polypurine tract, RSE RNA
stability element, AAAA poly(A) tail
These experiments demonstrated that everything needed for reverse transcription to

proceed, except nucleotides, was contained within the virus particle (Boone and
Skalka 1980). The molecular steps involved in the process of reverse transcription
and the enzymatic activity of RT itself turned out to be more complex than originally
imagined. The overall reaction involves transformation of the linear, single-stranded,
positive-sense viral genomic RNA into the linear double-stranded proviral DNA.
Importantly, the proviral DNA differs from the genomic RNA template at its 5 and
3 termini, which contain sequence known as the long terminal repeats (LTRs)
(Fig. 28.2). Only portions of the LTR sequences are present in the genomic RNA.
Oddly enough, the sequence that serves as the proviral promoter to drive viral transcription is located at the 3 end of the genomic RNA. Therefore, one of the primary
goals of reverse transcription is to copy the promoter from the 3 end of the RNA and
move it to the 5 end of the provirus to enable transcription of the viral genes by
cellular RNA polymerase II. The polyadenylation signal at the 3 end of the provirus
is also created through the process of reverse transcription. The molecular gymnastics
required to properly synthesize the LTR-containing proviral DNA harnesses several
different enzymatic activities of RT: RNA-dependent and DNA-dependent DNA
synthetic capabilities and an RNA digestion activity (RNase H) that specifically
degrades the RNA component of RNADNA hybrids (Telesnitsky and Goff 1997).
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The importance of the discovery of reverse transcription by Temin and Baltimore

cannot be overstated. As stated in the press release from the Nobel Assembly at the
Karolinska Institute announcing the 1975 Nobel Prize in Physiology or Medicine:
Temin postulated that the genetic information of an RNA virus capable of giving transformation could be copied into DNA, and that this DNA in a manner similar to that described
for a DNA tumor virus could become integrated into the genetic material of cells. This
proposal by the overall majority of scientists was considered as heresy since it was in conflict with the central dogma accepted in the field of molecular biology in those days. This
dogma implied that information transfer in nature occurred only from DNA to RNA and
not in the other direction. Temin accumulated certain indirect evidences supporting his
theory but the major breakthrough occurred in 1970 when simultaneously Temin and also
David Baltimore showed the occurrence of a specific enzyme in RNA tumor virus particles
which could make a DNA copy from RNA. This enzyme was called reverse transcriptase
(The Nobel Prize in Physiology or Medicine 1975 Press Release 1975).

Nobelprize.org. 1 Aug 2010 http://nobelprize.org/nobel_prizes/medicine/laureates/1975/press.html.
The Process of Reverse Transcription

The steps involved in proviral DNA synthesis are depicted in Fig. 28.3, and several
reviews present a more detailed description of the process (Goff 1990; Katz and
Skalka 1990; Whitcomb and Hughes 1992; Telesnitsky and Goff 1997; Wilhelm
and Wilhelm 2001; Hizi and Herschhorn 2008; Gotte et al. 2010; Herschhorn and
Hizi 2010). Briefly, the primer tRNA-tryptophan is annealed to the primer-binding
site (PBS) near the 5 end of the RNA genome. RT binds to the viral RNA and
begins the synthesis of DNA using the RNA as a template. DNA synthesis continues
until the 5 end of the RNA is reached, creating a short product called the minusstrand strong stop DNA. The RNAse H component of RT degrades the RNA strand
of the RNADNA hybrid located upstream of the PBS. A molecular gymnastics
event known as the first strand transfer occurs next, when the minus-strand strong
stop DNA jumps to the opposite end of the RNA and hybridizes to a complementary repeated sequence, R. RNA-dependent DNA synthesis proceeds from R through
a region known as the polypurine tract (PPT), then continues to the PBS at the end
of the RNA strand. The PPT is not digested efficiently by RNase H and remains as
the final vestige of the viral RNA template. The RNA PPT sequence forms a duplex
with the DNA PPT sequence and primes DNA synthesis using the nascent minusstrand DNA as a template. DNA-dependent DNA synthesis continues through the
end of the minus-strand DNA, a segment of the annealed tRNA primer, terminating
at the PBS, and forms the plus-strand strong-stop DNA. The primer tRNA is
degraded by RNase H, leaving the PBS intact. The next gymnastics event, the second
strand transfer, involves translocation of the minus-strand PBS to the opposite end
of the DNA duplex, annealing to the complementary plus-strand PBS sequence.
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Fig. 28.3 Diagram of the steps in the reverse transcription reaction. U3 unique 3 region, R repeat,
U5 unique 5 region, PBS primer binding site, PPT polypurine tract, AAA poly(A) tail. Figure
modified from (Spidel 2005) and used with the authors permission
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L.J. Parent
DNA-directed DNA synthesis continues until both strands of the provirus are fully
intact. In RSV-infected cells, synthesis of the double-stranded DNA provirus is not
finished until after the reverse transcription complex enters the nucleus (Fig. 28.3)
(Varmus et al. 1978; Lee and Coffin 1991). The completed products of reverse transcription may be integrated into the host genome or the blunt LTR ends may be
joined by host ligases to the form dead-end products, 2-LTR circles and 1-LTR
circles (Telesnitsky and Goff 1997) (Fig. 28.3).
The consequences of reverse transcription have significant implications for
retroviral pathogenesis, and carcinogenesis. The accurate execution of reverse
transcription, including the precise construction of the LTRs, is required for the
provirus to be integrated into the hosts cellular DNA. Integration is, by definition,
a mutagenic event that permanently alters the genetic make-up of the host. The
integration of proviral DNA may directly result in cancer by introducing the retroviral promoter upstream of a gene that regulates cell growth or other homeostatic
functions, leading to deregulation of vital cellular activities and subsequent tumor
formation. This process of inducing cellular gene expression driven by an integrated viral LTR promoter is known as insertional mutagenesis (Fig. 28.4). In addition, integration of the provirus near a growth-controlling gene may result in
incorporation of a portion of the cellular gene into the virus particle, resulting in
oncogene capture (Fine and Sodroski 2000). This latter mechanism accounts for
the tumor-producing properties of RSV.
Genome Organization of RSV

Early classification of RSV and its relatives was controversial and inconsistent.
Throughout the mid-1900s, these viruses were called Rousviruses, for obvious
reasons, Leukoviruses, because of their similarities to avian leukosis virus, or
Oncornaviruses because they caused tumors in animals (Temin 1971). Members
of this virus family are enveloped 80100 nm diameter particles containing a nucleocapsid core that lacks apparent icosahedral symmetry. The discovery that ultimately
unified this virus family occurred in 1961, when density gradient-purified RSV virions
were shown to contain almost exclusively RNA and very little DNA (Crawford and
Crawford 1961). This finding led to the classification of RSV and related viruses as
RNA tumor viruses, to distinguish them from the families of DNA tumor viruses
such as polyoma viruses. This terminology continued to be used until Temin and
Baltimore discovered that RNA tumor viruses RSV and Rauscher murine leukemia
virus contained a polymerase with reverse transcribing activity, which was encoded
by the virus. Reverse transcription of the viral RNA genome into DNA was considered
the hallmark of this class of viruses; hence, they were renamed retroviruses, and
RSV was considered the prototype. RSV is presently classified as a member of the
Retroviridae family, Orthoretrovirinae subfamily, and Alphoretrovirus genus, to
denote it as belonging to the first group of retroviruses discovered.
The RSV genome is a diploid, single stranded RNA molecule of approximately
9.3 kb in length (Schwartz et al. 1983). The genome is synthesized by the cellular
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enzyme RNA polymerase II using the integrated proviral DNA as a template (Rabson
and Graves 1997). The viral RNA shares structural features with cellular mRNA,
bearing a 5 methylguanine cap and a poly(A) tail at the 3 end (Vogt 1997) (Fig. 28.2).
The two genomic RNAs in each virion are linked at their 5 ends through a noncovalent interaction that involves sequences in the dimer initiation site and additional
nucleotides included in the dimer linkage site. Several regulatory elements are contained
within the 5 and 3 untranslated regions (UTRs). The first nucleotide is at the start of
the R region, a short sequence repeated at the 3end of the genome. The 5 R region
is followed by a unique sequence denoted U5. The primer binding site (PBS), positioned
between R and U5, serves as the binding site for the primer of reverse transcription.
In RSV, this primer is the tryptophan-tRNA, which binds to a complementary
sequence in the PBS (Harada et al. 1975). The psi sequence, a cis-acting element
required for incorporation of the genomic RNA into virions, is located within the
5UTR, and extends seven codons into the gag region (Swanstrom and Wills 1997;
Vogt 1997). Genetic separation of the viral psi packaging element from the genes
encoding the viral structural proteins was a key discovery that ultimately led to the
development of viral vectors for gene therapy (Linial et al. 1978; Linial 1981).
The three genes absolutely required for replication are arranged in the same order
in all retroviruses: 5-gag-pol-env-3 (Rabson and Graves 1997; Vogt 1997). In RSV,
the PR-encoding sequence pro is located at the 3 end of the gag gene, although in
other retroviruses, pro is contained within the pol gene or in a distinct pro reading
frame (Swanstrom and Wills 1997). The src gene, unique to RSV, is dispensable for
virus replication but is required for cellular transformation, and it resides downstream of env (Schwartz et al. 1983). The src coding region is flanked by short direct
repeat sequences known as DR1 and DR2. The DR elements have pleiomorphic
effects on virus replication; they have been implicated in viral RNA packaging,
virus assembly, as a cis-acting signal for nuclear export of the unspliced viral RNA
(Sorge et al. 1983; Ogert et al. 1996; Simpson et al. 1997, 1998). A regulatory element
called the RSV stability element (RSE), recently discovered, lies just downstream
of the src gene and prolongs the half-life of the unspliced mRNA (Weil and Beemon
2006; Weil et al. 2009). There are three polyadenylation signals in the 3UTR, as
well as the R region and a unique region called U3, which contains the viral promoter
(Wang et al. 1975; Coffin and Billeter 1976). As discussed in the previous section,
during reverse transcription, the U3 region is copied and translocated to the 5 end
of the proviral DNA to recreate the promoter in its proper location, upstream of the
viral genes (Hughes et al. 1978; Shank and Varmus 1978).
Identification of the src Viral Oncogene

From studies of the cellular effects associated with RSV variants, Temin concluded
that viral genes controlled the morphology of transformed cells, and this idea
led to the hypothesis that transformation is the result of the action of viral genes
(Temin 1976). While this idea might not seem radical or insightful today, the
underlying cause of cancer was debated during the first part of the twentieth century.
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L.J. Parent
Thus, the study of RSV-induced tumors was the genesis for the hypothesis that
alterations in cellular DNA caused transformation into the malignant state.
One of the most intriguing observations made during this time was that different
RSV mutants varied widely in their ability to promote cellular transformation.
As discussed earlier, the morphf mutants described by Temin induced a different
morphology in infected cells compared to the wild-type virus (Temin 1960). Based
on these observations, Temin proposed that the virus becomes equivalent to a
cellular gene controlling cell morphology (Temin 1960), setting in motion the hunt
for the transforming viral gene (Martin 2004). He proposed that viral oncogenes, or
virogenes, were integrated into the host chromosome and caused cancer to arise in
these cells. His work laid the foundation for the virogeneoncogene hypothesis,
discussed in more detail below (Huebner and Todaro 1969). Another significant
step forward was the description of the Bryan strain of RSV, which was replicationdefective but caused cellular transformation, highlighting the idea that virus replication and tumor formation were genetically separable (Bryan et al. 1955; Hanafusa
et al. 1963; Temin 1963b). This idea was strengthened by the existence of irradiationinduced and naturally occurring mutants of RSV that were replication-competent
but nontransforming (Vogt 1971; Martin and Duesberg 1972). Thus, it became
apparent that the transformation gene of RSV was deleted or defective in these
nontransforming variants.
A series of experiments that followed in the late 1960s were instrumental in
establishing the identity of the gene carried by RSV that was responsible for cellular
transformation. The most critical advance was the description of an infectious,
temperature-sensitive (ts) viral mutant that caused transformation when cells were
grown at the usual temperature; remarkably, when shifted to a higher temperature,
the cells reverted to normal morphology (Martin et al. 1971). Martin concluded that
a protein encoded by the virus was required to maintain cellular transformation, and
the loss of the transformed state was due to failure of the protein to function at the
elevated temperature. The ts mutant reafffirmed the notion that virus replication and
oncogenesis were genetically segregated. As a result of Martins findings, the quest
for the oncogenic viral gene was undertaken by a number of investigators using the
emerging molecular biology methods being developed at the time (Varmus 1990;
Martin 2004).
To identify the transforming gene associated with RSV, the genomes from transformation-competent and transformation-defective (td) strains were compared to
determine whether there were any extra sequences present in the transforming
genome (Vogt 1971). It had previously been shown that the RSV genomic RNA
isolated from virus particles consisted of a 70 S RNA that could be reduced to two
35 S RNAs upon denaturation (Duesberg 1970). To determine whether the RNA
isolated from nontransforming td viruses had a lower molecular weight than that of
transforming viruses, Duesberg and Vogt metabolically labeled viral genomes with
radioactive uridine or orthophosphate during their synthesis in infected cells and
then compared the electrophoretic profiles of the 70S RNA species isolated from
purified virions (Duesberg and Vogt 1970). They found a correlation between
transformation activity and the presence of viral RNA of greater molecular weight.
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Subsequent analysis using RNA footprinting of RNAs from paired samples of

different strains of RSV (Prague C and Schmidt-Ruppin A) and their transformationdefective counterparts revealed that the transforming genomes were approximately
1015% larger than those lacking transformation capabilities (Lai et al. 1973).
This key experiment led to the conclusion that the additional genetic information
contained in the longer RNA genomes of the transforming viral strains was responsible for the transformed state of infected fibroblasts.
It took four more years before three groups independently mapped the unique
region of RNA linked to transformation activity to sequences near the 3 end of the
viral genome by isolating polyadenylated RNA via oligo-dT chromatography (Wang
et al. 1976). Electron-microscopic studies that examined heteroduplexes that formed
upon hybridization of RSV Prague B RNA and complementary DNA synthesized
from spontaneously arising td viruses determined that the defective genomes indeed
contained deletions. These studies revealed that the src gene was approximately
2,000 nucleotides in length and was located 8003,000 bases from the 3end of the
genome (Junghans et al. 1977). This gene was referred to by many names, including
x, onc and sarc, but ultimately the designation src was widely adopted to
reflect the sarcomas in chickens caused by RSV strains harboring this gene. Because
the td strains were replication competent and efficiently produced virus particles
from infected cells, src was considered dispensable for infection. Instead, it was
postulated that the viral src gene encoded a protein whose sole function is the
generation of the transformed phenotype. (Kamine and Buchanan 1977).
Discovery of the Cellular Origin of v-Src:

The VirogeneOncogene Hypothesis
At the time of the discovery of the src oncogene in RSV, there was active controversy
about the mechanism of virus-induced oncogenesis. The virogeneoncogene
hypothesis was proposed in 1969, based on observations that RNA tumor virus
genetic material, and sometimes virus particles as well, had been detected in many
different types of vertebrate animals (Huebner and Todaro 1969). Huebner and
Todaro proposed that RNA tumor virus sequences (the virogene), which included
information that caused malignant transformation (the oncogene), were ubiquitously
present in cellular genomes and were transmitted vertically. In their view, the viral
oncogenes were present in a dormant or covert form but became activated by
environmental stimuli including irradiation, chemical carcinogens, DNA tumor
viruses, or the normal aging process. This idea was bolstered by the existence of the
DNA provirus of RNA tumor viruses, which meant that sequences derived from
retroviruses were permanently inserted into the cellular genome.
Harold Varmus and Michael Bishop became interested in testing the virogene
oncogene hypothesis. In support of the hypothesis, Varmus et al. (1972) reported
the detection of RSV DNA sequences in uninfected chicken cells. A critical question
to resolve was whether the RSV sequences they found in normal cells coded for
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only the structural proteins of the virus, or was the transforming gene src present as
well? A major challenge was to develop single-stranded radioactively labeled DNA
probes that were specific for src to use in hybridization experiments (Varmus 1990).
Varmus and Bishop, along with colleagues Dominique Stehelin and Peter Vogt,
took advantage of the td mutants, presuming that the src gene was deleted in its
entirety in these transformation defective strains, to obtain a src-specific probe
(Stehelin et al. 1976). They reasoned that they could apply subtraction hybridization
to remove the genes that were present in both the td and transforming genomes,
leaving only the src gene fragment since it had nothing to pair with in the td sequence.
To accomplish this feat, RNA isolated from RSV was reverse transcribed to produce
cDNA fragments that were used to anneal to viral RNA isolated from td RSV virions.
The heteroduplexes were removed using hydroxylappetite chromatography, resulting
in purification of a single-stranded DNA src fragment that hybridized selectively to
RSV but not td sequences. The probe was approximately 1,600 nucleotides in length
and corresponded to ~16% of the RSV genome, consistent with earlier studies that
mapped the transforming activity of RSV (Duesberg and Vogt 1970; Lai et al. 1973;
Junghans et al. 1977). This probe was the key reagent used to identify the cellular
homologue of the viral src gene.
To directly test the oncogenevirogene hypothesis, the src probe and the td-RSV
probes were hybridized with DNA from chickens and other avian species (Stehelin
et al. 1976). The src probe hybridized strongly to DNA from chicken, turkey, duck,
and quail; unexpectedly, there was also evidence of a sequence with significant
homology in DNA isolated from an Australian emu, which was genetically divergent
from the other bird species. By contrast, only chicken DNA contained a match for
the td sequences containing the remaining viral genes. The authors concluded that
the src transforming gene originated in chickens or a closely related species.
Their experiments confirmed the oncogene hypothesis, but refuted the virogene
component of the theory. They also speculated, correctly, that the cellular gene
corresponding to src was evolutionarily conserved in avian species and it served an
important role, possibly involving the normal regulation of cell growth (Stehelin
et al. 1976) based on the tumor activity of the viral src gene. It was proposed that the
cellular src gene became incorporated into the RSV genome by mechanisms involving
recombination or transduction (Stehelin et al. 1976). The slight differences in the
nucleotide composition of the cellular src homolog and the viral src gene were
proposed to have arisen during numerous rounds of virus replication during which
mutations in the viral gene were introduced.
Confirmation of c-src in Vertebrates: The First Protooncogene

Subsequent experiments showed that src was present in cells from virtually every
vertebrate species (Spector et al. 1978a, b). Furthermore, the src sequence was
not linked to the replicative genes of endogenous retroviral sequences (Padgett
et al. 1977; Hughes et al. 1979). Thus, the origin of the viral src gene (known as
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v-src) was almost certainly derived from a sequence present in the cellular
genome. Definitive proof was provided when the cellular src gene (referred to as
c-src) was cloned from normal chicken DNA (Parker et al. 1981; Shalloway
et al. 1981; Takeya et al. 1981). The gene was present in a single copy in the
chromosomal DNA spanning 33 kb, and the coding sequence was interrupted by
intervening sequences, or introns (Fig. 28.4a). By contrast, the v-src gene was a
contiguous sequence of 1,590 bp (Czernilofsky et al. 1980b). What mechanism
could explain how the spliced src gene became incorporated into the RSV coding
sequence?
Evidence supported the idea that incorporation of the c-src gene into the RSV
genome occurred after splicing; therefore, a simple DNA recombination event could
not have resulted in capture of the c-src oncogene. Instead, a more complex mechanism
needed to be invoked (Fig. 28.4b). Comparison of the sequences of v-src with the
cDNA encoding c-src revealed differences at both the 5 and 3 ends of the genes
(Swanstrom et al. 1983; Takeya and Hanafusa 1983). In v-src, a region of the c-src
coding sequence was deleted, intronic and untranslated sequences of the cellular
gene were introduced, and codons for the 19 C-terminal residues of c-Src were
replaced with a sequence coding for 12 different amino acids in v-Src (Swanstrom
et al. 1983; Takeya and Hanafusa 1983). Additionally, there were some minor
differences in how the v-src genes were arranged in different strains of RSV (Prague
C and Schmidt-Ruppin A), suggesting that acquisition of src might have arisen from
separate transduction events (Swanstrom et al. 1983). Even though capture of the
c-src gene might have occurred independently in various strains of RSV, the transduction of cellular genes by retroviruses is a rare event that has not been faithfully
recapitulated under experimental conditions.
Sequencing of the RSV genome and subsequent experiments that probed the
mechanism underlying retroviral transduction led to the development of two
models that explain how c-src is incorporated into the RSV genome (Swanstrom
et al. 1983; Takeya and Hanafusa 1983; Herman and Coffin 1987; Swain and
Coffin 1992) (Fig. 28.4b). In each case, the initial event required is insertion of
the provirus at or near the start of a cellular gene, just upstream of the coding
sequence. One of two possible events likely occurred next. In the first, a region
of the proviral sequence is deleted from the 3 long terminal repeat (LTR), bringing the beginning of the coding region of c-src just downstream of the viral env
gene. Transcription by the host RNA polymerase II (polII) enzyme creates a premRNA that contained viral sequences linked to the introns and exons of the src
mRNA. In the second potential mechanism, inefficient recognition of the proviral polyadenylation site by polII results in the synthesis of readthrough transcripts containing cellular DNA sequences linked to the provirus. The models
converge at the next steps, in which the pre-mRNA undergoes splicing to remove
the src introns. The spliced mRNA, which contains viral sequences merged with
the src gene, is copackaged with wild-type unspliced viral RNA genomes into
virions. During reverse transcription, the two viral RNAs recombine, even though
they lack significant sequence homology at their 3 ends, through a process
known as illegitimate recombination.
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L.J. Parent
Fig. 28.4 Potential mechanisms for transduction of the src oncogene into the genome of RSV.
(a) The c-src gene is shown with 12 exons and the intervening intronic sequences. After transcription and splicing, the mRNA is generated, as indicated by the poly(A) tail shown as (AAA)n.
(b) The proviral sequence of the retrovirus prior to acquisition of the src gene is shown. Integration
of the proviral DNA into the chromosomal DNA of the cell occurs into a region near the 5 end of
the src gene. Next, one of two possible mechanisms occurs (Swanstrom et al. 1983; Swain and
Coffin 1992). In the first possible mechanism (1), there is spontaneous deletion of the LTR after
integration; the fused DNA sequence undergoes transcription and splicing to generate a viral RNA
product that contains RU5, gag, pol, env, and src. This viral RNA serves as a genome and is incorporated into a virus particle (oval) along with an unmodified viral genomic RNA consisting of
RU5, gag, pol, and env. Through illegitimate recombination during reverse transcription,
the src gene becomes incorporated into the viral RNA genome just upstream of the LTR sequence.
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The end result is incorporation of the coding region of c-src at the end of the env
sequence, preceding the LTR (Schwartz et al. 1983, 1995; Swanstrom et al. 1983).
Because the src gene is located between the LTRs, it is carried with the RSV genome,
integrates into cells with the provirus, and is packaged into nascent virions with the
viral genomic RNA in all subsequent replication cycles. After integration, the v-src
gene is synthesized from a subgenomic, spliced mRNA to serve as the template for
pp60 v-Src protein synthesis to promote tumor formation in the infected cell (Rabson
and Graves 1997). RSV is unusual in that it acquired a cellular oncogene while
maintaining all of the sequences needed for virus replication. In most other oncoretroviruses, cellular gene transduction was concurrent with deletion of an essential
viral gene. In these cases, a helper virus was required to allow for transmission of
the virally induced tumor from cell to cell (Fine and Sodroski 2000).
The c-src gene became known as a protooncogene, a term coined by Bishop
to designate the cellular progenitor of the viral src oncogene (Bishop 1985, 1990).
Bishop was not in favor of the term cellular oncogene because the cellular homologues of viral oncogenes do not cause malignant transformation in normal cells.
Instead, the host genes encode proteins engaged in processes that regulate cell
growth and differentiation. Examples of protooncogenes include certain growth
factors and growth factor receptors, transcription factors, receptor and nonreceptor
protein kinases involved in signal transduction, chromatin modifiers, and regulators
of apoptosis (Croce 2008). A protooncogene can become oncogenic when its
expression level or activity is deregulated. This disturbance in function can arise
from a change in gene expression owing to insertion of a strong proviral LTR
promoter upstream of the transcription start site of the protooncogene. The abnormal
activation of protooncogene expression causes uncontrolled cell growth and malignant
transformation. Other nonviral mechanisms also induce protooncogenes to become
tumorigenic, including mutations that arise in sensitive coding regions that alter
protein function, chromosomal rearrangements that place alternative promoters or
enhancers nearby that stimulate gene expression, and gene duplications or amplifications that result in multiple copies of the protooncogene being expressed. Since
the c-src gene was identified in 1976, over 80 protooncogenes have been discovered,
providing critical tools needed to understanding how normal cell growth and
differentiation are regulated and how cancer arises when these control mechanisms
go awry (Cooper 1995; Vogelstein and Kinzler 2002).
Fig. 28.4 (continued) In the second possible mechanism (2), the cellular RNA polymerase II
reads through the end of the LTR, ignoring the viral polyadenylation signal, resulting in the
fudion of the src coding sequence at the 3 end of the viral RNA (Swain and Coffin 1992). This
mutated viral RNA then serves as a viral genome and become encapsidated into a virus particle
(oval) along with a wild-type genomic RNA. In the virus particle, reverse transcription occurs with
illegitimate recombination generating the positioning of src within the 3 LTR
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L.J. Parent
Isolation of the Protein Product of the Viral src Gene

Once the src gene was discovered, identification of the gene product of src quickly
followed [reviewed in (Martin 2001, 2004)]. Several groups used the 35 S viral
RNA isolated from td and nondefective (nd) variants of different strains of RSV to
synthesize viral proteins using a reticulocyte lysate in vitro translation system
(Purchio et al. 1977; Beemon and Hunter 1978; Kamine and Buchanan 1978;
Kamine et al. 1978; Sefton et al. 1978). A series of proteins ranging in size from
13 to 180 kilodaltons (kDa) were detected, with a proteins of 60 kDa identified in
the nd strains but not in the td strains. Brugge and Erikson (Brugge and Erikson
1977) had recently reported using serum from tumor-bearing rabbits infected with
Schmidt-Ruppin subgroup D RSV to identify a 60 kDa transformation-specific
antigen. The 60 kDa protein was detected in cells from two different species but not
from cells infected with nontransforming variants.
Ensuing studies of the viral Src oncoprotein determined that it possesses tyrosine
kinase activity (pp60v-src), which was surprising, as there were no previously known
tyrosine protein kinases (Collett and Erikson 1978; Levinson et al. 1978; Hunter
and Sefton 1980; Hunter et al. 1980). The Src protein is synthesized on cytosolic
ribosomes and transported rapidly to the plasma membrane by virtue of a short,
myristylated membrane-targeting sequence at the N-terminus (Buss and Sefton 1985).
The tyrosine kinase activation domain is located near the C terminus of the protein,
and several residues within Src are autophosphorylated (Hunter and Cooper 1985).
Tyrosine protein kinase activity is required for transforming activity (Bryant and
Parsons 1984; Wilkerson et al. 1985), and cells transformed by v-src exhibit an
approximately tenfold increase in total protein phosphorylation (Sefton et al. 1980).
Analysis of the targets of Src phosphorylation yielded insights into mechanisms
responsible for the transformed state (Martin 2001, 2004).
Characterization of the c-Src protein revealed that it is also a protein tyrosine
kinase, but it lacks robust transforming activity (Iba et al. 1984; Parker et al. 1984;
Shalloway et al. 1984; Johnson et al. 1985; Wang 1987). The tyrosine kinase activity
of c-src is tightly controlled, and in contrast to v-src, its overexpression does not
result in a dramatic increase in tyrosine phosphorylation of cellular proteins
(Iba et al. 1985). Furthermore, replacement of v-src with the c-src sequence in RSV
results in a loss of tumor formation (Iba et al. 1984) and the differences in activity
mapped to amino acid differences within the C-terminal sequence. Kinase activity is
mediated by the catalytic domain, which is activated through autophosphorylation of
a tyrosine residue, and mutants of v-Src that map to this region have temperaturesensitive phenotypes or lose transforming activity completely (Parsons and Weber
1989). The kinase domain of c-Src is functional, but its activity is regulated negatively by phosphorylation of its C-terminal tyrosine (Nada et al. 1991). Structural
studies of the active and inactive forms of Src revealed the elegant mechanisms by
which intermolecular interactions regulate phosphorylation and dephosphorylation
that dictate its kinase activity (Gonfloni et al. 1997; Weijland et al. 1997; Williams
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et al. 1997; Xu et al. 1997, 1999; Roskoski 2004). By contrast, v-Src C-terminal
tyrosine, and the kinase domain is, constitutively active (Parsons and Weber 1989),
accounting for its potent transforming activity.
Role of c-Src in Human Cancers

The cloning of the human c-src gene paved the way to explore the mechanism of
tumorigenesis in human tumors (Gibbs et al. 1985). Initial insights into the potential
signaling cascades controlled by c-Src were gleaned by studying the changes in cells
that expressed v-Src. These transformed cells exhibited uncontrolled cellular proliferation mediated by mitogen signaling, anchorage and growth factor-independent growth,
disruption of cell motility and cellcell adhesion, deregulation of integrins, and stimulation of a host of signal transduction pathways, including MAPK (mitogen-activated
protein kinase), PI3K (phosphatidylinositol 3-kinase), Ras, Stat3 and, FAK (focal
adhesion kinase) (Martin 2001; Guarino 2010; Maslikowski et al. 2010). Even though
many targets of v-Src were ultimately found to contribute directly to transformation
and proliferation, the challenge remained to determine whether c-Src played a central
role in the development of human cancers. Although beyond the scope of this chapter,
a large collection of data supports the critical role of Src in tumor progression, angiogenesis, and metastasis in human cancers of the colon, breast and prostate (Mao et al.
1997; Irby and Yeatman 2000; Frame 2002; Mayer and Krop 2010). Inhibitors of Src
and related tyrosine kinases have been used with great success to treat patients with
hematologic malignancies (Krause and Van Etten 2005; Baselga 2006), and Src-specific
antagonists are under development for clinical use for the treatment of various solid
tumors and bone metastasis (Araujo and Logothetis 2009, 2010; Aleshin and Finn 2010;
Lieu and Kopetz 2010; Mayer and Krop 2010; Rothschild et al. 2010). Many reviews of
the Src oncoprotein and its relationship to human cancers are available to provide more
in-depth information (Parsons and Weber 1989; Irby and Yeatman 2000; Martin 2001,
2004; Frame 2002; Alper and Bowden 2005; Guarino 2010).
The importance of RSV as the stimulus for the discovery of protooncogenes was
recognized by the Nobel Assembly in the press release that announced the winners
of the Prize in Medicine or Physiology in 1989: Michael Bishop and Harold Varmus
used an oncogenic retrovirus to identify the growth-controlled oncogenes in normal
cells. In 1976 they published the remarkable conclusion that the oncogene in the
virus did not represent a true viral gene but instead was a normal cellular gene,
which the virus had acquired during replication in the host cell and thereafter carried along. Bishops and Varmus discovery of the cellular origin of retroviral oncogenes has had an extensive influence on the development of our knowledge about
mechanisms for tumor development. (The Nobel Prize in Physiology or Medicine
1989 Press Release 1989) Press Release, The Nobel Assembly at the Karolinska
Institute, 9 October 1989, announcing the 1989 Nobel Prize in Physiology or
Medicine. Nobelprize.org. 1 Aug 2010 http://nobelprize.org/nobel_prizes/medicine/
laureates/1989/press.html
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L.J. Parent
RSV Virion Assembly and Structure

In addition to its critical role in establishing viruses as a cause of cancer, RSV was
also the initial retroviruses identified. RSV serves as the prototype for the family of
oncoretroviruses, leading the way in elucidating many fundamental features of the
virus replication cycle. As the fields of cancer biology, oncogenesis, and retrovirology have diverged, RSV has remained a mainstay in pioneering studies of viruscell
interactions, retroviral enzymology and virus assembly. Many of the seminal
discoveries arising from experiments using RSV have been applied directly to treatment strategies for human retroviral treatments and to the development of viral
vectors for gene therapy.
The virion structure of RSV shares many properties with all retroviruses, which
are spherical, enveloped particles (Fig. 28.1) [reviewed in (Vogt 1997)]. Virus
particles are composed of 65% protein, 35% lipid, and 12% RNA. Although the
majority of the protein and RNA species in the virus particle are derived from the
virus, many host proteins and RNAs are also incorporated into retrovirus particles.
The envelope is derived from the host plasma membrane when the virus particle
buds from the host cell [reviewed in (Wills and Craven 1991; Craven and Parent
1996; Swanstrom and Wills 1997; Demirov and Freed 2004)]. Envelope glycoproteins (made up of the SU, surface, and TM, transmembrane components) protrude
as spikes from the virion envelope and attach to specific receptors on the target
cell to permit virus entry [reviewed in (Hunter 1997)]. Virus particles are assembled
within the cell as immature particles, which are not infectious. The morphology of
immature particles is similar for all retroviruses, and consists of an electron dense
layer of Gag and GagPol proteins just under the lipid envelope (Swanstrom and
Wills 1997). Gag and Gag-Pol proteins are arranged in a hexameric lattice with
irregular defects to form a nearly spherical shell (de Marco et al. 2010). The arrangement of the viral RNA genome within the particle is not well understood.
Concurrent with budding, the immature virus particle undergoes maturation, a
process catalyzed by the retroviral PR (protease) enzyme, to generate the infectious
virus particle (Swanstrom and Wills 1997) (Fig. 28.1). Although the sequence
encoding PR is invariably located between the gag and pol genes in all retroviruses,
the presence of PR as a fusion with the Gag polyprotein is unique to RSV. During
processing, the viral protease dimerizes and cleaves the immature Gag polyprotein
into the MA (matrix), CA (capsid), and NC (nucleocapsid) proteins, common to all
Gag proteins, as well as the p2, p10, and SP (spacer) peptides, which are exclusively
present in RSV. The crystal structure of RSV PR was the first high resolution PR
structure obtained (Jaskolski et al. 1990). Importantly, the RSV PR structure was
used as a model to design inhibitors of HIV-1 PR, which have become principal
components of the anti-retroviral drug cocktail (Weber et al. 1989; Weber 1991;
Wensing et al. 2010).
With the processing of Gag and virus maturation, the appearance of the particle
undergoes a dramatic transformation, with the appearance of an electron-dense
irregular core structure near the center (Fig. 28.1) (Swanstrom and Wills 1997).
28
725
Fig. 28.5 Current model of RSV replication cycle. Infection is initiated with binding of the viral
glycoprotein (SU) to the cellular receptor (Tva), triggering fusion of the viral and cellular
membranes. The viral core enters the cytoplasm and reverse transcription occurs within the preintegration complex (PIC) in the cytoplasm. The PIC is translocated to the nucleus, and nuclear entry
occurs with nuclear envelope breakdown during mitosis and also through the intact nuclear pore in
nondividing cells. Integration into the chromosome is mediated by the IN enzyme and facilitated
by host factors. The proviral DNA is transcribed by polII and genome-length viral RNA are made.
A subset of these mRNAs undergoes splicing and exit the nucleus for translation into Env and Src.
Env is transported to the cell surface through the Golgi apparatus and secretory pathway. The remaining unspliced viral RNA has two fates. If it serves as the template for synthesis of Gag and Gag-Pol,
it exits the nucleus, likely using the host cofactors Tap and Dbp5. If it is packaged into virus particles,
there is evidence that the genomic viral RNA might interact with a fraction of the Gag protein that
traffics back into the nucleus after its synthesis in the cytoplasm. Gag has a nuclear export signal that
mediates exit of the viral ribonucleoprotein complex from the nucleus. In the cytoplasm, Gag-vRNA
complexes are targeted to the plasma membrane using host factors, combine with additional Gag and
Gag-Pol proteins, and deform the plasma membrane to release virus particle through budding.
As budding occurs, the maturation process occurs to generate a fully infectious virion
The hexameric array of Gag polyproteins is reorganized into a polyhexagonal shell

composed of the CA protein organized into hexamers and pentamers (Butan et al.
2008; Heymann et al. 2008; Cardone et al. 2009). The MA protein is arranged in an
irregular fashion under the lipid envelope near the transmembrane (TM) domain of
the envelope glycoprotein (Pepinsky and Vogt 1979). Presumably, the center of the
particle contains the NC protein bound to the viral genomic RNA. The specific locations
of other viral proteins, including p2, p10, SP, PR, RT, and IN are unknown.
The RSV replication cycle begins with binding of the SU component of the
envelope glycoprotein to its receptor, Tva, a relative of the low-density lipoprotein
receptor (Bates et al. 1993, 1998; Young et al. 1993) (Fig. 28.5). Binding triggers
726
L.J. Parent
fusion of the viral and cellular membranes, followed by entry of the viral core into
the cytoplasm (Barnard et al. 2006). Reverse transcription of the viral genome takes
place en route to the nucleus, but is not completed until after nuclear entry, when
proviral synthesis is completed (Varmus et al. 1978; Lee and Coffin 1991). Although
it was originally thought that nuclear entry of the RSV preintegration complex
required breakdown on the nuclear envelope during mitosis, more recent analysis
revealed that RSV integration does occur, albeit inefficiently, independently of cell
division (Hatziioannou and Goff 2001; Katz et al. 2002). The proviral DNA is
incorporated into the host chromosomal DNA by the viral IN enzyme, and integration
occurs preferentially in regions of the genome that encode genes transcribed by
RNA polymerase II (Brown 1997; Narezkina et al. 2004).
Following integration, RNA polymerase II transcription of the proviral DNA
results in the synthesis of a genome-length viral RNA, which is processed like cellular mRNAs to contain a 5 7-methylguanine cap and a 3 poly(A) tail (Rabson and
Graves 1997; Vogt 1997). A subset of the full-length viral mRNA undergoes splicing
to produce mRNAs that encode the Env and Src proteins, and these mRNAs are
exported from the nucleus, presumably by the usual route for spliced mRNAs that
involves deposition of exonexon junction machinery (Kim and Dreyfuss 2001).
By contrast, the unspliced viral mRNA has two potential fates, one as the template
for translation of the structural proteins Gag and Gag-Pol, and the other as the genome
for encapsidation into assembling virions (Butsch and Boris-Lawrie 2002).
The factors that govern whether the unspliced, genome-length viral mRNA is
translated or packaged are not well understood. However, retroviruses must export
their unspliced viral RNA from the nucleus to make Gag and Gag-Pol proteins to
begin the process of assembling new virus particles (Rabson and Graves 1997; Vogt
1997). To do so, the normal quality-control measures that prevent unspliced cellular
mRNAs from leaving the nucleus prematurely must be circumvented. For RSV, the
cellular Tap/Dbp5 complex is reported to play a role in export of the unspliced viral
RNA by binding to the cis-acting DR (direct repeat) elements that flank the src gene
(Paca et al. 2000; Leblanc et al. 2007).
Assembly of new virions is mediated by the Gag polyprotein, which is sufficient to
drive particle production and release (Wills and Craven 1991; Craven and Parent
1996; Swanstrom and Wills 1997; Pincetic and Leis 2009) (Fig. 28.6). To initiate virus
assembly, the Gag NC region selects the viral RNA for encapsidation by binding to
the psi sequence in the RNA sequence (Jaskolski et al. 1990; Rein 1994; Swanstrom
and Wills 1997; Jewell and Mansky 2000; Dsouza and Summers 2005; Zhou et al.
2005, 2007). Recent evidence supports a model whereby the RSV Gag polyprotein,
which undergoes transient nuclear trafficking, may capture its unspliced viral RNA
genome in the nucleus, thereby circumventing cellular mechanisms that retain
unspliced mRNAs in the nucleus (Scheifele et al. 2002; Garbitt-Hirst et al. 2009).
The RSV Gag protein has a dynamic trafficking pathway throughout the cell and
interacts with numerous host factors from its synthesis on ribosomes, through the
nucleus, and to the plasma membrane (Craven and Parent 1996; Scheifele et al.
2002; Butterfield-Gerson et al. 2006; Pincetic and Leis 2009). The regions of Gag
required for virus particle assembly have been identified as the M (membrane-binding),
28
727
Fig. 28.6 Functional domains and host partners of the Gag protein. The RSV Gag protein is
synthesized as a polyprotein, which is cleaved during virus maturation to generate the MA (matrix),
p2a, p2b, p10, CA (capsid), SP (spacer), NC (nucleocapsid), and PR (protease) proteins. Gag
contains two nuclear localization signals (NLS). The NLS in the N-terminal region of MA binds to
host import factors importin-11 and transportin-SR/transportin 3. The NLS in NC interacts with
the importin-alpha adaptor, which in turn binds to importin-beta to create a functional nuclear
import complex. The NC region also contains a zinc-knuckle domain composed of Cis-His boxes,
which are responsible for binding to the genomic vRNA for encapsidation. The CRM1 export factor binds to a nuclear export signal (NES) in the p10 region of Gag. The MHR, or major homology
region, of Gag is indicated and plays an important structural role. The assembly domains are illustrated below the Gag molecule. The M (membrane-binding domain) folds as an alpha-helical
domain that transports Gag to the plasma membrane through a poorly understood trafficking process. The I (interaction) domain in NC is present in two copies is made up of the Cis-His boxes
(Zn2+ knuckle domains indicated) and basic residues (red + containing circles) that are important
for NCvRNA and NCNC interactions. The L (late) domain is in the p2b region and interacts with
a complex of host factors including the ubiquitin ligase Nedd4 and several members of the
ESCRT-II and ESCRT-III families to facilitate the final pinching-off step of the budding process
I (interaction), and L (late) domains (Wills and Craven 1991). Analogous functional
assembly domains were subsequently identified in other retroviral Gag proteins
(Bennett et al. 1993; Weldon and Wills 1993; Wills et al. 1994; Zhou et al. 1994;
Parent et al. 1995; Verderame et al. 1996; Swanstrom and Wills 1997; Bowzard
et al. 1998; Sandefur et al. 2000; Pincetic and Leis 2009). The L domains of different
retroviruses contain sequence motifs that interact with a multitude of host factors
that belong to the ESCRT family of endosomal transport factors that mediate the
final release of particles from the plasma membrane (Parent et al. 1995; Demirov
and Freed 2004; Morita and Sundquist 2004; Pincetic and Leis 2009). Although
initially discovered in RSV and HIV, L domains and the family of host proteins with
which they interact are shared by many enveloped viruses (Freed 2002; Licata et al.
2003; Irie et al. 2004; Schmitt et al. 2005; Zhadina and Bieniasz 2010). Discovery
of this common pathway usurped by many unrelated enveloped viruses may lead to
the development of agents with broad antiviral activity.
728
L.J. Parent
Conclusions
RSV, a humble chicken virus, (Vogt 2009) has an important place in history for
the breadth and magnitude of its contributions to biomedical sciences and molecular
biology (Table 28.1). The scientists who studied RSV were rewarded for their
insights, not only by the awards and prizes they received for their achievements but
also with the excitement generated by the broad-reaching implications of their work.
Table 28.1 Timeline of landmark discoveries derived from studies of RSV
Year
Discovery
1910
A spindle cell tumor could be transferred from one Plymouth Rock hen to another
(Rous 1910)
1911
Tumor was transmitted by a filterable virus that fulfilled Kochs postulates, proving
infectious origin of the tumor (Rous 1911)
1938
Development of a method to measure the titer of infectious RSV (Keogh 1938)
1941
RSV-infected chicken embryo fibroblasts in culture demonstrate characteristic
spindle-shape morphology (Halberstaedter et al. 1941)
1955
Each RSV-infected cell produces virus particles, and the virus perpetuates malignant
state of the cell (Rubin 1955)
1956
Described the morphology of RSV using electron microscopy (Epstein 1956; Epstein
and Holt 1958)
1958
Development of the focus assay, which isolated colonies of individual cells infected
with RSV (Temin and Rubin 1958)
1959
RSV induces tumors in mammals (Schmidt-Ruppin 1964)
1960
Provirus Hypothesis proposed (Temin 1976)
1961
RSV virions contain an RNA genome (Crawford 1960)
1970
Discovery of reverse transcriptase activity in virions (Baltimore 1970; Temin and
Mizutani 1970)
1976
Discovery of cellular src gene in avian species (Stehelin et al. 1976)
1977
Development of an antibody to detect v-Src protein (Brugge and Erikson 1977)
1980
Nucleotide sequence of the v-src gene (Czernilofsky et al. 1980a; Takeya and
Hanafusa 1982; Czernilofsky et al. 1983)
1981
Cloning of chicken c-src gene (Shalloway et al. 1981)
1985
Cloning of human c-src gene (Gibbs et al. 1985)
Src oncoprotein discovered to have tyrosine kinase activity (Collett and Erikson 1978;
Levinson et al. 1978)
1978
Discovery of cis-acting packaging sequence sets stage for retroviral vectors
1985
Development of reverse transcriptase inhibitors for HIV treatment (Yarchoan et al. 1986)
1989
Crystal structure of RSV protease (Miller et al. 1989)
1991
Description of retroviral assembly domains (Wills and Craven 1991)
1995
Development of protease inhibitors for HIV treatment (Kitchen et al. 1995)
1997
Crystal structure of Src (Williams et al. 1997; Xu et al. 1997)
1999
Role of Src in human cancer demonstrated (Irby et al. 1999)
2004
Src-specific kinase inhibitors developed for human tumor treatment (Lombardo et al.
2004)
2009
Structure of the pentamer of mature polyhedral RSV capsids visualized (Cardone
et al. 2009)
28
729
Studies based on RSV launched entire new fields: tumor biology, cancer genetics,
oncogenes, retrovirology, tyrosine kinases, reverse transcription, cDNA cloning,
gene therapy vectors, and treatment of human cancers and AIDS. Ongoing research
into Src and its activities in normal and malignant processes continues to make
contributions to understanding the cell biology of cancer. Finally, the virus itself is
the basis for contemporary novel findings regarding fundamental mechanisms in the
retrovirus replication cycle.
Acknowledgements The author is grateful for support from the national institutes of health great
number ROI CA76534 and thanks members of her laboratory for eritical review of the manuscript.
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Chapter 29
Mouse Mammary Tumor Virus and Cancer

Susan R. Ross
Introduction
A key to understanding the transformation of normal cells is dissecting the various
genetic changes that lead to abnormal cell division. Since their discovery at the
beginning of the twentieth century, transforming retroviruses have been used to
study both the mechanism and progression of tumor development (Rous 1911).
Three general classes of transforming retroviruses have been described: acute transforming retroviruses, which encode oncogenes in their genome and thereby cause
rapid, polyclonal tumors of virtually all infected cells; nonacute transforming retroviruses, which cause dysregulated expression of cellular oncogenes upon integration
of the provirus into the host genome and usually cause monoclonal tumors with
latencies longer than those seen with acute retroviruses; and transactivating, transforming retroviruses, which encode proteins that contribute to transformation but
are not by themselves sufficient to induce full-blown cellular transformation (Coffin
et al. 1997). Several other chapters in this book cover transforming retroviruses that
cause cancer in humans as well as other mammals (see chapters on Jaagsiekte Sheep
Retrovirus and Lung Cancer, Avian and Murine Retroviruses, Rous Sarcoma Virus,
Human and Animal Retroviruses, HTLV-1 and HTLV-2). Here, I present a brief
review of the betaretrovirus mouse mammary tumor virus (MMTV), what is known
about how the virus induces tumors, and how this knowledge has been used to
develop a large number of mouse models for human breast cancer.
The recognition in the nineteenth century by mouse fanciers that mammary
tumors occur in mice, coincided with the breeding of mice for particular genetic
traits [for an excellent review of the history of mammary tumorigenesis in mice, see
Cardiff and Kenney (2007)]. In 1933, a landmark paper from scientists at the Jackson
S.R. Ross (*)

Department of Microbiology, Abramson Cancer Center, University of Pennsylvania
e-mail: rosss@mail.med.upenn.edu
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S.R. Ross
Laboratory demonstrated that there was maternal inheritance of mammary tumor

incidence; that is, the F1 female offspring of high tumor-incidence mothers mated
with low tumor-incidence fathers developed mammary tumors while the F1 offspring
of the reciprocal cross did not (Jackson and Little 1933). Shortly thereafter, J.J. Bittner
showed that high mammary tumor-incidence mice could be freed of a milk-transmitted
agent by foster nursing on low tumor-incidence mothers and suggested that this
agent might be a virus, representing the first identified mammalian tumor virus
(Bittner 1936). However, it was not until the 1960s that Bittner agent or MMTV
was shown to have an RNA genome like Rous sarcoma virus and thus was definitively classified as a virus (Duesberg and Blair 1966). In the three intervening
decades between Bittners observations and the definitive classification of MMTV
as a virus, much was learned about its in vivo transmission and the genetics of
susceptibility of different inbred strains of mice to infection (Nandi and McGrath
1973; Okeoma and Ross 2010). The use of recombinant DNA technology and the
advent of transgenic and knockout mice in the 1980s led to the identification of the
genes involved in susceptibility and resistance to virus infection and novel oncogenes
associated with MMTV-induced mammary tumors [recently reviewed in Okeoma
and Ross (2010) and Ross (2010)]. This in turn allowed the development of new
mouse models of human breast cancer, as described below.
MMTV Infection and Germline Transmission

Milk-transmitted MMTV is produced by the mammary epithelial cells of lactating
mothers and is most likely transmitted as cell-free virus to suckling mice during the
1st week of life (Nandi and McGrath 1973). In nursing pups, the virus first infects
dendritic cells in the gut and then spreads to B and T cells in the Peyers patches and
mesenteric lymph nodes. During the first 3 weeks of life, MMTV infection amplifies
throughout cells of the immune system, which then traffic to the mammary gland
and deliver virus to puberty-driven dividing mammary epithelial cells (Ross 2000).
MMTV infection of mammary epithelial cells also increases during pregnancyinduced cell division, but still depends on the presence of infected lymphocytes in
this tissue (Fig. 29.1) (Golovkina et al. 1998). Since MMTV is a nonacute, transforming retrovirus and causes transformation by integrating near-cellular oncogenes
and activating their expression, the more mammary cells infected, the more likely it
is that such an integration event will occur. Thus, virgin mice which have lower
levels of mammary epithelial cell infection have decreased tumor incidence compared
to mice that have gone through multiple rounds of pregnancy and the average timeto-tumor formation is longer in virgin than in multiparous mice (Nandi and McGrath
1973). Tumor induction is close to 100% by 1 year of age in mouse strains that are
susceptible to MMTV infection.
In addition to acquiring exogenous virus through milk, mice can also inherit
endogenous copies of the provirus, termed Mtv loci (Kozak et al. 1987). Indeed, most
commonly used laboratory strains have from one to six copies of MMTV in their
germline, the vast majority of which have one or more mutations in the viral genes
29

Infected lymphocytes
deliver virus to
mammary gland
741
Virions shed
in milk
int
Puberty and
initial
infection
Provirus integration
into chromosome
PregnancyAdditional
infection
Oncogene
activation
Transformation
Partruition
= apoptosis
Fig. 29.1 MMTV in vivo infection pathway. Lymphocytes in the small intestine become infected
by MMTV and virus spreads in these cells. The infected lymphocytes traffic to the pubescent and
lactating mammary gland and mammary epithelial cells get infected (Finke and Acha-Orbea 2001).
This results in mammary epithelial cells that secrete virus into milk which is transmitted to the next
generation. Proviral insertion occurs during puberty, but the higher virus load that occurs after
multiple pregnancies increases the probability that activating insertions next to cellular oncogenes
will occur, leading to transformation
and thus do not produce infectious virions. However, not all wild mice have endogenous
MMTV proviruses and thus it has been estimated that MMTV first infected mice ~10
million years ago, after their speciation (Baillie et al. 2004). There are also several
strains of inbred mice that inherit functional, fully infectious endogenous copies of
MMTVs, most likely representing more recent germline integrations; such mice cannot be freed of mammary tumor induction by foster nursing (Nandi and McGrath
1973; Michalides et al. 1978). At least 10 different exogenous and more than 30
endogenous MMTVs have been identified (Kozak et al. 1987; Imai et al. 1994;
Callahan and Smith 2000; Golovkina et al. et al. 1997).
MMTV Infection and Replication

Enveloped viruses, like MMTV, enter cells when the viral glycoprotein (Env) on
virions binds to receptors on the target cell surface. MMTV uses transferrin receptor
1 (TfR1) as its entry receptor (Ross et al. 2002). TfR1, whose normal function is to
mediate uptake of iron-loaded transferrin, is highly expressed on actively dividing
cells (Ponka and Lok 1999). Although virtually all cultured cells express large
amounts of TfR1, expression in vivo is limited to activated lymphocytes and a few
other tissues, including epithelial cells of the pregnant mammary gland (Brekelmans
et al. 1994; Schulman et al. 1989). Thus, milk-transmitted virus infection is largely
limited to these cell types in mice (Nandi and McGrath 1973).
Upon binding transferrin, TfR1 is endocytosed to the recycling endosome, a pH
6 compartment, where iron is released; the receptor then recycles to the surface
742
S.R. Ross
(Ponka and Lok 1999). However, MMTV requires both receptor binding and pH 5
to achieve entry into cells (Wang et al. 2008). MMTV binding to TfR1 redirects its
endocytosis to a late endosomal compartment from which the virus enters cells.
After entry into the cytoplasm, MMTV is reverse transcribed and the doublestranded DNA enters the nucleus and integrates into the chromosome; nuclear entry
of the preintegration complex, which consists of viral DNA and proteins as well as
cellular proteins, is believed to require cell division, similar to other retroviruses
with the exception of the lentiviruses (Ross 1997b). However, these steps in the
MMTV infection pathway have not been well-elucidated.
Transcriptional Control of MMTV

Retroviral long terminal repeats (LTRs) contain the transcription start site as well as
the regulatory sequences that determine proviral expression after integration into
the host chromosomes. In the case of MMTV, which infects both lymphoid and
mammary epithelial cells, there are sequences that specify expression in both cell
types as well as hormone-regulated virus transcription (Ross 1997b). One of the
earliest observations made about MMTV was that glucocorticoids and progesterone
increase the amount of shed virus in both mice and cell lines derived from MMTVinduced mammary tumors (McGrath 1971). The increased virus production is due
to enhanced transcription of MMTV, the result of direct interaction of the glucocorticoid or progesterone hormone receptors with sequences in the LTR (Payvar et al.
1983). More recently, it has been shown that other transcription factors, such as
FoxA1 and NF1, participate in the hormone-regulated control of MMTV transcription
(Fig. 29.2a) (Holmqvist et al. 2005; Vicent et al. 2010).
Regulatory elements that determine high levels of viral transcription in mammary
cells have also been identified in the LTR (Fig. 29.2a). These include recognition
sites for the STAT5 transcription factor, which is a target of prolactin receptor
signaling and is critical for the development of mammary alveolar epithelial cells,
the cell type most often the target of MMTV-induced transformation (Qin et al.
1999; Cardiff and Kenney 2007). Indeed, MMTV transcription in mammary epithelial
cells is induced by prolactin (Qin et al. 1999). While both MMTV proviruses
acquired by infection and many endogenous Mtv loci are expressed in virgin
mammary gland, pregnancy and lactation dramatically increase their transcription.
This is due in part to increased prolactin, glucocorticoid, and progesterone levels
and because another factor, the Cutl1/CCAAT displacement protein (CDP), which
represses MMTV transcription in virgin mammary glands, declines in late pregnancy
at the same time that there is an increase in the level of viral transcripts (Maitra et al.
2006). Additionally, there are other sequence elements that determine mammary
epithelial cell transcription for which the transcription factors have not been identified (Mok et al. 1992; Mink et al. 1992; Qin et al. 1999).
Because the MMTV LTR directs high levels of expression in mammary cells, it
has been extensively used to create transgenic mice that develop breast cancer.
29
MCF
743
STAT 5
b
Wnt, Fgf
(upstream/downstream; enhancer activation)
Notch
(exon; disrupt N terminus)
eIF3e
(intron; premature termination; truncated protein)
Fig. 29.2 The MMTV LTR contains transcriptional regulatory elements that control virus and
oncogene expression. (a) Diagram of the MMTV LTR and the relative position of the transcription
factor-binding sites that have been identified (see text). MCF mammary cell factor. (b) Possible
mechanisms by which MMTV insertion near a cellular oncogene can result in dysregulated oncogene expression. Known examples of each mechanism are shown
Indeed, the MMTV-c-myc transgenic mouse was the first mouse model of breast
cancer (Stewart et al. 1984). Since 1984, a large number of different genes have
been placed under the control of the MMTV LTR (ras, neu, simian virus 40 large
T antigen, polyomavirus middle T antigen, transforming growth factor b, etc.) and
used to study mammary gland differentiation and transformation in transgenic mice
(Cardiff 2001, 2003). In many of these models, the incidence and kinetics of mammary tumor induction are increased in multiparous versus virgin mice, most likely
the result of the increase in LTR-driven transcription. MMTV-cre-recombinase
transgenic mice have been crossed to mice with floxed genes to induce mammary
epithelial cell-specific deletion of tumor-suppressor and other genes (Wagner et al.
2001). Although MMTV-transgenic mice have been an invaluable tool for researchers,
in some models, high level transgene expression is dependent on pregnancy and
thus this system is not always amenable to studying early events in mammary cell
differentiation.
MMTV also infects lymphocytes and other cells of the immune system and there
are transcriptional regulatory elements in the virus that drive expression in these
cells (Ross 1997a; Arroyo et al. 1997; Reuss and Coffin 2000). It is, therefore, not
surprising that many MMTV-oncogene transgenic mice, including the original
MMTV-myc mice, develop lymphomas in addition to mammary tumors (Stewart
et al. 1984; Leder et al. 1986; Choi et al. 1987). MMTV expression levels are, however,
much lower in lymphoid than mammary tissue, so MMTV LTR-driven transgenes
generally have a much larger effect in the mammary gland. MMTV virus variants
744
S.R. Ross
have also been found in some T-cell lymphomas (Ball et al. 1985; Michalides 1983).
These T-cell tropic MMTVs have deletions/insertions in their LTRS that are believed
to create novel transcription factor regulatory sites that drive higher expression of
the variant viruses in T cells. Integration of the variant MMTVs in this cell type also
activates oncogenes and transformation (see below) (Yanagawa et al. 1993; Bhadra
et al. 2005).
MMTV Induction of Tumors

MMTV is highly efficient at inducing mammary tumors in mice that are genetically
susceptible to infection, with almost 100% of mice developing 12 independent
tumors by the age of 1 year (Nandi and McGrath 1973). MMTV-induced mammary
tumorigenesis occurs when the provirus integrates near-cellular oncogenes and
activates their expression (Fig. 29.1). Because this integration appears to occur
stochastically, the higher the level of infection, the more likely it is that such a transforming event will occur and as such, the kinetics and incidence of mammary tumor
induction can be used as measures of infection levels (Golovkina et al. 1993).
Retroviral insertion, which can occur >250 kb away from the cellular oncogene,
alters oncogene expression through several mechanisms (Akagi et al. 2004). These
include: enhancer activation, leading to de novo or increased expression of genes at
the provirus integration site; promoter insertion, whereby the viral promoter is used
in lieu of the normal promoter to initiate transcription; posttranscriptional dysregulation, which occurs when the virus integrates within the gene-coding region and
either through read-through transcription or altered splicing results in an mRNA
with an altered half-life; and gene disruption, where integration into the coding
region results in the loss of gene expression or the production of a mutant or
nonfunctional gene product [reviewed in Dudley (2003)]. Common integration sites
(CISs) are defined by proviral integrations into the same genomic region in more
than one independent tumor. Several groups have also defined CIS to include
integrations into different genes of the same pathway; as an example, in MLVinduced leukemia, integrations into multiple members of the Notch pathway (notch1,
notch2, jag1, lfng, dtx1, dtx2, tcfe2a, rbpsuh, hes1, hes2, hes5) have been detected
(Suzuki et al. 2002). MMTV most frequently causes enhancer activation, although
several examples of gene disruption/truncation have also been documented
(Fig. 29.2b) (Callahan and Smith 2008).
A large number of CISs (historically termed integration or int genes) have been
implicated in MMTV-mediated mammary tumors (Table 29.1) (Callahan and Smith
2000, 2008). In particular, Wnt1 was the first oncogene cloned by insertion site
analysis and Wnt family members and other genes in this pathway are frequently
targeted by MMTV in tumors (Nusse and Varmus 1982; Callahan and Smith 2008).
Numerous fibroblast growth factor genes, as well as their receptors, are also frequent sites of MMTV integration (Dickson et al. 1984; Theodorou et al. 2007;
Callahan et al. submitted). Finally, members of the R-spondin (Rspo) family have
29
745
Table 29.1 CIS in MMTV-induced mammary tumors in wild-type and transgenic mice
Mouse strain
Oncogene
Associated with HBC a
Wild type
Multipleb
Wnt1/Wnt10b
BALB/c
Wnt3/Wnt9B/Nsf
+
Multiple
Wnt3a/Wnt9a
+
Multiple
Fgf3
+
GR
Tpl2/Cot
+
BALB/c
Fgf4
BALB/c
Fgf10
+
BALB/c, C3H
Fgfr2
+
Czech II
Notch4
BALB/c
int-5/aromatase
Czech, BALB/c
Rspo2
BALB/c
Rspo3
+
Czech II
eIF3e-p48
C3H, Czech II
Pdgfra
BALB/c
Pdgfrb
+
Transgenic
MMTV-Wnt1
Fgf4, Fgf8
, +
MMTV-neu
Notch1
WAP-TGFb
Wnt1/Wnt3
, +
WAP-p53172H
Pdgfra
Data is from Nusse et al. (1984), Theodorou et al. (2007), Callahan et al. (submitted), Lowther
et al. (2005), Dickson et al. (1984), Erny et al. (1996), Theodorou et al. (2004), Gallahan and
Callahan (1997), Durgam and Tekmal (1994), Gattelli et al. (2006), Marchetti et al. (1995),
MacArthur et al. (1995), Shackleford et al. (1993), Dievart et al. (1999), Schroeder et al. (2000),
Chatterjee et al. (2002), and Meyers and Dudley (1992)
a
Association with human breast cancer (HBC) analysis of the Oncomine database, the Cancer
Genome Atlas, Cosmic (http://www.sanger.ac.uk/genetics/CGP/cosmic), and Broad Gene Ranker
databases
b
More than two mouse strains, such as C3H/He, BALB/c, Czech II, and MMTV transgenics
also been identified as MMTV CIS (Gattelli et al. 2006; Lowther et al. 2005).
These three gene families have been termed core CIS for MMTV. Several groups
have recently also used high-throughput analysis insertion site analysis and uncovered
genes not previously associated with MMTV-induced cancer (Theodorou et al. 2007;
Callahan et al., submitted; Kim et al. unpublished observations). The variant
MMTVs that induce T-cell lymphomas also integrate near-cellular oncogenes and
activate their expression. Notch1, c-myc, and retinoic acid receptor g (RORg) have
been identified as CISs in these tumors (Broussard et al. 2002, 2004; Yanagawa
et al. 2000).
MMTV-induced mammary tumors are thought to be the result of virus infection of
a single initial stem cell, and mammary cell transplantation studies have indicated that
oncogene integration events occur very early in transformation (Fig. 29.3) (Gattelli
et al. 2006; Kordon and Smith 1998). Most MMTV-induced tumors proceed through
at least two stages, the pregnancy-dependent hyperplastic alveolar nodule (HAN)
746
S.R. Ross
Infection of mammary epithelial stem or
progenitor cell
Random provirus
integration
env
Transcription/translation
of Env(and other viral
proteins)
Env expression on
cell surface
ITAM-mediated
signaling
CIS1
Oncogene activation
CIS2
Activation of
additional oncogenes
Other mutations
TRANFORMATION
Fig. 29.3 MMTV-mediated mammary tumorigenesis. MMTV infects mammary stem cells and
after proviral integration, the viral genome is transcribed and translated. The Env protein then
signals through its ITAM motif, leading to increased lobuloalveolar differentiation. After integration
of the MMTV provirus into one or more CISs, the cell becomes fully transformed and develops
into a tumor
followed by the hormone-independent mammary tumor (Callahan and Smith 2000).

Some studies have suggested that progression of tumors from a pregnancy-dependent
to a pregnancy-independent state depends on additional MMTV integration events
and indeed, the former frequently present as polyclonal populations while the latter
are generally monoclonal (Buggiano et al. 2002). Thus, integration events potentially
affect both the early and late steps of transformation. Moreover, because MMTV
infects stem cells, which can develop into ductal, alveolar, and epithelialstromal cells
within the mammary gland, insertional alteration of oncogene expression can occur in
any of these cell types, which could affect the final tumor phenotype.
29
747
Several other observations support the idea that the activation of multiple oncogenes
is required for MMTV-induced mammary tumorigenesis. Most MMTV-induced
tumors contain ten or more proviral integrations and a large percentage of mammary
tumors derived from MMTV-infected mice have integrations at multiple CISs, such as
Wnt1 and Fgf3 (Fig. 29.3) (Callahan and Smith 2000). Moreover, double-transgenic
mice, such as MMTV-Wnt1/MMTV-Fgf3, exhibit accelerated mammary tumor induction in comparison to their single-transgenic MMTV-Wnt1 or MMTV-Fgf3 parental
lines (Kwon and Weissman 1984). Similarly, MMTV infection of transgenic mice
with a genetic predisposition to mammary tumorigenesis (e.g., MMTV-Wnt1, MMTVc-neu, or MMTV-Tgfb) accelerates tumorigenesis, and several novel oncogene insertion
sites have been found in such mice (Dievart et al. 1999; Schroeder et al. 2000;
Shackleford et al. 1993). There are rare MMTV-induced tumors with only one MMTV
insertion site (Nusse and Varmus 1982); for these tumors, it has been suggested that
genetic changes in cellular oncogenes that are not induced by virus could participate
in transformation (Fig. 29.3) (Callahan et al. submitted).
There is also evidence that MMTV infection of mammary epithelial cells leads
to their dysregulated growth prior to the integration of the provirus into particular
CIS. Early studies that examined the histopathology of mammary glands from uninfected and MMTV-infected mice indicated that lobuloalveolar differentiation, the
result of increased cell division at terminal buds, was increased by viral infection
(Squartini et al. 1983). This phenotype has been recapitulated in MMTV-transgenic
mice (Ross et al. 2006a). More recent studies have indicated that this is due, at least
in part, to expression of the MMTV Env protein in infected cells. The MMTV Env
protein is found on the surface of infected cells and signals through an immunotyrosinebased activation motif (ITAM). ITAMs are highly conserved sequences found in
receptors involved in the activation, proliferation, survival, and differentiation of
hematopoietic cells (Grande et al. 2007). The tyrosine residues found in the canonical
motif DxxYxx(L/I)x6-12Yxx(L/I) are necessary and sufficient for signaling and after
phosphorylation by intracellular Src-family protein tyrosine kinases, the ITAMassociated tyrosines function as docking sites for SH2-containing signaling proteins,
such as Syk, which link receptor-initiated signals to downstream cellular responses.
Expression of the MMTV Env protein alone in cultured normal human or mouse
mammary epithelial cells leads to morphological transformation (Katz et al. 2005).
Moreover, infectious MMTV with mutations in the ITAM, while fully infectious in
mice, shows slower kinetics and decreased incidence of mammary tumor induction
(Ross et al. 2006b). Interestingly, a number of oncogenic viruses, some with tropism
for nonhematopoietic cells, encode ITAM-containing plasma membrane-associated
proteins that play a role in their ability to transform cells, including EpsteinBarr
Virus (EBV) LMP2A, Kaposis Sarcoma Virus K1, and Bovine Leukemia Virus
gp30 [reviewed in Grande et al. (2007)]. It has also been recently shown that the
C35 protein, which is part of the HER2 amplicon and overexpressed in many invasive
breast cancers, contains an ITAM and may be involved in transformation (Katz et al.
2010). These data suggest that ITAM-mediated signaling by the MMTV Env and
other proteins in infected mammary epithelial cells participates in the transformation
process (Fig. 29.3).
748
S.R. Ross
MMTV and Human Breast Cancer

MMTV-induced mammary tumors usually have different histopathological signatures
when compared to human breast cancers. For example, the most commonly seen
form of human breast tumors, invasive ductal carcinomas, are rarely found in
MMTV-infected mice. Instead, MMTV tumors are usually characterized as acinar
in origin (Cardiff and Wellings 1999). However, many, if not all, of the human
mammary lesions are thought to originate in the terminal ductal lobular unit and
atypical lobular type A (ALA) lesions are morphologically similar to the mouse
mammary HAN lesions (Callahan and Smith 2000). Moreover, the tumors that
develop in MMTV-transgenic strains can resemble more common forms of human
breast cancer. For example, tumors in MMTV-ErbB2 transgenic mice are very similar
to human ductal carcinoma in situ (Cardiff et al. 2000; Mikaelian et al. 2004; Rosner
et al. 2002).
The first identified MMTV CIS, Wnt1, has not been implicated in human breast
cancer. However, other Wnt family members and the downstream targets of the Wnt
signaling pathway (b-catenin, E-cadherin, cyclin D1) are mutated or deregulated in
many human cancers, including breast cancer (Table 29.1) (Li et al. 2000).
Additionally, several Fgf family members have been implicated in human breast
cancer (Meyers and Dudley 1992; Theodorou et al. 2004). In silico comparison of
genes identified in MMTV CIS screens with expression databases of human breast
cancers have uncovered new potential markers that may be useful for molecular
diagnosis (Theodorou et al. 2007; Callahan et al. submitted). Thus, MMTV target
site analysis in both wild-type and transgenic mice strains with tumor morphology
similar to that seen in humans may have potential for uncovering pathways relevant
to the human disease.
A long unresolved question is whether any virus similar to MMTV exists in
humans. Very early studies indicated the presence of MMTV-like proteins in human
breast cancer samples and milk, as well as antibodies to MMTV proteins in human
sera (Levine et al. 1984; Keydar et al. 1984). However, it is now believed that these
studies detected cross-reacting proteins and nonspecific antibodies (Dion et al.
1987; Goedert et al. 2006). More recently, several groups have also detected
sequences with 8595% homology to MMTV in human breast cancer biopsies using
nested polymerase chain reaction (Wang et al. 1995; Ford et al. 2003), although an
equivalent number of studies have failed to find such sequences (Mant et al. 2004;
Park et al. 2011). Similarly, there have been some studies indicating that the mouse
virus can infect human cells (Lasfargues et al. 1979; Indik et al. 2007), although our
group has been unable to show that MMTV can use human TfR1 as an entry receptor
(Ross et al. 2002; Wang et al. 2008).
If MMTV-like viruses do exist in humans, it is unclear how such viruses are transmitted. In contrast to MMTV-infected mice that develop mammary tumors, where
most if not all mammary cells are infected, a very low percentage of cells in human
breast cancer biopsies appear to contain these sequences and additionally, normal
breast tissue from the same individuals are negative for the viral DNA (Wang et al. 1995).
29
749
That only breast cancer and not the normal mammary epithelial cells carry MMTVlike sequences would, thus, argue against a milk-borne mode of transmission. In
mice, the major mode of transmission is through milk and horizontal spread in adult
mice housed in the same cage is very rare (Nandi and McGrath 1973). Moreover,
infection of adult mice with MMTV results in life-long, high-titer antibodies against
the virus (Luther et al. 1997), so if the virus was transmitted from mice to humans,
humans with breast cancer should have anti-MMTV antibodies, which, as discussed
in the preceding paragraph, have not been detected. Thus, the mechanism by which
the virus would make an efficient zoonotic leap from mice into humans is currently
unclear.
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Chapter 30
Jaagsiekte Sheep Retrovirus and Lung Cancer

Chassidy Johnson and Hung Fan
Introduction
Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious neoplasm
in sheep termed ovine pulmonary adenocarcinoma (OPA) previously known as sheep
pulmonary adenocarcinoma and ovine pulmonary carcinoma (Palmarini et al. 1997,
1999; Palmarini and Fan 2003). The first documentation of OPA was in the nineteenth century when a farmer from Cape of Good Hope, South Africa wrote to the
magistrate stating that he was losing many of his sheep to a disease he called
Jaagziekte. In Afrikaans, the word translated to Jaagsiekte (Jaag = chase; siekte = sickness) because the affected sheep had the appearance that they had been chased (Tustin
1969; York and Querat 2003). OPA has been reported on all continents with the
notable exception of New Zealand and Australia. JSRV infection and OPA is a veterinary concern and economic burden in high endemic regions (i.e. Europe and Africa),
and the possibility that it can act as a human pathogen has not been ruled out. OPA is
derived from lung secretory epithelial cells, type II pneumocytes and Clara cells, and
closely resembles bronchioloalviolar carcinoma (BAC) or adenocarcinoma of mixed
type with BAC component in humans (Perk and Hod 1982; Palmarini et al. 1997).
Compared to other types of cancers (i.e. leukemia, lymphoma and breast), model
systems to study naturally occurring lung cancers are limited. The similarities
between OPA and BAC make JSRV and OPA a useful model system to study human
lung carcinogenesis. Furthermore, study of JSRV and enJSRV has proven to be an
elegant model system to study retroviral/host evolution.
C. Johnson H. Fan (*)

Department of Molecular Biology and Biochemistry, Cancer Research Institute,
University of California, Irvine, CA 92697, USA
e-mail: hyfan@uci.edu
755
756
C. Johnson and H. Fan
Retroviruses
The Retroviridae are a large family of enveloped and positive-stranded RNA viruses
that infect a wide range of vertebrate species including humans, birds, cats, fish,
sheep, and more. Retroviral infection can have pathogenic outcomes including
malignant, degenerative, and immunodeficiency diseases. Retroviruses have provided
useful tools to elucidate mechanisms of cancer. The fundamental genomic organization
of all retroviruses is (5)-R-U5-gag-pol-env-R-U3 (3) (Fig. 30.1). Gag encodes the
structural proteins that comprise the internal structure of the virions including the
matrix (MA), capsid (CA), and the nucleocapsid (NC) proteins. The pol gene
encodes the protease (PR), reverse transcriptase (RT), and integrase (IN) enzymes.
The env gene encodes the envelope surface glycoprotein (SU) and transmembrane
(TM) proteins. Gag, pol, and env are the genes required for the structure and replication of retroviruses. Simple retroviruses only encode these genes, whereas complex
retroviruses encode additional genes. Some retroviruses have also captured cellular
genes from the host that act as oncogenes. The virion structure of a simple retrovirus
is depicted in Fig. 30.2. A hallmark feature of retroviruses is that they can convert
the RNA genome into a DNA copy that becomes permanently integrated into the
chromosomal DNA of the infected cell.
Viral genome
5 CAP
gag
R
pol
AAA 3
env
U3
U5
Provirus
gag
U3 R U5
LTR
pol
env
U3
R U5
LTR
Fig. 30.1 Schematic organization of a simple retrovirus genome. The two forms of the genome
are depicted. The RNA genome contains gag, pol, and env genes and is capped at the 5 end and
polyadenylated at the 3 end. The viral RNA contains terminal repeats (R) and unique regulatory
sequences (U3 and U5) at either end. The RNA genome is reverse transcribed into double stranded
DNA by the viral reverse transcriptase. This process results in long terminal repeat elements
(LTRs) at either end of the viral DNA. The viral DNA is integrated into the host DNA by viral
integrase to form the provirus
30 Jaagsiekte Sheep Retrovirus and Lung Cancer
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TM
CA
RT
NC
SU
MA
IN
PR
Lipid bilayer
Fig. 30.2 Structure of a simple retrovirus. The virion of simple retroviruses contain two copies of
the RNA genome that are coated with nucleocapsid (NC). The capsid (CA) forms the inner core
that contains the genome, reverse transcriptase (RT), integrase (IN), and protease (PR) enzymes.
The virion is enclosed in a lipid bilayer that contains envelope protein subunits, surface (SU) and
transmembrane (TM). The matrix (MA) is located beneath the lipid bilayer
Retroviral Lifecycle
The lifecycle of a simple retrovirus is shown in Fig. 30.3. Following SU binding to
the cellular receptor, the virion is internalized and the nucleocapsid core is released
into the cytoplasm of the host cell. The RT in the core particle is then activated.
Reverse transcriptase is an RNA-dependent DNA polymerase that gives the virus
the ability to transcribe the single-stranded RNA genome into a double-stranded
DNA. The reverse transcribed copy of the viral RNA yields a slightly longer DNA
copy because it is flanked by long terminal repeat elements (LTRs) that are formed
during the duplication event (Fig. 30.1). The DNA copy is transported to the nucleus
where the virus-encoded integrase (IN) catalyzes integration into the cellular DNA
to form the provirus. The U3 region of the LTR contains the viral promoter and
enhancer elements; the provirus is transcribed by cellular RNA polymerase II to
give a primary transcript identical to virion RNA. Viral transcripts are exported to
the cytoplasm (with and without mRNA splicing) and translated into viral polyproteins, or are used as genomes for newly packaged virions. Following packaging, virions
are released at the cell surface by budding. After release, proteolytic processing of
the virion polyproteins occurs and the mature virion can now infect new target cells.
The fact that the virus does not kill the host cell is consistent with lifelong infection
and in many cases development of malignancies. Details specific to JSRV will be
discussed below.
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maturation
Receptor
binding
b
budding
Endocytosis
Env localization
to membrane
Virion assembly
g
Uncoating
Viral proteins
Genomic RNA
Reverse transcription
Transcription
Integration
Fig. 30.3 The retroviral lifecycle. The retroviral life cycle is shown as described in the text
Oncogenic Retroviruses
Oncogenic retroviruses have been known for over 100 years. The first retroviral
cancer was described in 1908 for avian myeloblastosis (Ellerman and Bang 1908;
Rous 1911). Many other oncogenic retroviruses were later described such as Rous
sarcoma virus (Rous 1911), murine mammary tumor virus (MMTV) (Bittner 1942),
and several strains of murine leukemia virus (MuLV) (Gross 1951). The oncogenic
retroviruses are largely animal viruses with human T cell lymphotropic virus
(HTLV) being the exception. Retroviruses can be subdivided into two classes of
retroviruses based on how fast they induce disease and the mechanism of transformation: acute transforming and non-acute retroviruses.
Acute-transforming retroviruses induce disease rapidly and resulting malignancies are typically polyclonal. Additionally, they can often transform cells in culture.
The oncogenic properties of acute transforming retroviruses are due to the presence
of virally encoded oncogenes that were acquired from cellular genes (proto-oncogenes)
by a process termed oncogene capture. Cellular proto-oncogenes encode proteins
whose normal functions are to stimulate controlled cell growth or division. Oncogene
capture typically renders the virus replication defective, so acutely transforming
retroviruses usually replicate as a mixture with a related replication-competent
helper virus. Retroviral oncogenes are typically altered from their cognate protooncogenes, expressing mutant proteins that can constitutively activate cellular growth
pathways.
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Non-acute retroviruses have a long latency to tumor formation (months to years),

do not transform cells in culture, and are replication competent. Non-acute retroviruses
do not encode oncogenes or any other genes to which the malignant potential can be
directly attributed. Rather tumor induction is due to insertional activation, where
integration of the provirus into host DNA results in activated expression of an
adjacent proto-oncogene (Fan et al. 1997). Proviral integration occurs at multiple
sites (almost randomly) throughout the genome. However, when proviral integration
occurs near a cellular oncogene, the viral promoter and/or enhancer elements can
initiate over-expression of the proto-oncogene, ultimately leading to development
of a tumor. The low probability of integration next to a proto-oncogene explains
why tumors induced by non-acute retroviruses require relatively long times (multiple
rounds of infection) to develop. In fact, non-acute retroviruses can be used to identify
genes that can function as oncogenes (Li et al. 1999; Hansen et al. 2000; Mikkers
et al. 2002). Although oncogene capture and insertional activation of proto-oncogenes
are the two primary mechanisms that retroviruses use to induce tumors, some viral
proteins can be directly transforming. For example, HTLV-1 and HTLV-2 express a
viral protein Tax that can transform T cells (Nerenberg et al. 1987; Grassmann et al.
1992; Akagi and Shimotohno 1993; Grossman et al. 1995).
Biology of OPA and JSRV

OPA
Clinical Signs of OPA
Animals that develop OPA show progressive signs of respiratory illness. The malignancy and fluid accumulation produced by the tumor cells make breathing difficult.
The normal function of type II pneumocytes and Clara cells is to secrete surfactant
and other fluids, and over-production of surfactant by the tumor cells leads to alveolar
obstruction. Production of large amounts of lung fluid is considered a signature of
OPA. Lung fluid can be collected by performing the wheel barrow test where the
hind-quarters are held up, causing the lung fluid to drain through the nose. Four
hundred milliliters per day have been collected by this method, although secretion of
1040 ml per day is more common and in some cases no fluid secretion is observed
(Cousens et al. 2009). Although OPA develops in sheep and goats, goats are less
susceptible to OPA (Sharma et al. 1975a, b; Sharp et al. 1986; Tustin et al. 1988).
Gross Pathology
OPA is classified as classical, atypical, or a mixture of the two types (Garcia-Goti
et al. 2000), although no molecular differences in JSRVs have been attributed to the
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different OPA classifications. Classical OPA presents with frothy fluid that fills the
trachea and exudes from the nares (Dungal 1938; Tustin 1969; Wandera 1971;
Beytut et al. 2009). OPA can occur in all parts of the lung, but most frequently in the
distal regions. Natural cases typically have one large tumor mass and experimental
cases show numerous small nodules that are widely disseminated throughout the
lung. When the tumor mass(es) are large, the normal architecture of the lung is
distorted. Multiple lobes of the lung may be affected with a defined boundary
between normal and neoplastic areas. Soft pockets of necrosis and abscessation are
frequently observed. Bronchial and mediastinal lymph nodes are often enlarged and
edematous and up to 10% contain metastase (Demartini et al. 1988; Rosadio et al.
1988b). Metastasis to liver, kidney, heart, skeletal muscle, or other extrathoracic
tissues has been observed, although rarely (Mackay and Nisbet 1966; Nobel et al.
1969; Hunter and Munro 1983).
Atypical OPA has only been reported in natural cases from Spain and Peru
(Garcia-Goti et al. 2000; De Las Heras et al. 2003). Fluid is absent in the bronchial
airway and the tumors are hard, pearly-white, and have a dry cut surface that can
resemble scars (Garcia-Goti et al. 2000; De Las Heras et al. 2003). Atypical neoplasias
are nodular in both early and advanced stages in contrast to classical tumors, which
are diffuse. Atypical OPA is typically located in the diaphragmatic lobe.
Histological Descriptions
Lung alveoli are comprised of two predominant epithelial cell types, type I and type
II pneumocytes. Type II pneumocytes are surfactant secreting, cuboidal cells, and
upon injury can act as progenitors for type I pneumocytes. Type I pneumocytes are
squamous and are more abundant than type II pneumocytes, comprising the majority
of the alveolar epithelia. Clara cells (found further up the airways in the bronchioles)
and type II pneumocytes secrete surfactant, a mixture of glycerophospholipids,
cholesterol, and proteins. Surfactant coats the surface of the alveoli where it has
multiple functions: decreases the surface tension at the airliquid interface to allow
proper airgas exchange, prevents alveolar collapse, and provides host defenses and
pulmonary immune function (Schmitz and Muller 1991; Sakai et al. 1992; Rooney
et al. 1994; Beytut et al. 2009). There are four surfactant proteins (A, B, C, and D)
that have different functional and structural properties. SP-C is exclusively expressed
in type II pneumocytes, whereas both type II pneumocytes and Clara cells can
express SP-A and SP-B. Generally, surfactant protein expression is increased in
OPA cells compared to normal lung cells, with SP-A levels being especially high.
OPA tumors are well differentiated and consist of acinar or papillary forms, or a
combination of the two. Acinar growth is most frequently observed in the lung
parenchyma and consists of cuboidal cells, whereas papillary growth consists of
columnar cells affecting intra-bronchiolar areas (Sharp et al. 1983; Rosadio et al.
1988b; Tustin et al. 1988; DeMartini and York 1997; Platt et al. 2002; Caporale
et al. 2006; Beytut et al. 2009). OPAs are typically papillary in appearance and
occasionally give the impression of solid growths. The neoplastic nodules compress
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the neighboring alveoli causing atelectasis (lack of gas exchange due to collapse)
and/or merge to form a larger mass.
OPA consists of malignantly transformed type II pneumocyte (82%), Clara (7%),
and undifferentiated cells (11%) (Platt et al. 2002; Wootton et al. 2006b). JSRV infection
has been detected in lung epithelial cells, CD4+ and CD8+ T cells, B cells, and cells
of monocyte/macrophage lineage (Holland et al. 1999). Transformed stem cells have
been reported to be the origin of many malignancies including breast, brain, colon,
ovary, pancreas, prostate, and possibly the lung (Al-Hajj et al. 2003; Singh et al. 2003;
Li et al. 2007; OBrien et al. 2007; Maitland and Collins 2008; Zhang et al. 2008).
A population of lung stem cells believed to be the origins of adenocarcinoma was
recently identified, termed bronchioloalveolar stem cells (BASCs) (Kim et al. 2005).
Despite the possibility that they are the targets for JSRV transformation in OPA, in
transgenic mice where the JSRV LTR is driving expression of a reporter transgene,
expression was not detected in BASCs (Dakessian and Fan 2008).
OPA tumor cells in early stages of malignancy have a low mitotic index and at
later stages, solid masses of pleomorphic cells with a high mitotic rate and scattered
foci of necrosis are often observed. Although tumor cells generally have a low
mitotic index, they express proliferating cellular nuclear antigen (PCNA), a marker
for proliferation. JSRV capsid protein is detected in the cytoplasm of tumor cells
with apical concentration in lung and lymph nodes (Palmarini et al. 1995; Palmarini
et al. 1999; Garcia-Goti et al. 2000; DeMartini et al. 2001; Palmarini and Fan 2001;
Palmarini and Fan 2003; Salvatori et al. 2004; Caporale et al. 2006). The fact that
JSRV capsid protein is detected in OPA cells indicates that JSRV continues to replicate
in these cells. Interestingly, capsid expression is not detected in all cells in OPA
tumors, nor in normal cells adjacent to tumor areas. Additionally, not all nodules in
multifocal OPA express capsid (Sharp et al. 1983; Palmarini et al. 1995; Platt et al.
2002). In contrast, JSRV SU has been detected in the cytoplasm of OPA cells and
proliferating cells located in the bronchioles whereas expression was not detected in
unaffected sheep (Payne and Verwoerd 1984; Salvatori et al. 2004).
Atypcial OPA is commonly acinar with stroma that is more heavily infiltrated by
inflammatory cells and connective fibers than classical OPA (De Las Heras et al.
1992; Garcia-Goti et al. 2000). Metastases in regional lymph nodes seem to be less
frequent and less JSRV-positive cells are observed (Garcia-Goti et al. 2000). Early
atypical lesions have numerous neoplastic polyps located in the terminal bronchioles
and alveoli that develop into more elaborated intrabronchiolar polyps (Wandera
1970; Sharp et al. 1983).
Transmission Studies Suggest That the Causative Agent

of OPA Is a Retrovirus
The first suggestion that a retrovirus could be the etiological agent of OPA was the
observation of particles resembling retroviruses in the lungs of sheep with clinical
OPA (Malmquist et al. 1972; Payne et al. 1983; Perk et al. 1974). Biochemical analysis
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confirmed reverse transcriptase activity, retroviral RNA and retroviral antigens in

tumor extracts (Perk et al. 1974; Verwoerd and de Villiers 1980; Sharp and Herring
1983). OPA was shown to be a transmissible malignancy by intratracheal inoculation
of sheep with particles containing reverse transcriptase activity isolated from OPA
(Martin et al. 1976), cytoplasmic fractions of tumor cells (Verwoerd and de Villiers
1980; Verwoerd et al. 1980) or pulmonary secretions (Sharp et al. 1983).
Molecular Biology of JSRV

Deduction of JSRV Sequence and Identification
of Endogenous JSRV
Characterization of JSRV was a major focus of research during the early 1980s.
JSRV could not be grown in culture, likely due to the fact that the LTR has highest
activity in differentiated distal airway epithelial cells, which do not maintain the
differentiated state in culture (Dobbs et al. 1985; Manzer et al. 2006; Wang et al.
2006). Therefore lung lavages of diseased sheep were used to purify the virus
(Verwoerd et al. 1983; York 1987; York and Querat 2003). A major breakthrough
was deduction of the sequence for a novel retrovirus (JSRV) in OPA lung fluids
(York et al. 1991, 1992). This was accomplished by partially purifying virus from
lung fluids, extracting RNA and using it as a template in in vitro reverse transcriptase
reactions. The resulting cDNAs were cloned, and sequence analysis of overlapping
clones allowed deduction of the JSRV sequence. The sequence analysis indicated
that JSRV is a member of the betaretrovirus family (see below), and it contains an
additional open reading frame termed orf-x, which is not present in other retroviruses. The assembled cDNA sequence was not infectious in vitro or in vivo likely
for technical reasons. Hybridization experiments with the isolated DNA led to the
discovery that in addition to the exogenous infectious JSRV in OPA, there are related
endogenous retrovirus (enJSRVs) present in all sheep genomes (York et al. 1992;
Hecht et al. 1994, 1996) (see below). Subsequently improved screening and cloning
approaches allowed isolation of a molecular clone of a full-length integrated JSRV
provirus from an OPA tumor from the UK, lJSRV21 (Palmarini et al. 1999). The JSRV
sequences in lJSRV21 closely resembled the deduced JSRV sequence (Fig. 30.4a)
(York et al. 1992; Palmarini et al. 1999).
JSRV Is a Betaretrovirus
Early clues that JSRV is a betaretrovirus came from the fact that extracts from lung
tumors and secretions from OPA-affected animals cross-reacted with antisera
against capsid for betaretroviruses mouse mammary tumor virus (MMTV) and
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Fig. 30.4 The JSRV genome. (a) Schematic representation of the genomic organization of the
JSRV21 genome. Typical of betaretroviruses, pro is in a different open reading frame from pol.
Note the presence of an accessory open reading frame (orfx) overlapping pol. (b) pJSRV21 and
pCMV2JS21 plasmid constructs. In pCMV2JS21, the U3 region of the proximal LTR was replaced
by the human CMV promoter. (c) Western blot of 300-fold-concentrated supernatant from 293T
cells transiently transfected with pCMV2JS21 and collected 24, 48, and 72 h post-transfection.
The filters were probed with a rabbit polyclonal antiserum against the major capsid protein (CA)
of JSRV (28). Lung secretions collected from an SPA-affected animal and concentrated in the same
way as the 293T supernatant were used as a positive control (LF). Concentrated supernatant from
mock-transfected 293T cells was used as a negative control (M). The 26-kDa CA protein is indicated. This figure is taken from Palmarini et al. (1999)
Mason-Pfizer monkey virus (MPMV) (Sharp and Herring 1983). Following cloning
of JSRV, sequence analysis revealed genetic similarity of the RT protein to other
betaretroviruses confirming that JSRV is a betaretrovirus. Based on Env and LTR
sequence homology, two genotypes of JSRV have been isolated: type I from South
Africa and France and type II from Scotland and Wyoming (Bai et al. 1996). It is not
known if the sequence differences between the two subtypes result in functional
differences. To date, three stains of JSRV have been isolated and sequenced. JSRV-SA
is from South Africa (York et al. 1991, 1992) and JSRV21 (Palmarini et al. 1999)
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and JSRVJS7 (DeMartini et al. 2001) are from the UK. Other members of the
betaretrovirus genus include mouse mammary tumor virus (MMTV), MasonPfizer
monkey virus (MPMV) and squirrel monkey retrovirus (SMRV). JSRV is closely
related to enzootic nasal tumor virus (ENTV), the etiological agent of a contagious
nasal adenocarcinoma of the mucosal glands affecting sheep and goats, which will
be discussed later.
Assembly of betaretrovirus viral cores occur in the cytoplasm in contrast to other
retroviruses where core assembly occurs during viral budding at the plasma membrane
(Coffin 1992). Distinct JSRV core structures have been described for intracytoplasmic,
budding, and extracellular virions. Intracytoplasmic virions, located below the apical
membrane, are described as type-A particles that are doughnut or ring shaped with
a small electron-lucent core, electron dense inner core, and double external membrane
that range in size from 60 to 100 nm (Hod et al. 1977; Payne et al. 1983). Virions
budding from the microvilli or into the intracytoplasmic vacuoles are complete
round particles (not crescent shaped) containing a core that is more electron-dense
than intracytoplasmic particles. JSRV particles observed outside the cell (type B/D)
are round or pleomorphic, ranging in size from 90 to 120 nm in diameter (Verwoerd
et al. 1980; Sharp et al. 1983).
JSRV Molecular Clones Are Infectious and Tumorigenic

Transfection of a cloned full-length JSRV provirus into various cell lines did not result
in production of virus, likely because the LTR has transcription specificity for differentiated airway cells (Palmarini et al. 2000a). To overcome this, the U3 region in the
upstream LTR was replaced with the CMV immediate early enhancer/promoter to
give pCMVJS21 (Fig. 30.4b). This plasmid would express native JSRV RNA from the
CMV promoter that is highly active in many cells. Viral particles were efficiently
produced in 293T cells transfected with pCMVJS21 (Fig. 30.4c). The resulting viral
particles were concentrated and used to intratracheally inoculate newborn lambs. Two
of four inoculated lambs developed OPA (Fig. 30.5), which was the first formal proof
that JSRV was necessary and sufficient to induce OPA (Palmarini et al. 1999).
Transcription Specificity of the JSRV LTR

Retroviral LTRs contain the viral promoter and enhancers that bind cellular transcription factors; therefore they dictate the cells in which the virus can be expressed.
The cell-type specificity of the JSRV LTR was determined using LTR-luciferase
reporter assays. The JSRV LTR had the highest activity in murine MLE-15 and
mtCC1-2 cells (Palmarini et al. 2000a). These cells were derived from lung tumors
of transgenic mice and they have retained differentiation characteristics of type II
pneumocytes and Clara cells, respectively (Malkinson et al. 1997). JSRV LTR activity
was low in human lung epithelial lines (A549, H358 and H441) and the sheep BAC
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Fig. 30.5 Induction of SPA in JSRV21infected lambs. (ac) Lung tumor tissues from lambs inoculated with JSRV21 produced from transfected 293T cells. Hematoxylin and eosin-stained lung
tumor sections are shown. (a) Low-magnification micrograph (bar, 380 mm) showing many neoplastic foci in the microscopic field (some are indicated by arrows). (b) High-magnification
micrograph (bar, 40 mm) of a neoplastic nodule with a clear papillary pattern (asterisk). Myxoid
tissue-containing cells with elongated or round nuclei are present in the interstitium of the neoplastic
tissue. (c) Papillary proliferation (asterisk) occluding the lumen of a bronchiolus (bar, 40 mm).
(de) Immunohistochemistry for JSRV CA antigen (brownish stain). (d) A neoplastic focus (asterisk)
is shown. No staining is present in the cells infiltrating the tumor or in adjacent normal cells.
(e) Rabbit preimmune serum staining; no staining in the tumor nodule (asterisk). (f) A lung section
from an uninoculated lamb stained for JSRV CA antigen. This figure was taken from Palmarini
et al. (1999)
cell line (JS7). The latter cell lines have not retained epithelial differentiation, indicating that maintenance of differentiation of lung cells may be required for JSRV
LTR activity.
JSRV LTR sequences important for activity in MLE-15 have been determined by
mutational analysis and in vivo footprinting of an M-MLV driven by an LTR
containing JSRV enhancers (McGee-Estrada and Fan 2006). A map of putative and
confirmed transcription factor binding sites is shown in Fig. 30.6. The NF-1, HNF3b, and C/EBP binding sites in the JSRV LTR are critical for activity in MLE-15
cells although not for the low level activity in other cell lines examined (McGee-Estrada
et al. 2002; McGee-Estrada and Fan 2006).
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Fig. 30.6 In vivo footprinting of the JSRV enhancer. A composite of in vivo DMS footprinting for
both strands of the JSRV enhancers in the LTR of DMo+JS Mo-MuLV-infected MLE-15 cells is
shown. Factor-binding sites where strong footprints were observed are shown in bold and italics.
Protected bases are indicated by arrows pointing away from the sequence, and hypermethylated
bases are indicated by arrows pointing toward the sequence. Filled and open arrowheads indicate
strong protection and hypersensitivity, respectively. Canonical binding sites that have been
analyzed by EMSA are shown in bold. A shaded rectangle indicates boundaries for the putative
Gfi-I binding site. This figure was taken from McGee-Estrada and Fan (2006)
LTR Binding Sites

HNF3s (Hepatocyte Nuclear Factor 3, aka FOXA2) are a family of nuclear proteins
expressed in the liver and lungs and are critical in development of these tissues
(Overdier et al. 1994; Costa et al. 2001). HNF-3a and -b are highly expressed in
both type II pneumocytes and Clara cells (Clevidence et al. 1994; Kaestner et al.
1994) where they are important for expression of SP-B, CC-10, and other lungspecific genes (Sawaya et al. 1993; Bohinski et al. 1994; Clevidence et al. 1994;
Bingle et al. 1995; Bruno et al. 1995; Margana and Boggaram 1997). Two putative
HNF-3 binding sites have been identified in the JSRV LTR. The upstream HNF-3
binding site, which binds HNF-3b, is critical for LTR activity in MLE-15 cells and the
downstream site is not important. Results were different in NIH-3T3 and mtCC1-2
cells where the HNF-3 sites were not required. These results indicate that there are
cell-type specificities for LTR activity and sites other than HNF-3 can mediate
expression of the JSRV LTR. However, binding sites identified in cell lines such as
NIH-3T3 where the LTR only shows low basal activity may not be as important
as those identified in differentiated type II pneumocytes (e.g. MLE-15).
767
C/EBP (CCAAT/Enhancer binding protein) has three isoforms (a, b and d) that
are expressed in many tissues (Birkenmeier et al. 1989; Sugahara et al. 1999).
Isoforms a and d are highly expressed in the bronchiolar epithelium, while b is
mostly found in alveolar macrophages (Sugahara et al. 1999). C/EBPa and b bind
to a C/EBP binding site in the JSRV LTR, which is critical for activity (McGee-Estrada
and Fan 2006). NF-1 (nuclear factor protein-1) controls expression of many target
genes, including the surfactant protein C promoter (Bachurski et al. 1997). There
are at least four mammalian NF-1 genes (A, B, C and X), with multiple isoforms as
a result of splicing (Rupp et al. 1990; Chaudhry et al. 1997). NF-1A has been shown
to transactivate the SP-C promoter in HeLA cells (Bachurski et al. 1997). The NF-1
binding site in the JSRV LTR is important for LTR activity, although not as critical
as other sites such as those for C/EBP and HNF-3b. The core binding sequences of
the two HNF-3b and C/EBPab binding sites are conserved in type I and type II
JSRV LTRs (McGee-Estrada and Fan 2007), illustrating the importance of these
sites for JSRV LTR activity.
LTR Elements Are Determinants of Disease Outcome

Env and the LTR are the main determinants of disease for many retroviruses. Env
determines cell specificity at the level of viral entry and the LTR dictates where the
virus can be expressed transcriptionally. For JSRV, several findings indicate that
the LTR plays a more important role in disease specificity than Env. First, the JSRV
receptor (hyaluronidase 2; Hyal2) is expressed in many cell types throughout the
body and viral DNA is found in many different cell types including PBMCs, macrophages, B lymphocytes, CD4+, and CD8+T lymphocytes in lymph nodes (Holland
et al. 1999; Miller 2003). Despite the presence of viral DNA in the different cell
types, expression is highly restricted to OPA tumor cells in vivo and differentiated
type II pneumocytes and Clara cells in vitro (Palmarini et al. 1995). Indeed lung
epithelial cell lines that have not maintained the differentiation state do not support
activity of the LTR (Palmarini et al. 2000a). Second, when mice are infected intranasally with adeno-associated virus expressing JSRV Env in the absence of the
LTRs, lung adenocarcinomas develop even though the vector transduced multiple
cells and tissues in the airways (Wootton et al. 2005). Third, JSRV, ENTV, and
enJSRV are closely related betaretroviruses with high sequence similarity, with the
most differences in the LTR (McGee-Estrada and Fan 2007). All three JSRV isolates
contain two conserved HNF-3 binding sites in U3, while the ENTV and enJSRV U3
regions do not. The C/EBP binding site of JSRV is interrupted in enJSRV but conserved
in ENTV LTRs. Although several other binding sites are conserved between the
JSRV and ENTV LTRs, the ENTV-1 LTR is less active than the JSRV LTR in
MLE-15 cells. The transcriptional specificities of the enJSRV and exogenous JSRV
LTRs differ because enJSRVs respond to progesterone and they do not respond to
lung-specific factors (Palmarini et al. 2000b). Indeed enJSRV is highly expressed in
the reproductive tract of ewes and at low levels in lungs.
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Rej
Retroviral RNA is transcribed as genome-length RNA. Nuclear viral RNA can be
spliced in the nucleus to form mRNA for envelope protein. Spliced viral mRNA
is exported to the cytoplasm for translation. Alternatively, unspliced viral RNA is
transported to the cytoplasm where it is packaged into virions or translated as the
mRNA for Gag and Gag-pol polyproteins. For cellular mRNAs, splicing is typically
a prerequisite for RNA export to the cytoplasm. Retroviruses have evolved two
mechanisms to export unspliced RNA: virally encoded trans-acting proteins or cisacting RNA elements (Cullen 2003). For example, human immunodeficiency
viruses (HIVs) encode Rev that facilitates export of unspliced RNA to the cytoplasm
(Cullen 2003). Rev binds to a Rev response element (RRE) on the viral RNA, and
it facilitates nuclear export via the cellular protein Crm1 (Cullen 2003). Simple
retroviruses do not encode accessory proteins; rather they export RNA via cis-acting
constitutive transport elements (CTEs), secondary stem-loop structures that directly
bind cellular RNA export proteins (e.g. TAP/NXF-1 for MPMV).
Recently, the mechanism for export and translation of unspliced RNA for JSRV
has been described. A trans-acting factor Rej or JSE-SP is encoded in the 5 end of
env (Caporale et al. 2009; Hofacre et al. 2009). The protein is generated by translation of a multiply spliced mRNA and/or by cleavage of the signal peptide from the
full-length Env precursor polyprotein (Hofacre et al. 2009). JSRV Rej is analogous
to regulatory proteins of other betaretroviruses, mouse mammary tumor virus
(MMTV), and human endogenous retrovirus K (HERV-K) (Rem and Rec; respectively) (Lower et al. 1995; Indik et al. 2005; Mertz et al. 2005). In human 293T cells,
Rej is required for Gag expression and it appears to act by facilitating export of
unspliced RNA, mRNA translation and post-translational modifications (Caporale
et al. 2009; Hofacre et al. 2009). However, in other cells, Rej is not required for
export of unspliced viral RNA but it is still essential for expression of Gag protein; it
apparently facilitates translation of the cytoplasmic unspliced viral RNA (Hofacre
et al. 2009) an unusual mechanism for the Rev-like proteins. A cis-acting Rej
response element (RejRE) is contained in the 3 of Env, and this is embedded in a
larger JSRV RNA expression and export element (JREE) that also contains a CTE for
export of unspliced viral RNA in cell lines other than 293T (Nitta et al. 2009). Export
of unspliced viral RNA is CRM1 and B23-dependent but not TAP-dependent
(Caporale et al. 2009; Nitta et al. 2009). A core stem-bulge-stem structure in the
RejRE is required for activity and secondary structure of the RejRE core is important
(Nitta et al. 2009). While Rej plays a role in viral RNA export only in 293T cells, Rej
binds to the RejRE in all cell lines tested (Nitta et al. 2009). Thus Rej binding to the
RejRE may also be important for translation of unspliced viral RNA.
Orf-X
The JSRV genome contains an additional open reading frame termed orf-x. Orf-x
overlaps with the 3 end of the pol gene and the codon usage differs from the rest of
769
the virus (York et al. 1992). Based on sequence analysis, orf-X shares sequence
similarity to the adenosine 3A receptor and is predicted to encode a very hydrophobic
accessory protein of 166 amino acids containing four putative transmembrane
domains (Bai et al. 1999). ORFs Homologous to orf-x are not found in other betaretroviruses and it has been debated if it truly represents an accessory gene for JSRV
(York et al. 1992; Bai et al. 1999; Rosati et al. 2000). Orf-x is not required for Envmediated transformation in vitro (Maeda et al. 2001) or in vivo (Wootton et al. 2005;
Caporale et al. 2006; Cousens et al. 2007). Evidence that orf-x plays a role in the
JSRV lifecycle is based on the fact that the sequence is strongly conserved among
different JSRV isolates and endogenous JSRV, suggesting a selective pressure to
continue carrying it (York et al. 1992; Bai et al. 1996, 1999; Rosati et al. 2000; York
and Querat 2003). Additionally, orf-x mRNA has been detected in JSRV-infected
cells and lung tumors (Palmarini et al. 2002). However, a mutant of JSRV in which
the orf-X reading frame was mutated by insertion of two stop codons induced OPA
with the same efficiency as wild-type virus (Cousens et al. 2007). Thus while OrfX
might be important for some aspect of JSRV infection in vivo, it is not required for
oncogenesis by JSRV.
The JSRV Receptor: Hyal2

The cellular receptor for JSRV was mapped and cloned from human cells by a combination of somatic cell hybrid, molecular cloning, and gene transfer experiments (Rai
et al. 2000, 2001). The JSRV receptor was found to be hyaluronidase 2 (Hyal2)
(Rai et al. 2001). Initial studies reported that Hyal2 is a lysosomal hyaluronidase
(Lepperdinger et al. 1998), but it was later found that it is a glycosylphosphatidylinositol
(GPI)-anchored cell surface receptor with very little hyaluronidase activity (Rai et al.
2001; Liu et al. 2003a; Duh et al. 2005; Van Hoeven and Miller 2005; Miller et al.
2006). Hyal2 is located on human chromosome 3p21.3, and it is interesting that this
chromosomal region may encode a tumor suppressor gene, because it is in a region of
loss of heterozygosity (LOH) in human lung cancer (Zabarovsky et al. 2002). Hyal2 is
present on the surface of many cell types, which explains how JSRV DNA can be
detected not only in lung cells but also lymphocytes, macrophage, and other cell types
of sheep with OPA (Holland et al. 1999; Miller 2008; Rai et al. 2001).
Mouse cells cannot be infected by retroviral vectors pseudotyped with JSRV Env
because murine Hyal2 does not bind JSRV Env (Rai et al. 2001). Likewise rat cells are
only slightly permissive for infection (Liu et al. 2003a). The relevance of these results
to the potential role of Hyal2 in JSRV-induced malignancies will be discussed later.
Mechanisms of JSRV Oncogenesis

The mechanism by which JSRV induces tumors has been of considerable interest.
Considering the existing paradigms for oncogenic retroviruses, it was important to
determine if JSRV is an acute transforming retrovirus or a non-acute retrovirus.
770
The rapid time course to disease and multifocal neoplasia were consistent with JSRV
being an acutely transforming retrovirus, i.e., that it carries an oncogene. However,
the JSRV genome does not contain any open reading frames with high homology to
cellular proto-oncogenes the hallmark of acute transforming retroviruses. On the
other hand, the viral orf-x reading frame of JSRV and related viruses does not have
an obvious role in viral replication, so it might potentially be an oncogene although
it is contained in non-oncogenic enJSRVs as well (York et al. 1992; Bai et al. 1999;
Rosati et al. 2000). However, a JSRV mutant in which orf-x was mutated shows the
same rapid disease induction as wild-type virus (Cousens et al. 2007).
At the same time, there is no concrete evidence that JSRV uses insertional activation in induction of malignancy. In an OPA-derived cell line containing only one
copy of JSRV DNA, the provirus is integrated into the structural gene for pulmonary
surfactant protein A (SPA), as opposed to a proto-oncogene (DeMartini et al. 2001).
Another study found only two out of 70 OPA tumors with insertions in the same
locus (chromosome 16) (Cousens et al. 2004). Identification of more OPA tumors
with common insertion sites will be needed before a role for insertional activation
in JSRV oncogenesis could be established.
The JSRV Genome Can Transform Cells

Given the rapid rate of JSRV oncogenesis, we tested if the JSRV genome might
contain an oncogene using a functional assay. NIH-3T3 cells are contact-inhibited,
but they are highly susceptible to morphologic transformation and they have been
used to detect both viral oncogenes and activated cellular proto-oncogenes (Shih
and Weinberg 1982). Transfection of pCMVJS21 DNA into NIH-3T3 cells resulted
in formation of transformed foci; cells in the foci could grow in soft agar while
control NIH-3T3 cells cannot (Maeda et al. 2001). Thus, the JSRV genome contains
a gene that is capable of transformation, i.e. an oncogene. This observation has been
confirmed by other laboratories (Rai et al. 2001).
Env Is the Oncogene

Further experiments indicated that expression of JSRV Env was sufficient to transform NIH-3T3 cells and in some studies env alone was more efficient at transformation than the entire JSRV molecular clone (Maeda et al. 2001; Rai et al. 2001).
This was the first report that a native retroviral envelope protein could transform
cells. JSRV Env has been shown to transform various cell lines including murine
NIH-3T3 fibroblasts (Maeda et al. 2001), rat 208F fibroblasts (Rai et al. 2001;
Maeda et al. 2005), avian DEF and DF-1 fibroblasts (Allen et al. 2002; Zavala et al.
2003), human bronchial BEAS-2B epithelial cells (Danilkovitch-Miagkova et al.
2003), MDCK canine kidney epithelial cells (Liu and Miller 2005), and RK3E rat
kidney epithelial cells (Maeda et al. 2005).
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JSRV Env not only can transform cell lines in culture, but it can also induce lung
(and other) tumors in animals. This has been shown in transgenic mice expressing
env transgenes (Dakessian et al. 2007; Chitra et al. 2009), and in mice infected with
an adeno-associated viral vector (Wootton et al. 2005). A JSRV-based retroviral vector
expressing Env also induces lung tumors in sheep (Caporale et al. 2006). Together,
these results indicate that Env is an oncogene and it alone is sufficient to induce OPA.
Additional viral genes and/or viral spread are not required for oncogenesis.
Domains of Env Required for Transformation

The domains of JSRV Env involved in transformation have been studied extensively
(Palmarini et al. 2001b; Chow et al. 2003; Hofacre and Fan 2004; Hull and Fan
2006). Initial attention was focused on the cytoplasmic tail (Lander et al. 2001) of
the TM protein, since sequence comparisons between exogenous (oncogenic) JSRV
and endogenous (non-oncogenic) enJSRV Env proteins indicated relative conservation
between the proteins except for the TM CT (Palmarini et al. 2001b). Chimeras
exchanging the CTs between these two Env proteins indicated that the CT of exogenous JSRV is essential for transformation (Palmarini et al. 2001b). There is a
tyrosine residue in the JSRV CT at position 590 while the CT of enJSRV lacks
tyrosines, suggesting that this residue is important for Env transformation. This was
confirmed when mutation of the tyrosine to phenylalanine (Y590F) or aspartic acid
(Y590D) abolished transformation in NIH-3T3 cells (Palmarini et al. 2001b).
Additionally, when JSRV virions containing the Y590D mutation were inoculated
into sheep, the virus could not establish infection or induce disease (Cousens et al.
2007). Thus Y590 in the CT is important for JSRV Env transformation. The amino
acid sequence surrounding Y590 is YRNM, and if Y590 is phosphorylated it could
potentially bind cellular proteins with SH2 domains. YXXM and YXN are putative
binding motifs for the SH2 domains of the PI3K regulatory subunit (p85) and
growth factor receptor binding protein-2 (Grb-2), respectively (Songyang et al.
1993). Mutation of the YXXM motif (e.g. M593T) abolished or decreased transformation in cellular transformation assays (Palmarini et al. 2001b; Allen et al. 2002;
Liu et al. 2003a, b). On the other hand, mutation of the YXN (e.g. N592T) did not
abolish transformation, and in fact this increased transformation potential (Palmarini
et al. 2001b). Taken together these results suggested that the YXXM motif in the
JSRV CT might bind PI3K, leading to downstream signaling. Indeed, JSRVtransformed cells show constitutive phosphorylation (activation) of the downstream
kinase Akt (Palmarini et al. 2001b).
Alanine scanning mutagenesis on the JSRV CT has been conducted (Hull and
Fan 2006). The CT is 44 amino acids in length, and the N-terminal 14 residues form
an amphipathic helix that is likely embedded in the lipid bilayer of the cell membrane. The alanine scanning mutations indicated that the C-terminal 1012 residues
are not essential for transformation. In contrast, mutations in the amphipathic helix
and downstream amino acids generally affected transformation. For some residues,
772
alanine substitution resulted in complete inhibition of transformation, for others

there was partial inhibition, and for four residues there was enhancement. These
mutations could affect either the global structure of the CT, or binding of cellular
proteins involved in transformation to the CT. They will be useful in future studies
on the mechanism of JSRV transformation.
While the TM CT is essential for JSRV transformation, other regions of Env
protein are also important. Deletions within SU (from the signal peptide to the junction between SU and TM) also abolished transformation (Hofacre and Fan 2004),
indicating that SU is also important for Env transformation. The role of SU in transformation is independent from that of the TM CT, since co-transfection with two
transformation-defective JSRV env mutants with mutations in SU and in the TM CT
results in transformation (Hofacre and Fan 2004). The mechanism by which SU acts
in JSRV transformation has not been determined.
The potential role of the extracellular portion of the TM protein (the ectodomain)
has also been investigated. The ectodomains of endogenous and exogenous JSRV
TMs are highly conserved, with only four amino acid differences (Palmarini et al.
2000b). Sequential mutation of the exogenous JSRV TM ectodomain amino acids
to those of enJSRV TM partially reduced transformation efficiency (S. Hull and H.
Fan, unpublished). Thus the ectodomain of TM may also contribute to JSRV transformation, although in a limited fashion.
Signaling in JSRV Transformation

PI3K-Akt-mTor Pathway
Class I phosphatidylinositol-3-kinases (PI3K) are cellular lipid kinases consisting of a
regulatory and catalytic subunit. They are further subdivided into class IA and class IB.
Activation of cellular receptor tyrosine kinases and/or Ras proteins mediate recruitment to the plasma membrane and activation of class IA PI3Ks, whereas class IB
PI3Ks are activated by G protein-coupled receptors (Stephens et al. 1994, 1997). At the
plasma membrane activated PI3K phosphorylates phosphatidylinositol 4,5 bisphosphate (PIP2) forming the secondary messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn recruits proteins with pleckstrin homology (PH) domains
such as the serine-threonine kinases Akt and 3-phosphoinositide-dependent kinase 1
(PDK) by binding pleckstrin homology (PH) domains in those proteins (Corvera and
Czech 1998). Once at the plasma membrane, PDK1 activates Akt by phosphorylation
at threonine 308 (Alessi et al. 1997). Activated Akt can phosphorylate numerous downstream targets that regulate many cellular pathways, including those leading to oncogenic transformation. mTor is one of the major downstream targets of Akt and is a
master regulator of cell growth, size, and protein synthesis (Lawlor and Alessi 2001;
Abraham 2002; McManus and Alessi 2002); dysregulation of the PI3K-Akt-mTor
pathway has been described for many cancers (Bellacosa et al. 2004).
773
As discussed above, the requirement of the YXXM motif for transformation

suggested that JSRV Env protein directly binds the PI3K regulatory subunit (p85)
to activate signaling through the PI3K-Akt-mTOR pathway. Indeed, Akt is activated
in many different Env-transformed cell lines including rodent fibroblasts (NIH-3T3
and 208F) (Palmarini et al. 2001b; Alberti et al. 2002; Chow et al. 2003; Liu et al.
2003b; Maeda et al. 2003; Hofacre and Fan 2004; Maeda et al. 2005; Hull and Fan
2006), chicken embryonic fibroblasts (CEF and DF-1) (Allen et al. 2002; Zavala
et al. 2003), and kidney epithelial cells (MDCK and RK3E) (Liu and Miller 2005;
Maeda et al. 2005). Akt activation was found to be PI3K dependent because PI3Kspecific inhibitors (LY294002 and/or wortmannin) reverted a transformed phenotype, inhibited transformation, or inhibited kinase activity (Palmarini et al. 2001a, b;
Maeda et al. 2001; Liu et al. 2003b; Maeda et al. 2003; Zavala et al. 2003). A positive
correlation between the levels of Akt activation and degree of Env transformation
has also been reported (Liu et al. 2003b). Thus PI3K appears to play a role in Akt
activation during Env transformation.
At the same time, increasing evidence indicates that Env can also activate Akt
independently of PI3K. First, Env efficiently transforms cells and activates Akt in
cells where the PI3K regulatory subunit (p85) was inactivated by a dominant negative p85 protein or in cells derived from p85a/b double knockout mice (Maeda et al.
2003). However, other regulatory subunits for class IA PI3Ks potentially could be
compensating for the loss of p85. Second, PI3K inhibitors (LY294002 or wortmannin)
did not inhibit Env-mediated transformation or revert the transformed phenotype of
NIH-3T3 cells (Maeda et al. 2003). Third, phosphorylation of Env at Y590 would be
required for p85 binding, and this has not been detected in JSRV-transformed cells.
Moreover, in vivo binding of p85 and JSRV Env has not been observed (Liu et al.
2003b; Liu and Miller 2005) although the lack of detection could have been technical.
Fourth, in some studies cells could be transformed by an Env with a mutant YXXM
motif, and the resulting transformed cells still showed Akt phosphorylation (Liu
et al. 2003b; Zavala et al. 2003; Liu and Miller 2005). Finally, Akt activation is not
always observed in Env transformed cells or OPA tumor cells. One study found that
activated Akt was not detected in Env-transformed (DF-1) cells or lung sections from
OPA (Zavala et al. 2003). In another study, only 37% of late-stage OPA tumors
showed activated Akt (Suau et al. 2006). Thus Akt activation may not always be
required for Env transformation and other pathways may compensate.
The role of the PI3K-Akt-mTOR pathway in JSRV transformation has also been
assessed by performing transformation assays in the presence of the mTOR inhibitor
rapamycin (Maeda et al. 2005). Rapamycin generally inhibits JSRV transformation,
although inhibition is partial. Moreover, depending on the cell line, the effects
differ. For instance, rapamycin has a modest effect on JSRV transformation in
NIH-3T3 fibroblasts (3040% reduction), while it has a stronger effect in RK3E
epithelial cells (~70% reduction). Cell-type specific signaling also has been reported
for other viral oncogenes (Aftab et al. 1997).
In summary, signaling through Akt and mTOR is observed in JSRV-transformed
cells, and its relative importance for transformation varies among cell lines. In
JSRV-transformed cells activation of Akt appears to result from both PI3K-dependent
774
and -independent mechanisms. Despite the importance of the YXXM motif in Env
for transformation, it is probably not directly binding and recruiting PI3K to the
plasma membrane. Other as-yet-unidentified mechanisms are likely responsible for
activation of this signaling pathway.
Ras-Raf-MEK-MAPK Pathway
MAP kinases are serine/threonine protein kinases that respond to extracellular
stimuli, and they regulate many cellular functions including mitosis, differentiation,
proliferation, and cell survival vs. apoptosis (Pearson et al. 2001). There are five
(and likely more) MAPK protein families that are each activated by a cascade of
upstream regulatory kinases. Each cascade includes two activating kinases:
MAPKKK, MAPKK and MAPK. The extracellular signal-related kinase I and 2
(ERK1/2, aka p44/42) are prototypical MAPKs. They connect extracellular signals
at the cell membrane (e.g. activated receptor tyrosine kinases) to activation (phosphorylation) of transcription factors in the nucleus, thus regulating gene expression
(Ballif and Blenis 2001). Dysregulated signaling to ERK1/2 is one of the most
important pathways in cancer. In MAPK signaling, extracellular mitogen stimulation
indirectly leads to activation of intracellular Ras proteins small G-proteins.
One effect of Ras protein activation is binding effector proteins, such as Raf
(a MAPKKK), which assists in activation at the plasma membrane. Activated Raf
phosphorylates (activates) MEK-1/2 (a MAPKK), which in turn phosphorylates
(activates) ERK1/2 (a MAPK). Activated ERK1/2 migrates to the nucleus where it
phosphorylates and activates transcription factors such as ELK-1. Other MAPK
families include c-Jun amino terminal kinase (JNKs 1, 2 and 3), p38 (isoforms a, b,
g and d), ERKs 3/4, and ERK-5.
The Ras-Raf-MEK-MAPK pathway has also been implicated in JSRV Env-mediated
transformation, although again the relative importance may be cell type and/or
species specific. In transformation of NIH-3T3 and RK3E cells, the Ras-Raf-MEKMAPK pathway is of major importance, since treatment with MEK-1 inhibitors
(e.g. PD98059) abolishes transformation (Maeda et al. 2005). In NIH-3T3 cells, the
predominant signaling is initiated through H or N-Ras, since the H/N-Ras inhibitor
FTI-277 also abolishes JSRV transformation (Maeda et al. 2005). FTI-277 can also
revert the transformed phenotype of JSRV-transformed NIH-3T3 cells (Maeda et al.
2005). On the other hand, in RK3E cells, FTI-277 only partially inhibits transformation, indicating that stimulation of Raf and MEK-1 in these cells is occurring through
alternate proteins; K-Ras is at least partially involved (Maeda et al. 2005). Despite
the evidence for Ras-Raf-MEK-ERK signaling being important for JSRV transformation, constitutive phosphorylation of ERK1/2 was not observed in NIH-3T3 and
208F cells (Liu et al. 2003b; Maeda et al. 2005), although it was in RK3E cells
(Maeda et al. 2005).
The p38 MAPK negatively regulates JSRV transformation in NIH-3T3 and RK3E
cells since the p38 inhibitor SB203580 substantially increases JSRV transformation
(Maeda et al. 2005). p38 appears to be inhibiting phosphorylation of MEK1/2 in
775
these cells, since treatment of JSRV-transformed cells with SB203580 results in

enhanced MEK1/2 and ERK-1/2 phosphorylation.
The Ras-Raf-MEK-MAPK pathway has been implicated in vivo because phosphorylated ERK1/2 has been consistently detected by immunohistochemistry in
naturally and experimentally induced OPA tissues (De Las Heras et al. 2000, 2003,
2006; Maeda et al. 2003). On the other hand, phosphorylated p38 was not consistently
detected in OPA tumors (Maeda et al. 2005).
In summary, signaling through the Ras-Raf-MEK-MAPK pathway is important
for JSRV transformation in the cell lines studied, and this is consistent with activation
of this pathway in OPA tumors. p38 is activated in JSRV-transformed cells, and it
may negatively modulate signaling through Ras-Raf-MEK-MAPK, at least in tissue culture. The details of which isoforms in this pathway are involved in signaling
may differ depending on the cell line, type or species of origin. Finding the cellular
protein(s) that interact with Env would provide insight into how Env activates the
PI3K-Akt-mTOR and Ras-Raf-MEK-MAPK pathways. A diagram of the signaling
pathways involved in JSRV transformation is shown in Fig. 30.7.
Other Pathways
Src is an intracellular non-receptor tyrosine-specific protein kinase that was the first
proto-oncogene discovered. Studies on JSRV CT mutants suggested that Env may
signal to Src, and treatment with the Src inhibitor PP2 inhibited transformation
(Hull and Fan 2006). Treatment of 208F cells with Src inhibitors also reverted or
inhibited Env transformation as did a dominant negative Src protein (Varela et al.
2008). Exactly how Src is functioning in Env transformation or how it is activated
is unknown.
Mutations in the epidermal growth factor receptor (EGFR) are frequently
observed in human BAC tumors, and treatment with EGFR inhibitors (gefitinib)
has significantly improved survival rates (Marchetti et al. 2005). Although OPA
resembles BAC, EGFR inhibitors do not affect Env transformation in vitro (Varela
et al. 2008).
Primary type II pneumocytes derived from primary OPA tissues had constitutively active Akt and increased levels of telomerase activity compared to normal
type II pneumocytes (Suau et al. 2006). This has suggested that Akt activation could
lead to telomerase activation and inhibition of senescence. Inhibition of telomere
shortening is believed to be required for malignancy.
It has also been suggested that Env could be regulating total Akt levels through
Hsp90, a molecular chaperone that functions in folding, assembly, maturation, and
stabilization of proteins (Varela et al. 2008). Hsp90 can affect several signaling proteins including Akt, c-Src, and p53 (Varela et al. 2008). Inhibition of Hsp90 reverted
and/or inhibited JSRV Env transformation of rodent fibroblasts at least partially by
inducing Akt degradation. Furthermore, Hsp90 was found to be expressed in naturally occurring OPA and inhibition of Hsp90 reduced proliferation of primary and
immortalized cells from OPA tumors (Varela et al. 2008).
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JSRV Env
SU
Hyal2
RON
TM
Src
RAS
PI3K
Rac1
Akt
R
Raf
MEK 1/2
p38
TSC1-TSC2
RheB
ERK1/2
ERK
mTOR
TRANSFORMATION
Fig. 30.7 Signaling Pathways for transformation by JSRV Env. Pathways and intermediates that
have been established in JSRV Env transformation are shown in solid arrows. Dashed arrows
indicate where the mechanisms of signaling to the pathway are not yet understood. Question marks
are where signaling has been established in other biological systems, but it is unclear if it is important for JSRV Env transformation. The two predominant pathways in Env transformation are the
Ras-Raf-MEK1/2-ERK1/2 and PI3K-Akt-mTOR pathways. Signaling from Hyal2-RON, Src and
Rac1 signaling has also been found to be important Env transformation in at least some cases.
Interaction between Env and the proteins in the indicated pathways has not been detected. It is
likely that additional intermediates are involved in some of these signaling pathways
Env Transformation in 3-D Culture

The JSRV Env transformation studies discussed above were performed in monolayer
culture. While these experiments have proven informative, there are limitations
because monolayer cultures do not recapitulate the polarized epithelial structures or
the cellcell and cellenvironment interactions that occur in the lung. An in vitro
model has been to suspend epithelial cells in extracellular matrix (e.g. Matrigel).
Under these conditions, MDCK cells readily form well-differentiated hollow
spheres termed acini, consisting of a single layer of polarized epithelial cells with
777
many of the properties of normal epithelia (OBrien et al. 2001, 2002; Lubarsky and
Krasnow 2003; Debnath and Brugge 2005). MDCK cells expressing Env that were
cultured in 3-D formed aberrant structures with multiple lumens (Johnson et al.
2010). The disruption was largely mediated by an increase in proliferation and also
by resistance to the proliferative suppression signal associated with lumen clearing.
On the other hand, JSRV Env did not disrupt establishment of cell polarity, tight
junctions, or apoptosis associated with lumen clearing.
The PI3K-Akt-mTor and Ras-Raf-MEK-MAPK pathways are important in
MDCK transformation but differences were observed in monolayer vs. 3-D culture
conditions. In monolayer culture, inhibition of PI3K reverted the transformed
phenotype more efficiently than did inhibiting mTOR. In contrast, in 3-D culture,
inhibition of mTor (rapamycin) more efficiently reverted transformation than did
inhibition of PI3K (LY294002). Thus in 3-D culture, Env signals to mTor in both
PI3K-dependent and -independent manners, but the PI3K-independent pathway
may be more important. This is reminiscent of the previous finding in monolayer
that JSRV Env can signal to Akt (upstream of mTOR) through PI3K-dependent and
-independent mechanisms (Maeda et al. 2005; Hull and Fan 2006).
Similar to other cell lines, inhibition of MEK (PD98059) inhibited Env transformation of MDCK cells in monolayer, indicating a positive role for MEK in this
process (Johnson et al. 2010). In contrast, inhibition of H/N-Ras (FTI-277) enhanced
transformation of Env-expressing MDCK cells, indicating that H/N-Ras has a negative role in MDCK transformation in monolayer. FTI-277 also enhanced transformation
of parental MDCK cells, indicating that H/N-Ras may have a general negative effect
on growth of MDCK cells in monolayer. In contrast, in 3-D culture inhibition of
both MEK and H/N-Ras enhanced transformation and the size of Env structures;
this was also true for control acini. These results indicate a general inhibition of MDCK
cell growth in 3-D culture by the H/N-Ras-MEK pathway, in contrast to a positive
role for MEK in transformation in a monolayer.
The studies on MDCK 3-D cultures emphasize the fact that the differentiation
state of the cells can influence signaling pathways involved in JSRV transformation.
In particular, conditions that maintain differentiation of lung epithelial cells
(e.g. 3-D culture) will be most informative about the mechanisms of JSRV lung
carcinogenesis.
The Hyal2/RON Pathway

As described above, the cell surface receptor for JSRV Env is Hyal2. Studies on the
minimally transformed human lung epithelial cell line BEAS-2B have indicated
that in these cells Hyal2 is complexed with a membrane-spanning growth factor
receptor RON (Danilkovitch-Miagkova et al. 2003). RON (also known as STK) is a
receptor tyrosine kinase that belongs to the Met proto-oncogene family. It is widely
expressed in human tissues, in particular those of epithelial origin and immune cells
(Comoglio and Boccaccio 1996). Interestingly, RON is overexpressed in a variety
778
of human tumors, and certain RON mutants induce tumorigenic transformation

in vitro; RON may also promote tumor metastasis (Santoro et al. 1996; Wang et al.
2003). Additionally, transgenic mice overexpressing RON develop lung tumors
(Wang et al. 2003), which supports a role for RON in lung cancer. Indeed RON is
overexpressed and constitutively activated in some human lung cancer cell lines,
and in particular those derived from BAC (Danilkovitch-Miagkova et al. 2003).
The facts that RON can associate with Hyal2, and that over-expression of RON can
lead to lung cancer, have suggested that JSRV transformation may involve the Ron/
Hyal2 axis in lung epithelial cells. RON is phosphorylated in Env transformed
BEAS-2B cells and expression of a dominant- negative kinase-dead RON mutant
blocked Env-mediated transformation (Danilkovitch-Miagkova et al. 2003). In normal
BEAS-2B cells, Hyal2 is constitutively associated with RON, inhibiting its ability to
bind its growth factor ligand (macrophage-stimulating protein; MSP) and activate its
tyrosine kinase. When JSRV Env is introduced into BEAS-2B cells, Env binds to
Hyal2, resulting in its degradation. This in turn frees RON to respond to MSP and
become activated; activation ultimately leads to phosphorylation of downstream
targets such as Akt (Danilkovitch-Miagkova et al. 2003). Thus, in effect, Hyal2 functions as a tumor suppressor in BEAS-2B cells by binding and blocking the mitogenic
activity of RON; Env counteracts Hyal2 by binding it and stimulating its degradation.
Although the Hyal2/RON interaction is likely involved in JSRV transformation
in BEAS-2B cells, it is not required for transformation in several other cell lines and
mouse models. First, JSRV Env does not bind murine Hyal2, yet Env transforms
murine cell lines and induces tumors in mice (Wang et al. 1995; Liu et al. 2003a, b;
Miller et al. 2004; Maeda et al. 2005; Wootton et al. 2005, 2006a, b). Additionally,
over-expression of Hyal2 or RON did not affect Env transformation or activation of
RON in any cell lines tested (Liu et al. 2003a; Miller et al. 2004). When the receptor
binding domain (RBD) of Env (that binds Hyal2) is deleted, the resulting Env can
still induce transformation of rodent cell lines (Rai et al. 2001; Liu et al. 2003a;
Miller 2003; Miller et al. 2004). Moreover, MDCK cells do not express RON (Wang
et al. 1994, 2004; Danilkovitch-Miagkova et al. 2001). Thus while the RON-Hyal2
pathway may contribute to Env transformation in human lung airway cells or
BEAS-2B cells, it is probably not absolutely required. It will be interesting to
explore the role of RON-Hyal2 in ovine lung epithelial cells that would express
RON as well as a Hyal2 that is a functional Env receptor.
Pathogenesis by JSRV
Natural History of OPA in Flocks
OPA was shown to be a transmissible malignant disease by intratracheal inoculation
of sheep with: tumor cells (Coetzee et al. 1976), tumor homogenate (Wandera 1970;
Verwoerd et al. 1980; Salvatori et al. 2004), OPA lung fluid, and finally JSRV produced
in culture (Palmarini et al. 1999; Cousens et al. 2007). In nature, a major route of
779
JSRV transmission appears to be by aerosols (Dungal 1938, 1946; Tustin 1969)

because lung fluid from OPA-affected sheep contain high concentrations of JSRV
(1071010 JSRV particles per ml) (Cousens et al. 2009), making spread through
aerosolized lung secretions plausible. The environment of the lung is rich in surfactant proteins, proteases, and denaturing agents that inactivate viruses as well as
other pathogens. However, JSRV can survive in this environment as well as outside
the host for several weeks (Cousens et al. 2009). The stability of the virus is largely
attributed to the Env protein. MuLV-based vectors pseudotyped with JSRV Env are
remarkably stable at room temperature and relatively resistant to the effects of
surfactant (Zsengeller et al. 1999; Coil et al. 2001). JSRV infection can also be
spread vertically (intra-uterine) and by suckling of colostrum from infected animals
(Salvatori et al. 2004; Grego et al. 2008).
Latency to Disease
Incubation time to development of OPA is 68 months when JSRV is introduced
into flocks of sheep where infection is not endemic (Dungal 1938). Although sheep
of all ages are susceptible, cases of OPA from natural infection rarely occur in
animals younger than 79 months of age (Dungal 1938; Tustin 1969; Hunter and
Munro 1983; Gonzalez et al. 1993). The disease latency following intratracheal
inoculation of lambs that are several months old is 512 months (Tustin 1969;
Wandera 1970; Martin et al. 1976). When very young lambs are inoculated, clinical
signs appear at 36 weeks or even more rapidly (46 days) (Sharp et al. 1983). It has
been suggested that younger lambs are more susceptible to OPA because they have
more proliferating type II pneumocytes and Clara cells (Salvatori et al. 2004;
Caporale et al. 2005).
Not all animals infected with JSRV develop OPA, (Salvatori et al. 2004; Caporale
et al. 2005), and some breeds may be more resistant to OPA than others. In 1933, an
outbreak of OPA in Iceland resulted from introduction of a JSRV-infected breeding
stock into the island (York and Querat 2003); up to 90% of Gottorp sheep died from
OPA compared to 10% of Adalbol sheep (Dungal 1938). Additionally, flock immunity
might theoretically provide protection to animals. Development of OPA was high
(20% or more) during the first few years following introduction of infection into a
flock and it decreased (less than 5%/year) after a few years (Dungal 1938; Shirlaw
1959; Wandera 1967; Tustin 1969; Sharp and DeMartini 2003). However, an
immune response to JSRV has not been detected in infected animals.
In natural cases, co-infection with the ovine lentivirus maedi-visna virus (MVV)
is frequently observed in OPA tumors (Markson et al. 1983; Snyder et al. 1983).
Animals also frequently present with secondary bacterial infections. In most studies,
OPA sheep co-infected with maedi-visna virus (MVV) present with more severe
clinical signs than those only infected with JSRV (Markson et al. 1983; Snyder
et al. 1983; Rosadio et al. 1988b). Development of OPA was also studied in
animals experimentally co-infected with JSRV and MVV (Hudachek et al. 2010).
780
OPA was observed along with the spontaneous regression of the tumors and regression of OPA was more common than progression; CD3+ T cells and antibodies
against JSRV were detected, which could have been responsible for the regression.
Thus under these conditions, MVV might stimulate the immune response, leading
to protection against progression of JSRV-induced OPA.
Immune Responses
JSRV-infected sheep rarely develop antibodies to JSRV. The general lack of detectable
cellular and/or humoral immunity in infected animals is a unique property of JSRV
infection (Holland et al. 1999; Summers et al. 2002; Sharp and DeMartini 2003;
Ortin et al. 2007). Expression of closely related endogenous JSRV-related proviruses (enJSRVs, see below) in the fetal thymus during T cell development is believed
to cause immunological tolerance to exogenous JSRV (Palmarini et al. 2001a;
Spencer et al. 2003; Palmarini et al. 2004). Despite this, OPA tumors generally show
a large influx of macrophages and downregulation of MHC I and II complex expression (Hunter and Munro 1983; Summers et al. 2005). Downregulation of MHC 1 in
OPA may prevent elimination of virally infected cells by virus-specific CD8+
T cells (Summers et al. 2005). The effects of the infiltrating macrophages are
unknown, but it is possible that they secrete IFN-g, which could modulate MHC I
and II levels. A recent study of natural OPA cases reported numerous CD3+ T cells
located in pulmonary tissue and surrounding the neoplastic foci (Beytut et al. 2009).
However, sheep with natural OPA have reduced CD4+ T cells in the peripheral
blood, increased circulating neutrophils or increased CD3+ T cells at neoplastic foci
(Holland et al. 1999; Summers et al. 2002; Sharp and DeMartini 2003). These
changes are not always observed in experimentally infected animals. Therefore, it is
believed that the presence of neutrophils, macrophage, lymphocytes, and plasma
cells in naturally infected cases are due to secondary infections (Rosadio et al.
1988a; Garcia-Goti et al. 2000; Summers et al. 2005).
Endogenous JSRV
ERVs
Retroviral integration is an essential step in the lifecycle resulting in integration of
proviral DNA into the host genome. If a retrovirus infects a germ cell progenitor,
this results in inheritance of the proviral DNA as a Mendelian element in resulting
progeny. Vertically transmitted retroviruses are termed endogenous retroviruses
(ERVs) (Boeke and Stoye 1997; Coffin 2004). The process of accumulating copies
of retroviruses into the germ line is termed endogenization. While endogenization
of retroviruses is relatively infrequent, over evolutionary time scales this has resulted
781
in accumulation of numerous ancient and modern (recently acquired) ERVs in

most eukaryotes. Since there is no mechanism for elimination of ERV DNAs from
cells, ERVs accumulate over time. For instance, approximately 8% of the human
genome consists of ERV proviruses. Most ERVs are replication defective, likely
reflecting selection against replication competent ERVs. However, some modern
ERVs can encode viral proteins, or in some cases complete viruses.
When expressed, ERVs can have various biological effects and in some cases
expression is selected. Biological effects of ERVs include immune suppression
(Golovkina et al. 1992; Gifford and Tristem 2003), resistance to exogenous retroviral
infection (Best et al. 1997), and facilitating early embryonic development (Villarreal
1997; Mi et al. 2000; Dupressoir et al. 2005). ERVs have also been implicated in
diseases such as autoimmunity (Nakagawa and Harrison 1996; Perron et al. 1997),
multiple sclerosis (Perron et al. 1997), and cancer (Lower et al. 1996) in humans,
although causal roles have not been definitively established.
Sheep are interesting because they carry copies of active ERVs that are highly
similar to the exogenous betaretroviruses JSRV and ENTV the enJSRVs. Indeed
endogenization of enJSRVs appears to be ongoing (Arnaud et al. 2007, 2008).
Studies in sheep by Palmarini and collaborators provide strong evidence of co-adaptation between exogenous betaretroviruses, enJSRVs, and their hosts.
enJSRV
enJSRVs have been integrating into the sheep genome for the last 57 million years.
Currently, there are at least 27 enJSRV proviruses in the sheep germline DNA
(Palmarini et al. 2000b; Arnaud et al. 2007), and five have intact proviruses with
uninterrupted ORFs (Arnaud et al. 2007). These five enJSRVs have 8589% sequence
identity to exogenous JSRV (Palmarini and Fan 2003). Regions of sequence divergence reside in the U3 region of the LTR and three regions in Gag and Env termed
variable regions 1, 2, and 3 (VR1-3) (Palmarini et al. 2000b). None of the EnJSRV
Envs contain the YXXM motif in the CT region of the Env TM domain and are
unable to induce transformation (Palmarini et al. 2001b; Arnaud et al. 2007).
enJSRV and exogenous JSRV LTRs differ because enJSRV LTRs are not preferentially active in lung epithelial cells (McGee-Estrada and Fan 2007). They also
may respond to progesterone, consistent with their high expression levels in the
female reproductive tract tissue (Palmarini et al. 2000b).
EnJSRV Restriction Factor

Certain EnJSRVs function as restriction factors for exogenous JSRV infection.
EnJSRV and JSRV use the same receptor, Hyal2, and expression of EnJSRV Env in
some tissues can block JSRV entry through receptor competition. In addition, two
782
recently acquired enJSRVs (56A1 and 20) express Gag protein that transdominantly
inhibit exogenous JSRV viral particle release through a mechanism termed JSRV
late restriction (JLR) (Mura et al. 2004). JSRV Gag normally coalesces into viral
cores in the pericentrosomal area, after which the cores engage the intracellular trafficking machinery for transport to the cell membrane. enJS56A1 Gag behaves as a
mis-folded protein, likely due to tryphophan at position 21 (W21); however, it can
associate with exogenous JSRV Gag forming multimers and/or chimeric viral particles
that are unable to traffic to the cell membrane. It has been suggested that the inhibitory effects of enJSRV on exogenous JSRV infection may have driven a switch in
tissue tropism for the exogenous virus from the genital tract (where enJSRV is
expressed) to the alveolar epithelium of the lung (no enJSRV expression) (Palmarini
et al. 2000b; Palmarini et al. 2004). Indeed, LTRs of the restrictive enJSRVs are not
active in differentiated lung cells (Palmarini et al. 2000b).
The Role of enJSRV in Placentation

The fusigenic properties of retroviral Env proteins have led to the proposal that
ERVs play a role in placental morphogenesis. Indeed, retroviral particles are
observed in the reproductive tract of many different species (Kalter et al. 1975;
Harris 1991), and the human proteins syncitin-1 and -2 that promote syncitial trophoblast fusion are HERV env proteins (Blond et al. 1999; Mi et al. 2000). Studies
in sheep are the first to provide in vivo evidence that ERVs play a physiological role
in conceptus and placental development (Dunlap et al. 2006a, b). EnJSRV expression
is most abundant in the female reproductive tract of sheep, including the vagina,
cervix, uterus, and oviduct (Spencer et al. 1999; Palmarini et al. 2000b; Palmarini
et al. 2001a; Dunlap et al. 2005). Env expression is temporally regulated during
conceptus development and placental morphogenesis (Dunlap et al. 2005). It has
been proposed that co-expression of Env and Hyal2 mediate cell fusion leading to
formation of multinucleated syncytial plaques that are critical for formation of the
placenta (Dunlap et al. 2006a, b). Indeed, it was shown that targeted downregulation
by siRNA of EnJSRV Env expression in the oviduct leads to failure of the sheep
conceptus to implant (Dunlap et al. 2006a, b).
ENTV
ENTV is the causative agent of contagious ovine and caprine nasal adenocarcinoma
(ONA and CNA), which arises from the secretory cells of the ethmoid turbinate
(Cousens et al. 1999; De Las Heras et al. 2003; Ortin et al. 2003). ENTV shares
~95% homology with JSRV. Two strains of ENTV, ENTV-1, and ENTV-2 have
been identified and are largely distinguished by their host tropism and dissemination
in infected animals. ENTV-1 infects sheep and is mainly confined to the tumor.
783
ENTV-2 infects goats and also establishes lymphoid infection (Cousens et al. 1996;
Ortin et al. 2003). As expected from the high degree of sequence homology, ENTV
shares many properties with JSRV. Specifically, Hyal2 is the cellular receptor for
entry, Env acts as an oncogene in in vitro and in vivo experiments (Alberti et al.
2002; Dirks et al. 2002; Wootton et al. 2006a) and the same signaling pathways are
involved in ENTV transformation (PI3K-Akt-mTor and Raf-MEK-MAPK pathways).
Although ENTV-1 employs Hyal2 for entry, additional factors are required because
Hyal2 is not sufficient for entry in some cell lines (Dirks et al. 2002).
JSRV and ENTV sequence are divergent in three regions: 3 of Env, the U3 region
of the LTR and Orf-x (Cousens et al. 1999). ENTV-1 has two stop codons within the
orf-x open reading frame, making it unlikely that orf-x protein is important for
ENTV-1 replication (Cousens et al. 1999). While ENTV-1 Env has been shown to
transform rodent fibroblast and epithelial cell lines (Alberti et al. 2002; Dirks et al.
2002; Liu et al. 2003a, b; Liu and Miller 2005) and induce lung tumors in mice
(Wootton et al. 2006a), there are significant sequence differences in the TM CT
region between JSRV and ENTV. However, utilization of the same pathways for
transformation suggests that the minimally essential amino acids and/or structure
are maintained. Specifically, residue Y590 in CT of JSRV Env is critical for transformation. This residue is conserved in ENTV-1 and shown to be required because
mutations ablate transformation (Alberti et al. 2002; Liu et al. 2003b).
Although JSRV and ENTV show an overall high sequence similarity, the U3
regions of the LTR are only 62% homologous. Most differences are observed in the
mid- and promoter proximal enhancer regions. A site critical for activity of the JSRV
LTR in lung epithelial cells, the upstream HNF-3b binding site, is absent in the
ENTV LTR and is believed to contribute to its lower activity compared to the JSRV
LTR in those cells (McGee-Estrada and Fan 2007). When expressed in an AAV vector,
ENTV-1 Env induces adenocarcinoma of the lung that closely resembles that induced
by JSRV Env. Thus ENTV Env is not the major determinant of disease specificity.
Rather, similar to JSRV, the ENTV LTR is likely the major determinant.
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Chapter 31
Small RNAs and Their Role

in Herpesvirus-Mediated Cancers
Sankar Swaminathan and Rolf Renne
EBV EBER RNAs

Structure and Expression of EBERs
The EBV-encoded RNAs (EBERs) are 166 and 172 nucleotide single-stranded
RNAs encoded by two genes separated by 161 bp in the EBV genome (Arrand and
Rymo 1982; Lerner et al. 1981; Rosa et al. 1981). They are the most abundant RNA
species in most EBV-infected cells, and are present at more than 5 106 copies per
cell (Lerner et al. 1981). EBERs are transcribed by RNA polIII and contain intragenic
control sequences typical of A and B boxes found in polIII transcripts (Jat and
Arrand 1982; Rosa et al. 1981; Howe and Shu 1989). However, both EBER 1 and 2
also contain upstream elements and TATA-like sequences typical of polII promoters
(Howe and Shu 1989). Although EBER transcription is primarily polIII dependent,
the upstream sequences are required for efficient transcription (Howe and Shu 1989,
1993). These upstream sequences are highly conserved in Herpesvirus papio, the
baboon herpesvirus homologous to EBV, suggesting that they are important in regulation of EBER expression (Howe and Shu 1988).
EBERs are expressed in nasopharyngeal carcinoma (NPC) and Burkitt lymphoma
(BL) biopsies and in BL cell lines and lymphoblastoid cell lines (LCLs) passaged
in vitro (Jat and Arrand 1982; Minarovits et al. 1992). Where transcription initiation
S. Swaminathan (*)
Division of Infectious Diseases, Department of Medicine, University of Utah School
of Medicine, Salt Lake City, UT 84132, USA
e-mail: sankar.swaminathan@hsc.utah.edu
R. Renne
Department of Molecular Genetics and Microbiology, UF Shands Cancer Center,
University of Florida, Gainesville, FL 32610-3633, USA
793
794
S. Swaminathan and R. Renne
rate has been directly measured in LCLs by nuclear run-on assay, EBERs are
transcribed at a rate at least ten times higher than that of the most highly transcribed
latency-associated EBV mRNA (Sample and Kieff 1990). EBERs are expressed in
all BL cell lines, but the relative level of EBER 1 and 2 varies among isolates
(Minarovits et al. 1992). In most cases, EBER1 is the predominant species, although
there are instances when EBER2 is expressed at higher levels. There appear to be
type-specific sequence differences in the EBER region among different EBV isolates,
although these variations are mostly in noncoding regions (Arrand et al. 1989).
The entire EBER locus is hypomethylated in BL cells in comparison to other regions
of the EBV genome (Banati et al. 2008; Minarovits et al. 1992), suggesting that
hypomethylation of the EBER locus may allow persistent EBER expression even
during restricted latency, when most latent gene expression is curtailed.
Although EBERs are detectable in cells in which EBV is undergoing lytic replication, they do not appear to play an essential role in the process. The steady-state level of
EBER RNAs does not decline significantly upon induction of lytic replication in latently
infected cells in culture (Weigel et al. 1985). Nevertheless, based on nuclear run-on
assays performed in BL cells in vitro, it is likely that EBER transcription declines during
lytic replication (Greifenegger et al. 1998). In lesions of oral hairy leukoplakia, an EBVassociated proliferative epithelial disease, lytic EBV replication predominates and
EBERs are not expressed at significant levels (Gilligan et al. 1990). These findings and
the ability of EBER-deleted EBV recombinants to replicate lytically (Swaminathan
et al. 1991) demonstrate that EBERs are dispensable for lytic replication.
EBERs form stable protein complexes with two cellular proteins, La and L22.
Based on folding algorithms and experimental evidence from chemical modification
and enzymatic cleavage, EBER RNAs are predicted to form stable secondary structures
with several stem-loops (Glickman et al. 1988; Rosa et al. 1981). Both EBERs
associate with the abundant cellular cytoplasmic autoantigen La and are immunoprecipitable with anti-La antibody (Lerner et al. 1981; Rosa et al. 1981). La binds to
many polIII transcripts but associates only transiently with such transcripts during
3 end maturation rather than forming stable complexes as it does with EBERs.
EBER1 also forms complexes with the ribosomal protein L22 (Toczyski and Steitz
1991). Each EBER1 molecule binds three molecules of L22 simultaneously at three
distinct stem-loops (Fok et al. 2006b; Toczyski and Steitz 1993). This binding leads
to a partial relocalization of L22 to the nucleoplasm from its usual nucleolar and
cytoplasmic locations (Toczyski et al. 1994). This interaction of EBERs with L22
may have important functional consequences (see below).
Functions of EBERs During Latent Infection

Several cellular functions have been proposed for EBERs but almost all remain
controversial. These functions may be broadly categorized into the following categories: preventing host cell translational shutoff, preventing apoptosis, enhancing
specific gene expression and facilitating B cell transformation (Fig. 31.1).
31 Small RNAs and Their Role in Herpesvirus-Mediated Cancers
795
Nucleus
IRF-3
RIG-I
Viral
Episome
Human
Genome
IFN, cytokines
EBER 2
EBER 1
Nucleolus
a PKR
Apoptosis
Apoptosis
Translation blockade
L22
Promoting
Oncogenesis
(d)
L22
Fig. 31.1 Interaction of EBV EBER RNAs with cellular proteins and proposed mechanisms of
action. (a) EBER1 is shown binding and inhibiting cellular kinase PKR, blocking PKR-mediated
apoptosis and PKR-mediated protein translation shutoff. (b) A PKR-independent role for EBERs
in preventing apoptosis. (c) EBERs are shown binding and activating RIG-1, leading to downstream activation of IRF-3 and transcription of IFN and cytokines, such as IL-10. Question marks
denote controversies or uncharacterized mechanisms: In (a) and (c), the localization of EBERs to
the cytoplasm is debated. The alternative mechanism of EBERs in preventing apoptosis is uncharacterized (b). How L22 sequestration may promote oncogenesis remains to be defined (d)
Preventing Protein Translation Shutoff

A potential role for EBER RNAs in protecting EBV-infected cells from translational
arrest was suggested by analogy to the small adenovirus RNAs, VAI and II, which
bear structural similarities to EBERs. During adenovirus replication, VA RNAs rescue
cells from inhibition of protein translation mediated by the cellular kinase PKR,
which is induced by interferon and activated by double-stranded RNAs produced
during replication of many viruses (Ghadge et al. 1994; Hovanessian 1989). Activated
PKR phosphorylates eukaryotic initiation factor eIF2-a, thereby inhibiting initiation
of peptide synthesis (Hershey 1991). Adenovirus mutants in which VA RNAs were
replaced by EBER1 could functionally substitute for VA RNAs (Bhat and
Thimmappaya 1983, 1985). Although rescue of adenovirus replication by EBER1
was only partial, it suggested that EBERs might play a role in counteracting the antiviral effects of interferon and PKR activation in EBV-infected cells. Consistent with
this model, several in vitro studies demonstrated that EBERs could directly bind
796
PKR and inhibit its activity. Both EBERs inhibit PKR kinase activity in peptide
phosphorylation assays and bind to PKR in vitro (Clarke et al. 1991; Sharp et al.
1993). When added to reticulocyte lysates at high concentrations, EBER1 prevented
inhibition of translation by double-stranded RNA (Clarke et al. 1990). When transfected into cells in culture, EBER plasmids enhanced overall protein synthesis;
however, PKR-negative cells from PKR knockout mice still exhibited the EBER
effect (Laing et al. 1995, 2002). Similarly, although EBER-transfected 3T3 cells
exhibited higher soft agar clonogenicity, there was little correlation with EBER
expression and EBER expression did not reliably predict tumorigenicity in nude
mice (Laing et al. 2002). Overall these data suggest that while EBER expression may
have effects on cell physiology, they are not due to PKR inhibition.
Whether EBERs actually interact with PKR and inhibit its activation during EBV
infection remains controversial. One of the main objections to EBERs acting as a
barrier to translational inhibition is their nuclear location. In situ hybridization studies
have demonstrated nuclear localization of EBERs (Barletta et al. 1993; Howe and
Steitz 1986). A subsequent report described EBERs in a perinuclear location, consistent with endoplasmic reticulum and Golgi localization, by confocal microscopy, but
this is the only such report (Schwemmle et al. 1992). A recent study demonstrates
that under conditions where UI small nuclear RNAs undergo nucleocytoplasmic
shuttling in heterokaryons, the EBERs do not shuttle (Fok et al. 2006a). In addition,
EBERs injected into Xenopus oocyte nuclei remained confined to the nucleus
whereas tRNAs underwent rapid cytoplasmic export. EBER1 was shown to have a
half-life of 2530 h and was more stable than RNAs that did undergo shuttling, indicating that rapid cytoplasmic degradation was not responsible for the inability to
detect shuttling. A nuclear location for the EBERs is clearly difficult to reconcile
with a role in modulating the cytoplasmic function of PKR in blocking translation
initiation. Another line of evidence suggesting that EBER RNAs are not critical in
protection from the effects of interferon comes from studies performed using LCLs
transformed by EBER-deleted recombinant EBV (Swaminathan et al. 1991).
Replication of VSV, which is highly sensitive to inhibition by interferon, is not
quantitatively impaired in EBER-negative LCLs treated with interferon, nor is the
growth of EBER-negative LCLs inhibited by interferon (Swaminathan et al. 1992).
Lymphomagenesis and Protection from Apoptosis

EBERs are able to enhance survival of Burkitt lymphoma cells under certain
experimental conditions. However the mechanism of this effect remains to be fully
explained. EBV-infected Burkitt lymphoma cells examined shortly after biopsy
usually express EBER RNAs and EBNA1, a nuclear protein required for episomal
EBV maintenance. Several cell lines derived from Burkitt lymphoma cells maintain
this restricted pattern of EBV gene expression in vitro. One such cell line, Akata,
spontaneously loses EBV genomes in culture (Shimizu et al. 1994). The resulting
EBV-negative cells are noticeably less robust, undergo spontaneous apoptosis, are
more serum-dependent, and are less able to form colonies in soft agar or tumors in
797
nude mice (Komano et al. 1998; Ruf et al. 1999). Reinfection of the EBV-negative
clones with EBV restores the parental phenotype, indicating that EBV gene expression
contributes to the oncogenic phenotype (Komano et al. 1998; Ruf et al. 1999).
Transfection of EBERs partially restores resistance to both spontaneous and
interferon-induced apoptosis (Komano et al. 1999; Ruf et al. 2000). Interferon
treatment and PKR activation can induce apoptosis by multiple mechanisms, including
eIF2-phosphorylation. Whether PKR inhibition is actually the mechanism by which
EBERs protect against apoptosis has remained controversial. Studies from independent
laboratories measuring the effect of EBERs on PKR kinase activity, on either PKR
itself or eIF2-a as a substrate, have yielded conflicting results (Nanbo et al. 2002;
Ruf et al. 2005; Wang et al. 2005). When considered in combination with the data
on nuclear EBER location, it appears that while EBERs may inhibit apoptosis, it is
unlikely that inhibition of PKR is the primary mechanism for this effect.
EBERs and Cellular Gene Expression

Several cell signaling pathways have been implicated in EBER effects in protection
from apoptosis and enhancement of B cell survival. Expression of a variety of
cytokines and growth factors is enhanced in several types of EBER-expressing cells.
In EBV-negative B lymphoma, T cell lymphoma, or gastric carcinoma cell lines,
stable expression of EBERs is associated with IL-10, IL-9, or insulin-like growth
factor 1 (IGF1) induction, respectively (Iwakiri et al. 2003, 2005; Kitagawa et al.
2000; Yang et al. 2004). In the case of gastric carcinoma, 515% of cases are
reported to be EBV-positive. Infection of EBV-negative gastric carcinoma cell lines
with EBV led to expression of a limited number of EBV genes including EBERs
and was correlated with increased IGF1 production, as was transfection of EBER
genes (Iwakiri et al. 2003). Similarly, infection of EBV-negative NPC-derived cell
lines with EBV led to increased IGF1 production and enhanced growth at low serum
concentrations. Increased IGF1 levels were associated with increased IGF1 mRNA,
implying induction of transcription or transcript stabilization by the EBERs. EBV
infection and EBER expression in EBV-negative subclones of two BL cell lines,
Akata and Mutu, have been correlated with increased expression of human IL-10
(Kitagawa et al. 2000).
A model for induction of IL-10 transcription by EBERs has been proposed based
on findings that transfected EBERs interact physically and functionally with RIG-I,
a cytoplasmic protein important in interferon induction by double-stranded RNA
[for review, see Bird (2007)]. According to this model, EBERs may bind and induce
the downstream activity of RIG-I, thereby affecting transcription of a number of IFN
target genes. Several lines of evidence support the interaction of RIG-I with EBERs,
including the use of dominant negative forms of RIG-I and the ability of RIG-I to
coimmunoprecipitate with EBER RNA (Samanta et al. 2006, 2008). Transfection of
RIG-I led to production of type I IFNs in EBV-positive BL cells but not in EBVnegative cells (Samanta et al. 2006). Transfection of RIG-I siRNA also decreased
798
IL-10 expression in BL cells but this effect was not observed in EBV-negative cells
or BL cells infected with the EBER-negative recombinant (Samanta et al. 2008).
Silencing IRF-3 expression also led to decreased IL-10 expression, suggesting that
RIG-I may activate the IL-10 promoter through IRF-3 induction. There is, thus, a
large body of experimental evidence that EBERs induce expression of several cytokines
that are capable of enhancing cell growth. This model, although attractive, still must
contend with the preponderance of evidence that EBERs are confined to the nucleus
whereas the RIG-I interaction must occur in the cytoplasm.
EBERs Role in B Cell Transformation and Lymphomagenesis

The ability of EBV to efficiently transform primary B lymphocytes in vitro is dependent on several EBV genes expressed during latent infection. Because of the virtually
ubiquitous expression of EBER RNAs in EBV-associated tumors, and different types
of latent infection and their conserved expression among different primate lymphocryptoviruses, it was thought that they might be important for the process of
transformation. The generation of EBER-deleted EBV recombinants in the early
1990s demonstrated that they were not essential for transformation of B lymphocytes
in vitro (Swaminathan et al. 1991). EBER-negative recombinants were generated by
transfection of cosmid DNA deleted for EBERs into a BL cell line (P3HR-1) harboring an EBV genome which has lost the essential transforming gene EBNA2. Virus
replication was induced and recombinants which had acquired both EBNA2 and the
EBER deletion were selected by virtue of their ability to transform and immortalize
primary B lymphocytes. EBER-deleted recombinants were isolated and arose at a
frequency (>15%) that suggested no intrinsic defect in their transforming capacity.
Furthermore, outgrowth of EBER-negative LCLs was not slower than that of EBERpositive LCLs, again suggesting that EBERs did not provide a significant growth
advantage. Nevertheless, it remained possible that the LCLs were initially coinfected with P3HR-1 virus, which provided EBERs in trans and were subsequently
lost prior to analysis. However, pure EBER-negative EBV was passaged from these
EBER-negative LCLs and was capable of immortalizing primary B lymphocytes
de novo. Immortalized cell lines derived from EBV deleted for EBERs also
maintained latent infection and a growth phenotype typical of EBER-positive
LCLs, indicating that in vitro, EBERs are not essential for maintenance of the transformed phenotype (Swaminathan et al. 1991). EBER-deleted recombinants were
also unimpaired in their ability to enter the lytic phase of replication (Swaminathan
et al. 1991).
By contrast, EBER-negative recombinants generated by another method suggest
that EBERs may provide a quantitative advantage in transforming ability (Yajima
et al. 2005). Using the Akata BL cell line, recombinant EBER-negative viruses were
derived by using drug selection and cre recombinase to remove the EBER genes
from the resident EBV genome. EBER-positive revertant knock-ins were also
generated by transfection of wild-type EBER plasmids and reselection of a stable
799
transfectant carrying EBER-positive recombinants. Transformation assays performed

with high titers of recombinant EBV generated from the EBER knockout and revertant strains revealed that the EBER revertant possessed approximately 20-fold more
transforming ability than the EBER-negative recombinant. Further, growth of the
EBER-negative LCLs was impaired compared to that of the revertants under low
serum conditions. Although these data suggest that EBERs play a role in B lymphocyte
growth and transformation, the results were based on comparisons of recombinants
ultimately derived from single clones of serially selected transfectants, which may
vary considerably in growth rates and phenotype.
Most recently, EBER-negative recombinant EBV and revertant EBER-positive
viruses derived from B95-8 EBV have been generated using bacmid technology.
When a panel of these viruses was tested for their ability to transform primary B
lymphocytes in vitro, and the transforming efficiency was quantitatively measured,
no differences were observed between EBER-negative recombinants and revertants
(Gregorovic G, et al. 2011). These data overall indicate that while strain-specific
differences may account for the varying reports, EBERs are not required for the
in vitro transforming ability of EBV.
The dispensability of EBERs for transformation in vitro suggests that they may
play an important role latent infection in vivo and possibly in tumorigenesis. Some
clues to their in vivo function may come from known cellular EBER-binding proteins. EBERs interact with two cellular RNA-binding proteins, La and L22; the
latter is a component of the large ribosomal subunit (Fok et al. 2006b; Toczyski
et al. 1994). La is known to be important in the biogenesis and maturation of polIII
transcripts, and is possibly involved in enhancing the stability or translation of some
mRNAs (Wolin and Cedervall 2002). Although EBERs are present at high copy
numbers, the levels of La in the cell are high enough that sequestration of La does
not seem to be a likely mechanism for EBER effects (Wolin and Cedervall 2002).
L22 is also an RNA binding protein and each EBER1 RNA binds three L22 molecules (Fok et al. 2006b). EBER binding causes relocalization of L22 to the nucleoplasm from the nucleolus and cytoplasm (Toczyski et al. 1994). Thus, a significant
amount of L22 may be sequestered by EBERs although depletion of L22 from ribosomes has not been observed to occur. A recent report on the association of a
Mareks disease virus (MDV) small RNA that also associates with L22 suggests that
relocalization and/or RNA binding of L22 may have oncogenic effects in vivo
(Kaufer et al. 2010). MDV expresses two copies of a ~450 nt RNA highly homologous to the telomerase-associated RNA (TR) that is an essential component of the
telomerase complex (Fragnet et al. 2003). MDV TR (vTR) is fully functional as a
telomerase component and is essential for efficient lymphomagenesis in chickens as
deletion of the vTR genes leads to decreased and delayed tumor formation (Trapp
et al. 2006). Importantly, MDV with a mutation of MDV vTR that abrogates telomerase binding is still lymphomagenic, indicating that the vTR has other functions
essential for lymphomagenesis (Kaufer et al. 2010). EBER-1, vTR and the host
chicken TR have similar effects on L22 localization as does the vTR mutant which
does not bind telomerase. Significantly, in another recent study, when EBER1 was
mutated so that it no longer bound L22, the ability of EBER1 to enhance BL cell
800
growth and colony formation in soft agar was significantly impaired (Houmani et al.
2009). Both EBV and MDV small RNAs, although very different in their structure
and transcriptional regulation appear to target the same cellular protein. How the
interaction with L22 might mediate growth effects remains to be determined.
Herpesvirus saimiri HSURs

H. saimiri infection leads to aggressive T cell leukemia and lymphoma in a variety of
New World primates and transforms T lymphocytes from marmosets in vitro into
continuously proliferating cell lines expressing T and NK activation markers [for
review, see Ensser and Fleckenstein (2005)]. H. saimiri infected tumor cells and cell
lines express seven small RNAs similar to human snRNAs that form snRNPs containing Sm proteins (Biesinger et al. 1990; Lee and Steitz 1990). They are assembled into
snRNPs in the cytoplasm by the SMN complex which performs this function in cellular U snRNP assembly (Golembe et al. 2005). During latent infection, HSURs are
found in the nucleus and colocalize with cellular snRNPs (Golembe et al. 2005).
HSUR1 and HSUR2 are the most highly conserved HSURs among HVS isolates and
are the only HSURs found in the closely related H. ateles (Albrecht 2000). HSUR1 is
the most abundant HSUR and the most abundant latent H. saimiri transcript, expressed
at approximately 20,000 copies per cell (Lee et al. 1988; Murthy et al. 1986).
The level of other HSURs is about 2,000 copies per cell. Despite their abundance,
conservation, and expression in H. saimiri-related tumors, HSURs are dispensable
for H. saimiri lytic infection and transformation in vitro (Ensser et al. 1999; Murthy
et al. 1989). They are thus similar to EBERs and likely to be important in vivo to
ensure viral persistence and infected cell growth and survival. Their mechanism of
action is similarly incompletely characterized although recent studies have provided
tantalizing clues to their function and interactions with other small RNAs.
Three of the HSURs, namely, HSUR1, HSUR2, and HSUR5, contain AU-rich
elements (AREs) in their 5 region, similar to those present in the 3 UTRs of many
cytokine mRNAs and growth factors. Several cellular proteins (HuR, hnRNPD
(AUF1), TTP, and BRF1), which either stabilize or destabilize ARE-containing
mRNAs, bind specifically to the ARE. HSUR1 and HSUR2 have been shown to
bind HuR and hnRNPD in transformed cells and HSUR1 binds to TTP (Cook et al.
2004). Although the presence of AREs in HSURs had led to the hypothesis that
HSURs might regulate the levels of host ARE-containing mRNAs, extensive and
thorough analysis of cellular mRNAs in cells transformed by HSUR1- and HSUR2deleted HVS failed to demonstrate changes in the levels of mRNAs with AREs
(Cook et al. 2004). However, the levels of HSUR1 itself appear to undergo regulation by the ARE-mediated pathway as mutation of the ARE in HSUR1 increased its
steady-state levels (Cook et al. 2004; Fan et al. 1997).
Despite the lack of any discernible effect on ARE-containing mRNAs, HSUR1
and HSUR2 appear to enhance expression of a specific set of genes important in
NK and T cell activation (Cook et al. 2005). When the transcriptional profile of
marmoset T lymphocytes transformed by mutant HVS deleted for HSUR1 and
801
HSUR2 was compared to cells transformed by wild-type HVS, HSUR expression

correlated with significantly increased levels of T cell receptor b and g chains, the T
cell and natural killer (NK) cell-surface receptors CD52 and DAP10, and intracellular proteins SKAP55, granulysin, and NKG7. Expression of these genes could be
rescued by exogenous HSUR1 and HSUR2 expression. The mechanism by which
HSUR1 and HSUR2 exert these effects remains to be defined.
Recently, another intriguing mechanism by which HSUR1 and HSUR2 may
modulate host cell gene expression has been identified. These two HVS small RNAs
were found to contain sequences complementary to miR-142-3p (HSUR1 and
HSUR2), miR-27 (HSUR1), and miR-16 (HSUR2) (Cazalla et al. 2010). This suggested that HSUR 1 and HSUR2 may sequester or inactivate these miRNAs, leading
to enhanced accumulation of the miRNA target transcripts. Both HSURs were found
in Ago2 complexes from HVS-transformed marmoset cells, whereas other cellular
snRNAs and other HSURs were not, indicating that HSUR1 and HSUR2 are selectively incorporated into miRNPs. Cell lines generated by transforming primary cells
with either wt HVS or HVS specifically deleted for HSUR1 and HSUR2 were then
used to assess the effect of HSUR1 and HSUR2 on the levels of these miRNAs and
to investigate their association with each other. All three miRNAs, miR-142-3p,
miR-27, and miR-16, were immunoprecipitated with anti-Sm serum, but only in wt
HVS-transformed cells, demonstrating that they interact specifically with HSUR1
and HSUR2 snRNPs. Furthermore, the level of miR-27 was markedly higher in
HSUR-deleted HVS transformed cells, suggesting that HSUR1 leads to downregulation of miR-27. Knockdown of HSUR1 confirmed the negative effect of HSUR1
on miR-27 accumulation and mutation of the miR-27 complementary sequence in
HSUR1 abolished the interaction and the negative effect on miR-27 levels.
Significantly, levels of FOXO1, a miR-27 target protein, were reduced in cells transformed by HVS lacking HSUR1 and HSUR2, indicating that the effect of HSUR1
on miR-27 has functional consequences on miR-27 target gene expression. Although
these findings suggest a mechanism of action for HSUR1, they raise several questions.
First, the implications of interactions with miR-16 and miR-142-3p are unclear, as
steady state levels of these miRNAs do not appear to be affected by HSURs, nor do
the levels of some of their potential target mRNAs. Second, miR-27 targets were not
identified among the set of activation genes described earlier that were identified by
microarray comparison of HSUR-deleted and wt HVS-transformed cell lines. Thus,
there are clearly additional mechanisms by which HSURs selectively enhance gene
expression that remain to be identified. Finally, as for EBV EBERs, the in vivo role
of HSURs in lymphomagenesis remains to be fully elucidated in animal models.
Viral MicroRNAs
In 2004, Tuschl and colleagues discovered the first viral encoded miRNAs in EBV
infected Burkitts lymphoma cells (Pfeffer et al. 2004). To date, the miRNA registry
miRBase (http://www.mirbase.org/) (Griffiths-Jones 2004; Griffiths-Jones et al. 2006)
contains 173 herpesvirus-miRNAs, encoded by members of all three herpesvirus
802
subfamilies [Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae,

recently reviewed in Boss and Renne (2010) and Umbach and Cullen (2009)].
Here, we summarize the current knowledge on miRNAs encoded by the two
g-herpesviruses EBV and KSHV that are associated with human cancer.
MicroRNA Biogenesis and Function

There is no inherent difference between expression and maturation of viral and
cellular miRNAs. EBV and KSHV miRNAs, are expressed from polII transcripts
(Ambros 2004; Bartel 2004; Bogerd et al. 2010; Diebel et al. 2010) and processing
begins with formation of an imperfect stem loop with a hairpin bulge that forms
within a miRNA precursor termed the pri-miRNA. The dsRNA region of the
pri-miRNA is recognized by DGCR8, which recruits the endonuclease Drosha to
cleave and release a 6080 nt long hairpin. This pre-miRNA is then exported into
the cytoplasm via the Exportin 5/RAN-GTPase pathway, where it is recognized by
Dicer and cleaved at the bulge. One strand of this dsRNA product is loaded into the
RNA-induced silencing complex (RISC). The remaining strand, known as the star
(*) strand, is degraded but in many cases can also be loaded into RISC with variable
efficiency [for review see: Ambros (2004) and Bartel (2004)].
RISC functions by guiding miRNAs to semicomplementary sites within the
3UTRs of target transcripts and induces translational silencing and/or degradation.
The 5 end of the miRNA, specifically nucleotides 28 termed the seed sequence, is
critical for determining mRNA target binding, but there are rare cases of miRNA
binding sites that have little seed binding, but significant 3 compensatory complementarity instead. Transcript 3UTRs have multiple binding sites for a specific
miRNA, and it is also common that one gene is targeted by multiple miRNAs. Due
to the flexible requirements for target recognition, a single miRNA can regulate
many targets and as a result miRNAs form large posttranscriptional regulatory
networks (Grimson et al. 2007).
After binding of RISC to the 3UTR of the target transcript, silencing is accomplished
through an incompletely deciphered mechanism(s). Current evidence suggests several
different mechanisms: inhibition of translational initiation by interfering with the
interaction of eIF4E, eIF6E, and the poly A binding protein, premature termination
of translation by inducing ribosomal drop-off after initiation, and messenger RNA
degradation by relocation of the RISC to cytoplasmic processing (P)-bodies, which
contain the RNA degradation machinery (Filipowicz et al. 2008).
EBV- and KSHV-Encoded miRNAs

After the initial identification of five EBV miRNAs (Pfeffer et al. 2004), a combination of tiled arrays, cloning, and bioinformatic approaches identified 18 additional
EBV miRNAs, located within the 12 kb deletion specific to the B95-8 strain analyzed
803
B95-8 deletion
BHRF1 BFLF2
BILF2
LF3
LF2
BALF5
EBV
BHRF1- 1
23
BART- 1 5 16 17
Cluster 1
ORF69
Cluster 2
K12
v-Flip
v-Cyclin
LANA
KSHV
KSHV-miR-K12-12 10
9 8 7 11 6 5 4 3 2 1
Fig. 31.2 Schematic representation of EBV and KSHV miRNAs. EBV and KSHV genomes are
represented with black arrows for ORFs, and black bars or rectangles for repeat sequences.
MiRNA locations are indicated with red arrows. Genomes are not drawn to scale. Figure was
compiled from Burnside et al. (2006), Cai and Cullen (2006), Cai et al. (2005), Cui et al. (2006),
Dunn et al. (2005), Grey et al. (2005), Grundhoff et al. (2006), Pfeffer et al. (2004, 2005), Samols
et al. (2005), and Yao et al. (2007)
in the original report (Pfeffer et al. 2004) and three more within the BART region
outside of the B95-8 deletion (Cai et al. 2006; Grundhoff et al. 2006). Recently, two
additional BART miRNA genes have been identified in EBV-positive NPC tissue
samples (Zhu et al. 2009). This brings the total of EBV miRNA genes to 25. Since
B95-8 immortalizes human B cells, none of the 18 miRNAs within the deletion are
required for immortalization in vitro however, as discussed below this does not rule
out a contributing role to pathogenesis and/or tumorigenesis in vivo. Like their metazoan counterparts, EBV miRNAs are expressed in a tissue specific fashion. BHRF1
and BART miRNAs are differentially expressed in lymphoid and epithelial cells and
are latency program specific. BART miRNAs are predominantly expressed in epithelial cells, whereas BHRF1 miRNAs are only expressed during latency program III
(when most latency-associated genes are expressed). A subset of EBV miRNAs is
induced during reactivation and some EBV miRNAs are expressed early after
de novo infection of B cells suggesting a role in the establishment of latency (Cai et al.
2006; Cosmopoulos et al. 2009; Edwards et al. 2008; Xing and Kieff 2007). These
temporal and spatial expression differences may point to cell-type-specific regulation
of host genes in EBV-associated NPCs versus lymphomas.
In 2005, four laboratories cloned miRNAs from Kaposis sarcoma-associated
herpesvirus (KSHV)-infected primary effusion lymphoma cells (PEL) and identified
a total of 12 miRNA genes giving rise to 18 mature miRNAs (Cai et al. 2005;
Grundhoff et al. 2006; Pfeffer et al. 2005; Samols et al. 2005; Umbach and Cullen
2010). All KSHV miRNAs are located within the major latency-associated region
of the genome with 10 of the 12 miRNAs organized in a cluster in the intragenic
region between v-Flip and the K12/Kaposin gene. Two additional miRNAs were
found to be located within the K12/Kaposin gene (Fig. 31.2). In PEL cells all KSHV
miRNAs are highly expressed and induction of lytic replication leads only to a
moderate increase of some KSHV miRNAs (Boss and Renne 2010; Cai and Cullen
2006; Pearce et al. 2005). PCR-based miRNA profiling showed that all KSHVinfected tumor cells of both lymphoid and endothelial origin express KSHV miRNAs,
albeit at different levels. KSHV miRNAs are also detectable early after de novo
804
infection of endothelial cells (OHara et al. 2009; Karlie Plaisance and Rolf Renne,
unpublished results). The fact that all KSHV miRNAs are expressed in both KS and
PEL suggests a role in pathogenesis and or tumorigenesis for this novel class of
latency-associated posttranscriptional regulators. Additional support for this notion
came from phylogenetic studies. Marshall et al. (2007) analyzed clinical samples
from AIDS-KS and KS patients of different geographical origin and found that most
miRNAs were highly conserved, suggesting in vivo selection for functional miRNA
genes. However, this study also identified a number of miRNA polymorphisms, and
a recent study suggests a linkage of such polymorphisms with pathogenesis (Whitby,
in press, Journal of Infectious Disease). Molecular studies revealed that KSHV
miRNA polymorphisms can affect both miRNA maturation and targeting in infected
cells (Gottwein et al. 2006; Han and Renne, unpublished results).
Identifying Targets and Functions of Viral miRNAs

Understanding the function of viral miRNAs, and more specifically their contribution
to tumorigenesis, will ultimately require comprehensive and cell-type specific target
gene identification. Conceptually, viral miRNAs can target cellular transcripts to
modulate the host environment, and/or target viral transcripts to regulate viral gene
expression. Examples of both types of regulation have been identified for at least one
viral miRNA from each human herpesvirus. This field is relatively new, and technologies for miRNA target identification are rapidly evolving from bioinformatic predictions
and individual target gene analysis to genomic and proteomic scale analysis (Chi et al.
2009; Hafner et al. 2010). However, studies over the past 6 years strongly suggest that
these novel viral posttranscriptional regulators modulate important biological processes
including oncogenesis, proliferation and cell survival, innate and adaptive immunity,
and control of latent/lytic infection, all of which clearly have implications for viral
persistence and pathogenesis (Table 31.1 and Fig. 31.3).
KSHV and EBV miRNAs Targeting Host Cellular Genes

To date, most efforts on identifying cellular genes targeted by viral miRNAs have
been focused on KSHV and EBV. The first cellular target genes for KSHV viral
miRNAs were identified by gene expression profiling of HEK 293 cells stably
expressing a miRNA cluster containing ten miRNAs (Samols et al. 2007). A total of
65 genes were downregulated in miRNA expressing cells. Among these, thromobospondin 1 (THBS1) was verified as an miRNA target using luciferase reporter
constructs containing THBS1 3UTRs. Moreover, protein levels of THBS1 were
decreased greater than tenfold in KSHV miRNA-expressing cells. THBS1, a strong
tumor suppressor and anti-angiogenic factor, had previously been reported to be
downregulated in KS lesions (Taraboletti et al. 1999). The 3UTR of THBS1 contained

Table 31.1 Experimentally verified KSHV and EBV miRNA targets
Viral targets miRNA
Gene target Gene function
miR-K12-9*
RTA
Replication and
KSHV
transcriptional
activator
EBV
miR-BART2
BALF5
DNA polymerase
miR-BART22
LMP2A
Viral oncogene in NPC
miR-BART 1-5p LMP1
Viral oncogene
miR-BART16
miR-BART17-5p
Cellular targets
References
Bellare and Ganem
(2009)
Barth et al. (2008)
Lung et al. (2009)
Lo et al. (2007)
Lo et al. (2007)
Lo et al. (2007)
References
HCMV
miR-UL112-1
MICB
NK cell ligand
KSHV
miR Cluster
THBS1
EXOC6
ZNF684
CDK5RAP1
miR-K12-1
IkBa
p21
miR-K12-3
LRRC8D
NHP2L1
miR-K12-3
miR-K12-7
miR-K12-4-3p
miR-K12-5
C/EBPb
(LIP)
GEMIN8
BCLAF1
Angiogenesis inhibitor
SEC15 gene family
Zinc finger protein
Regulation of neuronal
differentiation
NF-kB inhibitor
Inducer of cell cycle
arrest
Immune cell activator
U4 snRNA nuclear
binding protein
Transcriptional
activator
Required for splicing
Proapoptotic factor
EBV
805
miR-K12-6
miR-K12-11
miR-K12-7
Rbl-2
MAF
Rb-like protein
MICB
NK cell ligand
miR-K12-11a
BACH1
Transcriptional
suppressor
miR-BHRF1-3
CXCL11
miR-BART2
MICB
Chemokine,
T-cell attractant
NK cell ligand
miR-BART3
miR-BART5
miR-BART16
IPO7
PUMA
TOMM22
Nuclear import protein

Proapoptotic factor
Mitochondrial
membrane protein
a
Shown to have seed sequence homology with human miR-155
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et al. (2007)
Samols et al. (2007)
Dolken et al. (2010)
Lei et al. (2010)
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Gottwein
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Nachmani
et al. (2009)
Choy et al. (2008)
806
nucleus
Viral
Episome
Human
Genome
Host Cellular Targets
Viral Targets
e Latent/ Lytic
Promoting
Oncogenesis
Control
BALF5
LMP1
LMP2A
RTA
(BCLAF1)
(IkBa)
(Rbl-2)
C/EBP beta
MAF
p21
Proliferation &
Cell Survival
BACH1
THBS1
BCLAF1
PUMA
Innate & Adaptive

Immunity
MICB
CXCL11
IkBa
Host
miRNA
Function
Fig. 31.3 Themes of viral miRNA gene regulation. Virus infected cells can produce miRNAs which
target both viral and cellular genes. (a) Inhibition of genes that may promote viral oncogenesis.
(b) Targets involved in promoting proliferation and cell survival. (c) Gene targets important in
innate and adaptive immunity. (d) Modulating host cell miRNA expression and function. Experiments
in MCMV show that viral miRNA synthesis completely overtakes host cell miRNA production
early after infection (Dolken et al. 2007). Viral miRNAs may hijack Drosha processing or RISC
loading leading to impaired function of host miRNA, leading to global de-repression of cellular
miRNA targets. (e) The maintenance of latency is a common function of viral miRNAs that target
immediate-early or early lytic viral genes. BCLAF1, IkBa, and Rbl-2 are host genes that contribute
to the latent/lytic switch by either sensitizing cells to reactivate or functioning to maintain latency.
Known viral miRNAs and target genes, including all references, are listed in Table 31.1
seed sequence binding sites for multiple KSHV miRNAs, suggesting that viral
miRNAs in clusters coordinately regulate host cellular target genes. Additionally,
osteopontin (SPP1) and PRG1, genes involved in cell-mediated immunity and
apoptosis, were identified as KSHV miRNA targets by similar techniques. These
initial findings demonstrated that KSHV-encoded miRNAs contribute to viral
pathogenesis by promoting angiogenesis (a hallmark of KS tumors) and by inhibiting
cellular immunity and apoptosis (Samols et al. 2007).
Using an elegant tandem-array approach, The Ganem group identified several
KSHV miRNA targets that were either induced by miRNA knockdown in latently
infected PEL cells or inhibited in uninfected B cells ectopically expressing KSHV
miRNAs (Ziegelbauer et al. 2009). This analysis revealed that three KSHV miRNAs
807
(miR-K12-5, K12-9, and miR-K12-10b), target Bcl-2-associated factor (BCLAF1).

BCLAF1 is a transcriptional repressor that can promote apoptosis. However, BCLAF1
expression in latently infected PEL cells can also inhibit KSHV replication.
Antagomir-based inhibition of KSHV miRNAs miR-K12-5, K12-9 and miR-K12-10b
resulted in sensitizing latently infected endothelial cells for lytic reactivation.
These data suggest that KSHV miRNAs can contribute to latency control by targeting
both the viral RTA gene (as discussed below) and cellular genes such as BCLAF1
(Ziegelbauer et al. 2009).
Viral miRNAs can also mimic cellular miRNA function. Two groups showed
that miR-K12-11 and human miR-155 shared complete seed sequence identity
(Gottwein et al. 2007; Skalsky et al. 2007). Mir-155 is aberrantly expressed in many
human malignancies and when overexpressed in mice causes lymphoproliferative
disease (Garzon and Croce 2008). This led to the question of whether miR-K12-11
and miR-155 target a common set of genes. Prediction programs revealed that the
BACH1 gene contains four binding sites for both miR-K12-11 and miR-155 within
its 3UTR. BACH1 is a transcriptional repressor affecting expression of hemeoxygenase 1 (HMOX1), a protein that promotes cell survival and proliferation
(Igarashi and Sun 2006). Luciferase reporter assays confirmed miRNA regulation of
BACH1 and BACH1 protein levels were decreased in miR-K12-11 and miR-155
expressing cells. Gene expression profiling also revealed that miR-K12-11 and
miR-155 can regulate a common set of genes (Gottwein et al. 2007; Skalsky et al.
2007). In addition, Qin et al. (2010a) showed that miR-K12-11-dependent regulation of BACH-1 not only affected oxidative stress responses but also led to increased
expression of xCT, an amino-acid transporter, which has previously been shown to
function as a fusion receptor for KSHV.
Qin et al. (2010b) also showed that KSHV-encoded miRNAs induce IL-6 and
IL-10 secretion in murine macrophages and human myelomonocytic cells. C/EBPb,
a known regulator of IL-6 and IL-10 transcription, was shown to be targeted by the
KSHV miRNA cluster. Specifically, miR-K12-3 and miR-K12-7 inhibited the LIP
isoform of C/EBPb, which functions as transcriptional suppressor. These data suggest
that KSHV-encoded miRNAs directly regulate cytokine secretion of latently infected
cells (Qin et al. 2010b).
KSHV infected endothelial cells undergo transcriptional reprogramming, expressing
markers for both lymphatic and blood endothelial cells (Carroll et al. 2004; Wang
et al. 2004). Hansen et al. have recently demonstrated that KSHV miRNAs regulate
this reprogramming by targeting the cellular transcription factor musculoaponeurotic
fibrosarcoma oncogene homolog (MAF). MiR-K12-6 and miR-K12-11 together target
the 3UTR of MAF, thereby inducing endothelial cell differentiation, and possibly
contribute to KSHV oncogenesis (Hansen et al. 2010). Interestingly, miR-K12-11,
the ortholog of miR-155 is also involved in B cell differentiation and proliferation
in vivo (Boss and Renne, unpublished).
Two recent studies addressed the role of KSHV miRNA within the context of the
viral genome by generating a recombinant KSHV miRNA virus (Lei et al. 2010;
Lu et al. 2010). Lei et al. reported inhibition of NF-kB in 293 cells infected with the
mutant virus which was accompanied by a moderate increase in lytic replication.
808
IkBa, the NF-kB repressor, was subsequently shown to be targeted by miR-K12-1

(Lei et al. 2010). Gottwein and colleagues reported another target for miR-K12-1,
which like NF-kB is crucial for cell survival and proliferation. Several lines of
evidence demonstrate that miR-K12-1 directly targets p21, a key inducer of cell
cycle arrest and tumor suppressor (Gottwein and Cullen 2010).
Using a similar miRNA knockout virus, Lu et al. also observed a moderate
increase in lytic replication but identified entirely different mechanisms. In addition
to a moderate inhibition of RTA by miR-K12-5 Lu et al. observed a drastic genome
wide inhibition of DNA methylation after deleting the miRNA cluster. Retinoblastoma
(Rb)-like protein 2 (Rbl-2), a potent inhibitor of DNA (cytosine-5-)-methyltransferases (DnmT1, 3a, and 3b) was shown to be targeted by several KSHV miRNAs.
These data showed for the first time a role of viral miRNAs in epigenetic regulation
of latency (Lu et al. 2010).
The latest technique to identify miRNA targets utilizes cross-linking and immunoprecipitation of RISCs followed by microarray or high-throughput sequencing
analysis of the RISC-bound miRNA targets (Chi et al. 2009; Hafner et al. 2010).
Using this technique, Dolken et al. (2010) were able to confirm a significant number
of the above discussed targets and in addition determined six novel targets of KSHV
miRNAs and two targets of EBV miRNAs. KSHV miR-K12-3 was shown to target
LRRC8D, thought to be involved in proliferation and activation of lymphocytes and
macrophages, and NHP2L1, a nuclear protein that binds to U4 snRNA. MiR-K12-4-3p
targets GEMIN8, which is required for spliceosomal snRNP assembly in the cytoplasm
and pre-mRNA splicing in the nucleus. The KSHV miR-cluster was also found to
target EXOC6, ZNF684, and CDK5RAP1; however, no functional studies have
been presented on these novel target genes (Dolken et al. 2010).
For the more than 25 EBV-encoded miRNAs, few cellular targets have been
identified. Dolken et al. showed that EBV miR-BART16 target TOMM22 and miRBART3 targets IP07, both involved in cellular transport processes. However, their
role in EBV biology has not been determined (Dolken et al. 2010). Previously, EBV
miRNAs were shown to regulate PUMA and CXCL11. PUMA modulates apoptosis
through p53 upregulation and is targeted by miR-BART5 (Choy et al. 2008).
CXCL11 is an IFN-inducible T-cell chemoattractant and is targeted by miRBHRF1-3, thereby inhibiting T-cell recognition (Xia et al. 2008). In this context,
EBV miR-BART2 also suppresses the major histocompatibility complex 1-related
chain B (MICB) (Nachmani et al. 2009). The first viral miRNA targeting a host gene
involved in immune response was reported by Stern-Ginossar et al. (2007) who
identified MICB targeted by miR-UL112-1 in HCMV-infected cells. MICB is a
stressed-induced ligand that is essential for natural killer (NK) cell recognition of
virus-infected cells. Using elegant genetics approaches, they demonstrated that
targeting MICB significantly reduced NK cell killing of HCMV infected cells
(Stern-Ginossar et al. 2007). Later, two more g-herpesvirus-encoded miRNAs were
found to target MICB. Nachmani et al. (2009) showed that KSHV miR-K12-7 and
EBV miR-BART2 both directly target MICB mRNA and reduce its expression by
utilizing three different sites within the MICB 3UTR. Interestingly, MICB is also
targeted by the HCMV encoded UL16 protein and the KSHV MIR3 and MIR5 proteins
809
(Areste and Blackbourn 2009).This coevolution suggests that targeting MICB to

inhibit NK cell function is a critical step for herpesviral persistence in vivo.
Viral miRNAs Targeting Viral Genes

MiR-BART2 is encoded antisense to BALF5, a transcript encoding the EBV DNA
polymerase, and was initially hypothesized to function as siRNA (Pfeffer et al. 2005).
Barth et al. (2008) later confirmed that miR-BART2 does cleave BALF5 in a siRNAlike manner in EBV-infected cells. The first example of a viral gene targeted by viral
miRNAs through seed sequence binding was latent membrane protein 1 (LMP1) (Lo
et al. 2007). LMP1 is a viral oncoprotein required for immortalization of human
B-cells. However, overexpression of LMP1 leads to induction of apoptosis (Izumi and
Kieff 1997). The 3UTR of latent LMP1 contains potential binding sites for several
BART miRNAs. The BART cluster 1 miRNAs miR-BART16, miR-BART17-5p, and
miR-BART1-5p were shown to decrease LMP1 protein expression. MiRNA-dependent
targeting of LMP1 in latently infected cells provides a mechanism of fine tuning a
balance between proliferation and apoptosis (Lo et al. 2007). Recently, novel miRNAs
including miR-BART22 have been identified in EBV-associated NPCs. MiR-BART22,
which is expressed at high copy numbers in NPCs was shown to target the latent membrane protein 2A (LMP2A) and reduce its expression in NPC-derived cell lines, which
may facilitate NPC carcinogenesis (Lung et al. 2009).
To investigate whether KSHV miRNAs target KSHV immediate early transactivators, Bellare and colleagues utilized luciferase reporter assays in which the 3UTR of
the KSHV reactivation and transcriptional activator gene (RTA) was cotransfected with
individual KSHV miRNA mimics. Further analysis of miRNA knockdown using
antagomirs (sequence-specific miRNA inhibitors) in latently infected PEL cells showed
that miR-K12-9* modulates RTA expression at the protein level (Bellare and Ganem
2009). Lu et al. also found that KSHV miR-K12-5 can inhibit RTA expression.
However, this may reflect an indirect effect rather than direct targeting, since the 3UTR
of RTA does not contain a favorable miR-K12-5 seed sequence (Lu et al. 2010).
In summary, while the mechanisms by which herpesvirus-encoded miRNA function
may vary (miRNA-like versus siRNA-like for antisense control), it becomes clear
that all herpesviruses have evolved to utilize miRNAs as a means to regulate both
lytic and latent gene expression. Future work using appropriate in vivo models will
reveal how important these regulatory pathways are in the context of viral latency
and persistence.
Open Questions and Future Perspectives

The noncoding small RNAs and microRNAs expressed by EBV and KSHV are
likely to play important roles in the pathogenesis of both viruses for the reasons
810
outlined above. These RNAs are expressed in large amounts, are evolutionarily
conserved in many cases, and are expressed in a wide variety of cell types and
human cancers. The dispensability of the noncoding small RNAs of EBV and primate
rhadinoviruses for in vitro transformation despite these characteristics strongly suggests
important roles in either tumorigenesis or infected cell survival or immune evasion
in vivo. The molecular mechanisms of action of these small RNAs remain elusive
despite intensive investigation. The likelihood of EBERs playing a role in cell
proliferation and apoptosis resistance makes identification of their cellular target
pathways potentially important for devising novel therapeutic approaches for EBVrelated malignancies.
Another important question is whether KSHV and EBV miRNAs directly contribute
to viral tumorigenesis. A strong candidate for such a role in KSHV is miR-K12-11,
a functional mimic of human miR-155, which was shown to have oncogene activity
(Garzon and Croce 2008). This mechanism is conserved among viruses, since
MDV-1, an avian tumorigenic virus, also encodes a miR-155 homolog (Morgan et al.
2008; Zhao et al. 2009) and EBV strongly upregulates miR-155 (Cameron et al. 2008).
Very recently, Linnstaedt et al. (2010) have demonstrated that inhibition of miR-155
during immortalization can prevent the outgrowth of LCLs and even reduce proliferation of some EBV+ cell lines. Together, these data strongly suggest that
modulation of this central pathway may be a central mechanism in g-herpesviral
lymphomagenesis. Additionally, it has recently been discovered that miRNAs and
EBERs can be secreted by exosomes; hence, the regulatory effects of viral-encoded
small RNAs may not be limited to infected cells, but may also affect the tumor
microenvironment (Iwakiri et al. 2009; Pegtel et al. 2010; Valadi et al. 2007). In addition
to targeting pathways involved in apoptosis, proliferation, angiogenesis, and differentiation, KSHV and EBV miRNAs modulate innate immunity by targeting MICB
(Stern-Ginossar et al. 2007) and the control of viral latent and lytic replication by
targeting viral genes. While these additional functions are not directly related to
tumorigenesis, they contribute to a hallmark of herpesvirus infection, which is lifelong persistence, a prerequisite of viral oncogenesis.
Acknowledgments We thank Karlie Plaisance-Bonstaff for reading and editing the manuscript
and generating the figures.
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Chapter 32
Viral Malignancies in HIV-Associated

Immune Deficiency
Pankaj Kumar, Veenu Minhas, and Charles Wood
Introduction
There has been a large volume of experimental evidence that supports the idea that
immune system is capable of suppressing the growth of tumors. In fact, this concept
of immune surveillance mechanism against cancer was proposed a long time ago by
Paul Ehrlich. Clinical data that supports the importance of immune surveillance
as a mechanism for tumor prevention has been generated largely by two sets of
epidemiologic studies. First, studies that reported that long-term usage of immunosuppressive drugs following organ transplant is associated with higher risk for
developing malignant tumors. Second, with the advent of human immunodeficiency
virus (HIV) epidemic in the early 1980s, it was reported that HIV-positive patients
had an increased risk for developing certain malignancies. As early as 1982, the US
Center for Disease Control and Prevention (CDC) included Kaposi Sarcoma (KS)
and primary central nervous system lymphoma (PCNSL) as AIDS defining malignancies (CDC 1982; Anonymous 1992). Non-Hodgkin lymphoma (NHL) and invasive
cervical carcinoma were subsequently added as AIDS-defining conditions in 1987
(CDC 1987) and 1992 respectively (Anonymous 1992). The development of these
AIDS defining malignancies was considered sufficient to signify the progression of
HIV-infected patients to AIDS.
Interestingly, malignancies found in organ transplant patients have a common unifying feature with malignancies found in the HIV patients; both generally have a
viral etiology. Co-infection with other oncogenic viruses is considered a major risk
factor associated with the development of malignancies in HIV-infected individuals.
Although it is being debated whether HIV acts directly as an oncogenic agent or not,
it is generally accepted that HIV provides a dysfunctional immunologic background
P. Kumar V. Minhas C. Wood (*)

Nebraska Center for Virology, School of Biological Sciences, University of Nebraska-Lincoln,
4240 Fair Street, Morrison Center, Lincoln, NE 68583, USA
e-mail: cwood1@unl.edu
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for other oncogenic viruses to escape immune surveillance and induce tumors. Other
risk factors that are likely to play a role in the development of cancers in HIV-infected
individuals include the duration of HIV immunosuppression, longevity, especially in
those patients who are undergoing anti-retroviral therapy and therefore survive longer without the development of AIDS, family history of cancers, lifestyle, such as
smoking and exposure to sunlight. In addition to AIDS-defining malignancies listed
above, HIV-infected individuals have a higher incidence of non-AIDS defining tumors
compared to the general population. These non-AIDS defining tumors include
Hodgkins lymphoma, lung cancer, hepatocellular carcinoma, hematopoietic malignancies, cutaneous cancer, anal cancer, and various sarcomas other than Kaposis
sarcoma. The focus of this chapter is to give an overview on major malignancies
associated with viral etiology in the setting of HIV-associated immune deficiency.
AIDS-Defining and AIDS-Associated Malignancies

AIDS-Associated Lymphomas
AIDS-Associated Lymphomas (ARLs) are usually derived from B-cells as demonstrated by B-cell specific markers (such as CD19 and CD20) and rearrangement of
immunoglobulin (Ig) heavy-chain gene (Carbone et al. 2001). They present an aggressive clinical course with widespread involvement of one or more extranodal sites.
ARL is generally a late event in the course of HIV infection and prognosis is associated with the stage or extent of the disease, bone marrow involvement, age, severity of
the underlying immunodeficiency (measured by CD4 lymphocyte count) and the history of opportunistic infections prior to AIDS diagnosis (Levine et al. 2000). The
pathogenesis of ARL is probably multifactorial but ARL are very frequently associated with either Epstein-Barr virus (EBV) or human herpesvirus 8 (HHV-8) or both.
These viruses are closely related members of the gamma herpes virus family.
Abnormally high levels of IL-6 and IL-10 have been demonstrated in ARL. Both
cytokines function as autocrine growth factors. It is noteworthy that all three viruses
namely HIV, HHV8, and EBV have the ability to deregulate the cytokine network.
The World Health Organization (WHO) has classified ARLs into three categories
(Raphael et al. 2001); (a) lymphomas that also occur in immunocompetent patients and
includes Burkitts lymphoma, diffuse large B-cell lymphomas (DLBCL) and Hodgkins
lymphoma (HL); (b) lymphomas that occur more specifically in HIV+ patients such as
primary effusion lymphoma (PEL) and plasmablastic lymphoma of the oral cavity and; (c)
lymphomas that occur in other immunodeficiency states such as polymorphic B-cell lymphoma or post-transplant lymphoproliferative disorder (PTLD)- like B-cell lymphoma.
Burkitts Lymphoma
Burkitts Lymphoma (BL) was first described as an obscure tumor in African children
by Denis Burkitt almost 50 years ago (Burkitt 1958). Since its first description, BL
32 Viral Malignancies in HIV-Associated Immune Deficiency
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has generated a lot of scientific interest because of its unique epidemiological

features and has led to seminal discoveries including the discovery of EpsteinBarr
virus (EBV), the first virus to be associated with a human cancer. BL is genetically
characterized by a chromosomal translocation of the Myc gene to one of the three
immunoglobulin loci leading to high level of c-Myc expression (Ballerini et al. 1993;
Pelicci et al. 1986). Myc family of proteins are important transcriptional factors
that globally regulate multiple cellular processes including proliferation, differentiation, and apoptosis.
Three epidemiologically distinct forms are now recognized that differ in their
geographic distribution, sites of involvement, and frequency of association with
EBV (Diebold et al. 2001). The endemic form is present in areas of equatorial Africa
and Papua New Guinea where malaria is very common. It occurs mainly in children
and clinically involves the jaw or other facial bone although other organs can also
be affected. Nearly all cases of endemic BL are positive for EBV. Elsewhere, BL
occurs as a sporadic form affecting mostly the young adults. It usually presents as
an abdominal mass in the ileocecal region but can involve other organs such as the
endemic form. EBV is associated with about 20% of the sporadic cases. HIVassociated BL typically involves lymph nodes and bone marrow and has a high
association with EBV (~3040%) (Brady et al. 2007).
Histological picture of BL consists of homogenous infiltration of medium-sized
cells with round nuclei containing several nucleoli and very little basophilic cytoplasm.
Mixed within the tumor cells are numerous benign macrophages with engulfed cell
debris which confer a histologic pattern referred to as starry sky appearance. BL
cells express pan-B markers (CD19, CD20 and CD22), are positive for surface IgM
and Ig light chain and BCL6, but are negative for BCL2 and Terminal deoxynucleotidyl transferase (TdT), and have a high Ki67 proliferation index (Bellan et al.
2010). Microarray-based gene expression profiling studies have been employed to
establish the molecular signature of BL that would discern BL from diffuse large
B-cell lymphomas (DLBCL) (Hummel et al. 2006). Distinguishing BL from DLBCL
is critical as both tumors require different treatment strategies. BL is rapidly fatal if
untreated, but is curable with intensive chemotherapy that includes high doses of
cyclophosphamide and antimetabolites, as well as intrathecal chemotherapy. On the
other hand, treatment of DLBCL employs a less aggressive approach which typically
consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP),
with the monoclonal anti-B-cell antibody, rituximab. Accurate diagnosis helps the
clinicians to follow appropriate therapeutic regimen for treatment.
Although considerable progress has been made in understanding the role of EBV
in the transformation process, deciphering its exact role in the pathogenesis of BL
is still a work in progress. Apart from the endemic form where the virus can be
detected in virtually all cases; its association with other forms remains variable.
This observation gives a strong indication that EBV may be a cofactor and not the
initiating factor for tumorigenesis. Southern blot analysis of both: the c-Myc translocation and the fused terminal repeats of the EBV genome shows that the tumor
cells are monoclonal and probably result from expansion of a single infected
progenitor cell (Raab-Traub and Flynn 1986). Monoclonality of the tumors presents
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two possible scenarios; (1) EBV infection facilitates tumor initiation and argues for
EBV being the etiologic agent or; (2) EBV infection merely facilitates the malignant
progression by supporting unrestricted clonal expansion of B-cells that harbor a
Myc/Ig translocation and EBV clonality is a consequence of selective growth advantage conferred by viral episomes. The second scenario is particularly interesting as it
may explain that EBVs role can be replaced by other factors in tumors where EBV
is not detected, such as in sporadic BL (Moody et al. 2003).
Primary infection with EBV can have diverse outcomes ranging from asymptomatic infection, self-resolving infectious mononucleosis, to the development of
EBV-associated malignancies including Burkitts lymphoma, Hodgkins disease,
nasopharyngeal carcinomas, and B cell lymphomas in transplant recipients or AIDS
patients. EBV establishes lifelong latency in memory B cells by mimicking the
normal behavior of B-cells in response to an antigen. The current accepted model
for the establishment of EBV latency suggests that latent proteins expressed by
EBV during primary infection provide signals to the infected B cell to become
proliferating B-lymphoblasts (Young and Rickinson 2004). These activated lymphoblasts later differentiate into resting memory cells through a mechanism analogous to the germinal center reaction. Germinal centers are the sites within lymph
nodes where antigen-activated B-lymphocytes undergo isotype switching and
somatic mutation in their Ig genes to become memory B-cell. Isotype switching
enables the B cell to replace the class of antibody expressed while somatic hypermutation increases the affinity of B cell receptor for an antigen. The latently infected
memory cells express little or no genetic information and are therefore not recognized by the immune response. EBV displays distinct latency programs in various
malignancies or EBV-derived cell lines which are defined by the specificity of latent
gene expression (Young and Rickinson 2004). EBV positive cases of BL have a
highly restricted pattern of latent gene expression, only expressing EBNA-1 and
EBV-encoded small non-polyadenylated RNAs or EBERs (called latency-1).
EBNA-1 plays a crucial role in the maintenance and replication of the viral genome
(Rawlins et al. 1985) while EBERs possess anti-apoptotic activity and have been
shown to promote the tumorigenic phenotype (Nanbo et al. 2002). However, recent
studies report that some endemic cases of BL display alternate patterns of viral
latency characterized by the expression of EBNA3 proteins in addition to EBNA-1
and EBERs (Kelly et al. 2006). It was suggested that tumor evolution ultimately
selects these different forms of EBV latency that are compatible with high levels of
pro-apoptotic c-Myc expression provides a more detailed discussion on EBV
encoded latency genes and their contribution to B-cell transformation.
Diffuse Large B-Cell Lymphomas

Diffuse Large B-Cell Lymphomas (DLBCL) is one of the most common lymphomas
among ARLs accounting for about 70% cases. DLBCL are a heterogeneous group
of non-Hodgkin lymphomas that typically present as a nodal or extranodal mass
with fast tumor growth. Tumor cells are large with a nuclear size exceeding that of
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normal lymphocyte and display diffuse growth pattern that effaces the normal
lymph node architecture. Based on their histologic features, DLBCL are classified
into various subtypes that include centroblastic, immunoblastic, T-cell/histiocyterich, and anaplastic variants (Raphael et al. 2001). AIDS-related DLBCL is reported
to occur in younger patients and display immunoblastic morphology more often
than non-AIDS cases (Madan et al. 2006).
Certain subtypes of DLBCL defy clear-cut classification based on histopathology
reflecting the underlying clinical heterogeneity. In recent years, gene expression
profiling (GEP) studies have contributed significantly toward classifying tumors
based on differential patterns of gene expression. In a seminal study, Alizadeh et al.
classified DLBCL into two major molecular subtypes that seem to have derived
from distinct stages of B-cell differentiation: the germinal center B-cell (GCB) like
and the activated B-cell (ABC) like (Alizadeh et al. 2000). The gene expression
profile of GCB-DLBCL resembles to that of normal germinal center B-cells,
whereas the gene expression profile of ABC-DLBCL shares similarity to mitogenactivated B-cells. The genes overexpressed in GCB-DLBCL include GC markers
such as CD10, BCL-6, LMO2, A-MYB, and OGG1 while representative genes in
the ABC-DLBCL include IRF4/MUM1, FLIP, cyclinD2, FoX-P1, and BCL-2.
Interestingly, gene expression profile for GC or ABC subtypes of DLBCL does not
correlate with any of the previously described histopathological subtype. EBV has
been found in both subtypes of DLBCL, although less frequently in the GC subtype
(Chadburn et al. 2009). These distinct gene expression profiles have a prognostic
implication with some studies suggesting that patients of GCB subtype have a
significantly better overall survival than those of ABC-DLBCL (Alizadeh et al.
2000; Rosenwald et al. 2002).
Since AIDS-DLBCL have an aggressive course and poor clinical outcome
compared to DLBCL, few studies have sought to identify the differentially expressed
genes in AIDS-DLBCL versus DLBCL. Patrone et al. reported that gene expression
patterns in AIDS-related DLBCL was very similar to non-AIDS related DLBCL
with the exception of TCL1 (T-cell leukemia-1), a proto-oncogene that was highly
expressed in AIDS-DLBCL (Patrone et al. 2003). However, findings from a recent
report from Madan et al. suggest otherwise. They performed hierarchical cluster
analysis using scores of immunohistochemical stains for GC differentiation and
ABC markers on DLBCL from HIV-positive and HIV-negative patients and found
that the immunophenotypic profile of AIDS-related DLBCL was different as it
overlapped between the immunophenotypic profile of GC and ABC subtype of nonAIDS DLBCL suggesting a unique pathophysiology (Madan et al. 2006).
Biological mechanisms underlying DLBCL pathogenesis are complex and probably
involve multiple factors such as chronic antigen stimulation, immunosuppression,
cytokine deregulation, and several genetic alterations. Using comparative genomic
hybridization (CGH), several groups have identified distinct genetic alterations in
different subgroups of DLBCL (Tagawa et al. 2005; Bea et al. 2005). EBV has been
found to be associated more commonly with the immunoblastic variants (~90%) of
AIDS related DLBCL than the centroblastic variants (~30%). EBV-encoded transforming protein LMP-1 is found in 90% of the immunoblastic cases but is not usually
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detected in the centroblastic subtype. LMP1 is an integral membrane protein that

resembles constitutively active CD40, a glycoprotein belonging to tumor necrosis
factor receptor (TNFR) (Uchida et al. 1999). LMP-1 has been found to be transforming
in rodent fibroblasts (Wang et al. 1985). Transgenic mice expressing LMP-1 in
B-cells have increased propensity to develop B-cell lymphomas (Thornburg et al.
2006). LMP-1 activates a number of signaling pathways including NFkB, JNK, and
p38 pathways in a ligand-independent manner. LMP-1 also inhibits apoptosis in
latently EBV-infected cells by upregulating the expression of antiapoptotic proteins
BCL-2, BFL-1, and A20 while inhibiting the expression of pro-apoptotic BAX
protein (Soni et al. 2007).
Immunophenotyping using markers, BCL-6 and syn-1 have been used to define
the histogenesis of AIDS-related DLBCL. BCL-6 is a zinc finger transcription repressor
that is selectively expressed in B-cells located within GC while syn-1 is an integral
transmembrane glycoprotein and is a marker of post-GC B-cell differentiation.
The differential expression of BCL-6 and syn-1 segregate AIDS-related DLBCL into
two major phenotypic subsets: BCL-6+/syn-1 and BCL-6/syn-1+. These different
patterns of expression basically correspond to two physiologic stages of B-cell
development with B-cells within the GC displaying BCL-6+/syn-1 phenotype while
terminally differentiated post-GC B-cells showing BCL-6/syn-1+ phenotype.
Interestingly, among EBV-infected AIDS-related DLBCL, the expression of EBVencoded LMP-1 antigen can be detected only in BCL-6/syn-1+ phenotype which
correspond to post GC state and these tumors display immunoblastic morphology.
These findings suggested that the post-GC phenotype is the sole condition permissive
for LMP-1 expression in the context of AIDS-NHL or alternately, that LMP-1 expression forces post-GC maturation of these lymphomas (Carbone et al. 1998a).
Primary Central Nervous System Lymphomas (PCNSL) is a rare extranodal
variant of DLBCL that is associated with the advanced stages of HIV infection.
PCNSL was the most frequent brain tumor in AIDS patients till the introduction of
highly active antiretroviral therapy (HAART) in mid 1990s (Sacktor et al. 2001).
The confinement of the lymphoma within the CNS during the course of the disease
despite the fact that CNS lacks lymphatic system adds an intriguing feature to the
pathogenesis of PCNSL. It has been hypothesized that PCNSL originate from
B-cells derived from systemic lymphoid tissues that normally traffic in and out of
CNS. These B-cells are exposed to a germinal center environment outside the brain
and later, a malignant clone expressing specific adhesion molecule metastasize to
the brain (Uccelli et al. 2005). Experimental evidence seems to support this view as
mutations in BCL-6 5 noncoding regions, which are regarded as a marker for B-cell
transition through the GC, are frequently found in PCNSL (Larocca et al. 1998).
Gene expression studies demonstrate that PCNSL exhibit a distinct expression
profile compared to nodal lymphomas with an overlapping state of differentiation
that is characterized by an expression of both GCB and ABC genes (Rubenstein
et al. 2006). AIDS-related PCNSL is universally associated with EBV and frequently
express EBV-encoded LMP-1 protein. PCNSL is found in patients with very low
peripheral blood CD4 count. However, a recent study suggests that it is not the
absolute CD4+ T-cell count but the lack of EBV-specific CD4+ T-cells that account
825
for rapid progression in PCNS lymphomas in AIDS patients (Gasser et al. 2007).
Detection of high levels of EBV DNA from the cerebrospinal fluid (CSF) of
HIV-associated PCNSL has been shown to be a highly sensitive and specific marker
for the diagnosis and monitoring of the disease (De Luca et al. 1995).
Plasmablastic lymphoma of the oral cavity (PBL) is another variant of DLBCL
that is predominantly found in HIV-positive patients. It was first described in the jaws
and oral cavity of HIV-infected patients (Delecluse et al. 1997); although later it was
also reported from other extra-oral sites including gastrointestinal tract, soft tissues,
skin and heart of HIV-negative patients (Dong et al. 2005). The histopathological
picture is characterized by the presence of large round/oval cells with abundant
eosinophilic cytoplasm. The tumor cells have an eccentrically placed nucleus with a
single prominent nucleolus that is located in the center. The tumor cells have a terminally differentiated B-cell immunophenotype and are typically positive for plasma
cell markers such as CD38 and CD138. Although PBL is considered to be of differentiated B-cell lineage, they are negative for other common B-cell markers such as
CD20, CD79a, and PAX-5 (Vega et al. 2005). Monoclonal rearrangement of immunoglobulin heavy chain and positivity for post GC B-cell marker multiple myeloma-1
(MUM1)/IRF4 are indicative of their origin from post-GC (Gaidano et al. 2002).
An array-based comparative genomic hybridization of PBL indicates that these
tumors are more similar to DLBCL than the plasmacytomas (Chang et al. 2009).
While most PBL are positive for EBV, the prevalence of HHV8 remains somewhat
unresolved.
Hodgkins Lymphoma
Although not classified as an AIDS-defining malignancy, HIV-related HL is one of
the most common non-AIDS defining condition. Several studies have reported that
HIV-infected patients have a 318-fold increased risk of developing HL compared
to the general population (Biggar et al. 2006; Engels et al. 2008). The presence of
large mononucleated (Hodgkin) and multinucleated (Reed Sternberg) neoplastic
cells (HRS) within a reactive inflammatory cell background are the defining characteristic of HL. HL is unique among lymphomas because the neoplastic component
i.e., the HRS cells constitute <1% of the tumor mass (Brauninger et al. 2006). HL
tends to manifest more obvious clinical symptoms than non-Hodgkins lymphoma
(NHL) which often leads to early detection and favorable treatment.
HL is divided into two major entities that differ in their clinical and histopathologic
features: more commonly found (about 95%) classical HL (cHL) and not so frequent,
nodular lymphocyte-predominant HL (NLPHL). CHL has been classified into four
morphological subtypes: nodular sclerosis, mixed cellularity, lymphocyte rich, and
lymphocyte depleted (Stein 2001). There is considerable geographical and age
group variation within different subtypes of HD, e.g., young adults in the United
States and Europe are likely to have the nodular sclerosis variant, while mixed
cellularity or the lymphocyte-depleted subtype is more frequent in the developing
world (Cartwright and Watkins 2004). Most cases of HL in HIV patients are either
826
P. Kumar et al.
of mixed cellularity or the lymphocyte-depleted forms. In the United States, HL

follows a bimodal age distribution showing a peak at young age and second peak in
late life. Also white young adults with a history of infectious mononucleosis have a
higher incidence of HL.
The exact origin of the HRS cells has long been a controversial topic as these
cells display a phenotype that is not consistent with a defined hematopoietic cell
type. The HRS cells express CD15 and CD30 but are negative for CD45 and T-cell
markers (Schmid et al. 1991). CD30 is a membrane glycoprotein of the TNF receptor
family that is expressed in activated T and B cells. The B-cell markers, CD20,
CD79A, and B-cell-specific activator protein (BSAP) are variably expressed in a
subset of cases (Schmid et al. 1991). HRS cells show clonal immunoglobulin gene
rearrangement and thus support the idea that they represent clonal populations of
GC-derived tumor B cells. In an elegant study, Cossman et al. performed high
throughput gene expression studies from individual ReedSternberg cells isolated
from HL. This expression profile, closely resembled to that of GC B-cells and not
with dendritic cells, thus corroborated the generally held view that HRS cell is of
B-cell lineage (Cossman et al. 1999). A hallmark of HRS cells is the lack of a functional
B-cell receptor (BCR) (Marafioti et al. 2000). In a subset of cHLs, this lack of a
functional BCR has been attributed to crippling somatic mutations of the rearranged
immunoglobulin gene (Kanzler et al. 1996). However, in other cases of cHL with
functional gene rearrangements, the absence of Ig has been ascribed to downregulation
of B-cell specific transcription factors (OCT2, BOB1 and PU1) (Theil et al. 2001)
or epigenetic silencing of immunoglobulin gene transcription by promoter methylation (Ushmorov et al. 2004).
Normally, antigen-activated B-cells undergo somatic hypermutation of the BCR in
GC to generate B cells with higher affinity BCRs. These high affinity B-cells are
preferentially selected for survival and undergo clonal expansion and differentiation
into antibody-producing plasma cells. B cells in GC that lack B cell-receptor expression are eliminated by apoptosis. The HRS cells are an exception to this general rule
as they continue to proliferate in spite of having deleterious mutations which suggests
that somehow they are rescued from apoptosis by a transforming event. Several
different mechanisms have been proposed to explain how HRS cells avoid apoptosis
including genetic alterations of the bcl-2 family proteins, constitutive activation of the
NF-kB pathway and expression of EBV latent genes (Brauninger et al. 2006). Aberrant
signaling of NF-kB pathway has generated considerable interest. NF-kB family of
transcription factors plays a critical role in the regulation of apoptosis by upregulating
anti-apoptotic members of the Bcl-2 family and caspase inhibitors such as XIAP and
FLIP. NF-kB is constitutively active in HRS cells independent of the EBV status and
several factors have been found to contribute toward deregulated NF-kB pathway
(Krappmann et al. 1999). These factors include frequently found mutations that
inactivate NF-kB inhibitor kappa beta (IkB) alpha and epsilon subunits, gene amplification of c-rel, signaling through tumor necrosis factor receptors (CD30 and receptor
activator of NF-kB (RANK), and expression of LMP1 and LMP2A in EBV-positive
cases (Kuppers 2009). In addition, NF-kB-dependent upregulation of cellular
827
FLICE-inhibitory protein (c-FLIP) has been suggested to mediate resistance of HRS

cells to death receptors (CD95/TRAIL) induced death (Mathas et al. 2004).
Although EBV is not detected in all cases of HL, several lines of evidence favor the
idea that EBV has some role in the pathogenesis of HL. In the US, EBV can be frequently
detected in mixed cellularity variant of cHL but rarely in other subtypes. Strikingly, all
cases of HIV-related HLs are positive for EBV (Bellas et al. 1996). The association
between EBV and HL has been demonstrated by numerous seroepidemiological studies
in which antibody titers against viral capsid antigens (VCAs) have been consistently
found to be higher in HL cases compared to the general population (Besson et al. 2006).
The definitive proof revealing an etiologic association of EBV with HL was the detection
of the viral genome and virally encoded proteins in the HRS cells (Weiss et al. 1989).
EBV has been found to transform primary human B lymphocytes including GC B cells
that are incapable of expressing a functional immunoglobulin heavy chain due to crippling mutations introduced during somatic hypermutation (Mancao et al. 2005). HRS
cells positive for EBV display a type II form of latency with viral antigen expression
limited to EBNA1, LMP1, LMP2, as well as the EBER1, EBER2, and BamHI A transcripts (Deacon et al. 1993). The transforming potential of LMP-1 has been described
earlier. Unlike LMP-1, LMP2 is not required for transformation in B-cells. It is an integral
membrane protein with two isoforms, LMP2A and LMP2B. LMP2A can constitutively
induce several signaling cascades. The LMP2A amino-terminal cytoplasmic domain
contains an immunoreceptor tyrosine-based activation motif (ITAM) which is a conserved 6-amino acid sequence present in a number of proteins that serve as signal transducers for immunoreceptors. LMP2A functionally acts as a BCR mimic in vivo by
providing constitutive signaling required for B-cell development and survival (Mancao
and Hammerschmidt 2007). This was supported by studies using transgenic mice that
expressed LMP2A in cells of B cell lineage. The study reported that B-cells with nonfunctional BCRs that are normally eliminated by apoptosis were rescued by the expression of LMP2A (Caldwell et al. 1998). A follow-up study showed that oncogene Ras is
constitutively active in peripheral; BCR-negative B cells from LMP2A transgenic mice
(Portis and Longnecker 2004). By contrast, the function of LMP2B, a variant of LMP2A,
is still not resolved in the pathogenesis of EBV infections.
Primary Effusion Lymphoma

Primary Effusion Lymphoma (PEL) is one of the least common of the ARLs,
accounting for less than 14% of cases (Simonelli et al. 2003). PELs are almost
universally associated with KSHV infection and are found in patients with advanced
stages of AIDS. The prognosis of PEL is poor with a median survival of less than 6
months. PELs are recognized as clinical subtype of DLBCL where neoplastic cells
proliferate within major body cavities (pleural, peritoneal and pericardial), typically
without the formation of a grossly visible mass. However, a solid variant that does
not involve body cavities has also been recognized (Carbone et al. 2005). Mouse
model studies have shown some differences in the gene expression profile of both
variants with the solid tumors more likely to express adhesion molecules and structural
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P. Kumar et al.
proteins (Yanagisawa et al. 2006). In most cases, EBV coinfection is very frequent.
Although the pathogenetic role of EBV in PELs is not known, microarray studies
have shown differences in gene expression patterns between the EBV-positive and
EBV-negative cases (Fan et al. 2005).
Morphologically, PEL cells are large with abundant basophilic cytoplasm and
exhibit either immunoblastic (round nuclei with central prominent nucleoli) or
plasmablastic (eccentric nuclei with abundant cytoplasm) or anaplastic (very large
round cell with pleomorphic nuclei) features. PELs have aberrant immunophenotypic
characteristics with positive reactivity for CD45 (Leukocyte common antigen) but
reactivity for B-cell lineage markers like immunoglobulin (Ig), CD19, CD20, and
CD79a or T-cell specific markers like CD3, CD4, CD8 is absent (Nador et al. 1996).
Activation (CD30, CD38) and plasma cell markers (CD138, MUM-1/IRF4) are
typically present. PELs display rearrangements in immunoglobulin genes at the
genetic level suggesting that they are derived from the B-cell lineage (Matolcsy
et al. 1998). Gene expression profile analysis shows that PEL cells exhibit a gene
expression pattern that shares features of both immunoblasts and plasma cells, i.e.,
a plasmablastic profile (Klein et al. 2003). Nuclei of PEL cells stain positive for
KSHV LANA protein by immunohistochemistry, which is the standard assay to
establish diagnosis for KSHV infection. Although PEL cells are positive for EBV
(as detected by in situ hybridization for EBERs) in vast majority of cases, they are
negative for LMP1 expression (Horenstein et al. 1997).
The exact mechanism by which KSHV and EBV promote tumorigenesis in PEL
is not clear. KSHV is present in PEL cells as episomes and majority of the infected
cells express a latent pattern of gene expression; however, a small percentage (<3%)
of cell populations spontaneously undergo lytic reactivation (Renne et al. 1996).
PEL cell lines derived from PEL have been used extensively as a model to study
KSHV biology. Unlike KS tumor explants, PEL cell lines stably maintain viral episomes at high copy numbers (50150 copies per cell) (Ballestas et al. 1999). Among
the latently expressed genes, LANA-1, viral cyclin (v-Cyc), viral FLICE inhibitory
protein (v-FLIP), and LANA-2/viral interferon regulatory factor 3 (vIRF3) are
consistently expressed (Luppi et al. 1999). Another KSHV protein that is expressed
in a minority population of PEL tumor cells is viral interleukin-6 (vIL-6), a cytokine
that shares 24.7% amino acid identity with human IL-6. Studies have shown that
vIL-6 contributes some of the autocrine growth factor activity in PEL cell lines
suggesting that vIL-6 may help KSHV persist and spread in the human host
(Jones et al. 1999). Inoculation of NIH3T3 cells expressing vIL-6 into nude mice
resulted in the development of tumors more rapidly than the control cells. Moreover,
vIL-6-positive tumors were more vascularized than controls and expressed increasing
levels of vascular endothelial growth factor (VEGF) expression (Aoki et al. 1999).
Chromosomal translocations seen in other non-Hodgkins lymphomas like those
involving BCL2, BCL6, and Myc loci are consistently absent in PEL (Carbone et al.
1998b). A recent study investigated the genomic profiles of 28 PELs and ten PEL
cell lines using comparative genomic hybridization (CGH) and identified selectin-P
ligand (SELPLG) and coronin-1C (CORO1C) as the targets of amplification at
chromosome 12 (Luan et al. 2010). SELPLG is critical for cell migration and
chemotaxis, while CORO1C regulates actin-dependent processes and is important
829
for cell motility. Overexpression of these two genes in PELs may play an important
role in its pathogenesis.
Kaposis Sarcoma
Kaposis sarcoma (KS) is a complex, angiogenic tumor characterized by angiogenesis,
infiltration of inflammatory cells, and the proliferation of spindle-shaped cells,
which are the hallmark cells of this neoplasm. KS lesions appearing in young homosexual men in New York and San Francisco were one of the first indications of what
later became the AIDS epidemic. KS is one of the three AIDS-defining malignancies.
It was originally described by Moritz Kaposi in 1872 as an idiopathic, multipigmented sarcoma of the skin (Kaposi 1872). Since then, four clinical variants of
KS (classic, endemic, iatrogenic, and AIDS-associated), which most likely represent different manifestations of the same pathologic process, have been described.
The histological features of all these variants are indistinguishable and after the discovery of Kaposis sarcoma-associated herpesvirus (KSHV) in 1994, it became
clear that this virus is associated with all variants of KS. Since then, various diagnostic assays have been employed to understand the epidemiology of KSHV. It has
been found that seroprevalence of KSHV is uneven and mirrors the incidence of
KS. Conclusive data on transmission of KSHV are still lacking but both horizontal
(sexual and non-sexual) and vertical modes of transmission have been reported.
Saliva is emerging as one of the important routes of transmission because the virus
is shed frequently from the oropharynx (Brayfield et al. 2004; Duus et al. 2004).
Classic KS is a rare disease and the geographic location, ethnicity, age, and gender
heavily influence the incidence rate (Iscovich et al. 2000). This is the least aggressive
form of KS. It manifests as a slow or non-progressing skin condition of the limbs
and occurs primarily among older men of Mediterranean and Jewish descent
(Iscovich et al. 2000; Reynolds et al. 1965). The male to female ratio has been
reported to be 15:1 with age of onset usually greater than 50 years of age and rarely
(48%) prior to 30 years of age (Biggar et al. 1984; Dictor and Attewell 1988;
Geddes et al. 1994). The incidence of classic KS in North America, Northern
Europe, and Asia is low (Iscovich et al. 1998a, b, 2000; DiGiovanna and Safai 1981;
Guttman-Yassky et al. 2006).
Endemic KS was identified in parts of Central Africa, where the highest incidence
is observed in a belt-like path stretching from Cameroon, the former Zaire, to Uganda
and downward through Tanzania, north and eastern Zambia, Zimbabwe, and northern
South Africa (Cook-Mozaffari et al. 1998). It is recognized to be one of the most
common neoplastic diseases in equatorial Africa (Lothe and Murray 1962; Oettle
1962; Olweny 1984). The male to female ratio is similar to that of classic KS and a
mean age of onset of 48 years but the lymphadenopathic form is seen mostly in
prepubertal children with no sex predilection. Rapid visceral involvement can cause
early death and skin lesions are sparse (Friedman-Kien and Saltzman 1990).
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P. Kumar et al.
Both the iatrogenic and AIDS-associated forms are linked to overt immune
suppression. The iatrogenic form of KS was recognized in 1960s and 1970s following
advances in transplantation medicine. With the introduction of immunosuppressive
regimens used to prevent graft rejection, post-transplant patients developed
disseminated KS as early as 2 months and up to 8 years after initiation of therapy
(Friedman-Kien and Saltzman 1990). This form of KS often resolves when
immune-suppressive therapy is stopped, calling attention to immune deficiency as
an etiologic cofactor. Among transplant recipients, KS develops mostly in those
with preexisting HHV-8 infection. Seropositive recipients have been reported to
have a 2875 fold higher KS risk than seronegative recipients (Mbulaiteye et al.
2006a; Parravicini et al. 1997). This type of KS is aggressive and involves lymph
nodes, mucosa, and visceral organs in about half of the patients, sometimes even in
the absence of skin lesions (Antman and Chang 2000).
At the beginning of the AIDS epidemic, the risk of developing KS among homosexual men with HIV infection approached 50%, and approximately 40% of the
patients who were diagnosed positive for AIDS also presented with KS lesions
(Biggar and Rabkin 1996). AIDS-associated KS is a very aggressive form that often
disseminates to visceral organs (Beral et al. 1990). It has been noted that among
HHV-8/HIV coinfected males, the 10-year risk of developing KS is 3950% (Martin
et al. 1998; OBrien et al. 1999). In Africa, the ubiquitous presence of the virus in the
pre-HIV era, combined with the HIV epidemic has led to a dramatic increase in KS
incidence (Feller et al. 2010). In developed countries, AIDS-KS incidence decreased
after HAART introduction in 1996, probably due to enhanced immune reconstitution
and perhaps a direct effect of protease inhibitors as anti-angiogenic factors.
There are three distinct histologic stages of KS; the patch, plaque, and nodular
forms. Though KS is most commonly localized to the skin, organ involvement can
occur and is common in AIDS-associated KS (Wong and Damania 2005). KS is not
a conventional neoplasm. In KS lesions, proliferation, inflammation, and angiogenesis
seem to occur in parallel. This is markedly different from other cancers where
proliferation precedes inflammation and neoangiogenesis (Ganem 2010). Also, KS
lesions are comprised of several cell types including spindle cells, infiltrating
inflammatory cells as well as extravasated erythrocytes. The percentage of spindle
cells in KS lesions increases with the stage of the disease. Most researchers believe
that the spindle cells originate from endothelial lineage as they express many markers
of the endothelial lineage including CD31 and CD34; however, several reports
show that spindle cells also express markers for smooth muscle cells, macrophages,
and dendritic cells (Huang et al. 1993; Nickoloff and Griffiths 1989; Weich et al.
1991). A report published by Dupin et al. (1999) showed clearly that VEGFR-3,
which is restricted to lymphatic endothelium of adult tissues, was extensively
expressed in early KS. Additionally, spindle cells cannot be stained for VW/PalE
and eNOS (specific markers for blood vessels). Together, these results suggested
that spindle cells are lymphatic in origin. Recent large-scale gene expression studies
have revealed that KSHV infection induces dramatic reprogramming of the endothelial
cell transcriptome with induction of lymphatic lineage-specific genes, including
831
PROX1, a master regulator of lymphatic development; and downregulation of blood

vascular genes (Carroll et al. 2004; Hong et al. 2004; Wang et al. 2004).
Like other herpes viruses, KSHV replication alternates between two phases:
latent and lytic. The latent phase is characterized by a restricted pattern of viral gene
expression that facilitates the evasion of immune surveillance. In the late stages of KS,
essentially all spindle cells are latently infected by KSHV and express latent genes.
The latency locus is comprised of three open reading frames (ORFs) which includes
latency-associated nuclear antigen (LANA), viral cyclin (v-cyclin), and viral FLICE
inhibitory protein (v-FLIP), all controlled by a single promoter. LANA is a chromatin-interacting transcriptional factor that has been found to be essential for viral
episome maintenance in proliferating cells. It tethers the viral genome to the host
chromosomes assuring their segregation to daughter cells during mitosis. LANA
modulates the cellular transcription program by altering the functions of various
transcription factors. It can also inhibit tumor suppressor genes such as Rb, p53, and
VHL leading to impaired apoptosis and activation of genes involved in angiogenesis, cell proliferation, and survival. v-Cyclin, a viral homolog of the cellular cyclin
D associates with cyclin-dependent kinases (CDKs) and promotes cell-cycle progression by phosphorylating specific target proteins such as pRb and Histone H1
(Godden-Kent et al. 1997). v-FLIP is a potent anti-apoptotic effector due to its ability to activate NF-kB and therefore, has been associated with cell survival, morphologic changes, and inflammatory activation. As mentioned earlier, majority of
the tumor cells in KS are latently infected; however, a small number of KS tumor
cells also undergo lytic infection. The lytic phase drives the replication cycle and
majority of viral genes are expressed in this phase. This phase mainly allows for the
spread of the virus in the infected individual. Since the cells with lytic viral replication will ultimately die, it was safely assumed that lytic genes are not likely to play
a significant role in oncogenesis. However, emerging evidence from several laboratories supports a key role for many of these lytic proteins which have key regulatory
functions. Among the lytic genes, the G-protein-coupled receptor (vGPCR) is considered an important candidate responsible for the initiation of KS. vGPCR is a
constitutively active member of the family of CXC chemokine G-protein-linked
receptors and its expression has been found sufficient to induce cell transformation
and VEGF-mediated angiogenesis in NIH3T3 cells (Bais et al. 1998). Transgenic
mice expressing vGPCR within hematopoietic cells develop angioproliferative
lesions in multiple organs that morphologically resemble KS lesions (Yang et al.
2000). Another lytic gene that may contribute to KSHV tumorigenesis is viral
homologue of human IL6 (vIL6) which has been described earlier.
Immune suppression is a major force in the development of KS as observed
during HIV infection and in transplant patients. But the mechanism associated with
this phenomenon is still being debated. HIV-associated KS patients present a more
aggressive clinical course than that of other immunosuppressed patients, suggesting
that HIV contributes more than just providing the immune-deficient state. Several
studies support a role of HIV-encoded tat (Trans Activating Factor) protein as a
cofactor in the pathogenesis of AIDS-associated KS (Aoki and Tosato 2004; Ensoli
et al. 1990). HIV tat is a small polypeptide comprising of 86101 amino acids that
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P. Kumar et al.
functions as a potent transactivator (Romani et al. 2010). Tat has been shown to
induce the expression of several cytokines and angiogenic factors including IL-6,
IL-10, and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and 2),
which play an important role in KS progression. Tat is released from HIV-1 infected
cells and Tat-containing supernatants promote the growth of KS infected cells (Ensoli
et al. 1990). Overexpression of tat gene in transgenic mice results in the formation of
skin lesions that closely resemble KS (Vogel et al. 1988). Tat can induce KSHV
replication in lymphoma cells that are infected with KSHV (Harrington et al. 1997)
and recent studies demonstrate that Tat activates KSHV lytic replication in part by
modulating Janus kinase (JAK)/signal transducer and activator of transcription
(STAT) signaling (Zeng et al. 2007). Tat has also been implicated in promoting
KSHV transmission by enhancing the entry of KSHV into endothelial and other cells
(Aoki and Tosato 2004). These studies highlight the importance of cross talk between
HIV-1 and KSHV in the pathogenesis of KS-associated malignancies.
Cervical Cancer
Cervical cancer is the second most common cancer among women worldwide, with
an estimated 529,409 new cases and 274,883 deaths in 2008 (IARC, GLOBOCAN
2008). Cervical cancer kills more women in the developing world than any other
cancer. More than 80% of these cases occur in developing countries with highest
incidence rates recorded in Sub-Saharan-Africa. In developed countries, the incidence
and mortality rates due to cervical cancer have been declining over the years due to
widespread screening and intervention. Over the past 10 years, it has been clearly
established that persistent infections with oncogenic human papilloma virus (HPV)
types is causally associated with almost all cases of cervical cancer. This association
is supported by epidemiological studies and the detection of viral DNA from nearly
all cases of cervical cancers worldwide (Bosch et al. 2002). However, HPV is not a
sufficient cause of cervical cancer; other cofactors including smoking, multiple pregnancies, and immunosuppression may also contribute to the development of cervical
cancer (Baseman and Koutsky 2005). Sexual intercourse is the primary route of transmission for HPV and sexual promiscuity is the most important risk factor associated
with acquiring genital HPV infections (Kataja et al. 1993). HPV is most common in
young women in their late teens and early 20s. Dunne et al. reported that in United
States, the prevalence of HPV infection in women increases from 14 years through 24
years, and then decreases at older ages (Dunne et al. 2007).
HPV is a member of the Papillomaviridae family of non-enveloped DNA viruses.
These viruses are associated with a variety of cutaneous and mucosal conditions
ranging from benign warts to several oropharyngeal and genital neoplasms. The virus
specifically infects the basal cell layer of the squamous epithelium that is exposed
as a result of minor abrasions. HPV infections are often subclinical and transient,
resolving spontaneously over time, 90% cases clearing the infection within 2 years
(Ho et al. 1998). Long-term viral persistence is required for the malignant progression
of cervical cancer. Most cervical cancers originate at the transformation zone,
833
an area of the cervix where columnar epithelium changes to squamous epithelium

by a process called squamous metaplasia. Cervical Intraepithelial Neoplasia (CIN)/
Squamous Intraepithelial Lesions (SIL) are commonly used clinical terms that
describe precancerous lesions or the abnormal growth of squamous cells observed
in the cervix. Histologically, 9095% of the cervical cancers are squamous cell
carcinomas, while adenocarcinoma constitutes less than 5% of cervical cancers.
HPV genome consists of approximately 8 Kb of double-stranded, circular DNA.
More than 100 different HPV types have been classified on the basis of their nucleotide sequence, of which about 40 infect the genital tract. Based on their oncogenic
potential, these viruses are grouped into high risk, intermediate risk, and low risk
types. Types 16 and 18 are considered high-risk (oncogenic) types and are associated
with aggressive forms of cervical cancers throughout the world (Clifford et al. 2003;
Munoz et al. 2003). HPV 16 is detected more often in squamous cell carcinomas
whereas HPV 18 is frequently detected in adenocarcinomas. Infection with low-risk
HPV subtypes such as 6, 11, and 42 is usually associated with benign warts (Lorincz
et al. 1992). Some studies have shown that there are geographical variations in HPV
type distribution, e.g., in Sub-Saharan Africa, the high risk HPV types associated
with high grade SIL (HSIL) in HIV-infected women include HPV 52, 58, 53, 35, and
45 in addition to types 16 and 18 (Blossom et al. 2007; Sahasrabuddhe et al. 2007).
For high risk HPV types, early viral gene products, E6 and E7, play critical roles
in the transformation of epithelial cells (Hawley-Nelson et al. 1989). These proteins
bind to and inactivate a number of cellular proteins involved in regulating cell cycle
progression and apoptosis. One of the best characterized function of E6 protein is its
ability to promote the degradation of p53, an important tumor suppressor that regulates G1/S and G2/M cell cycle check point in response to DNA damage. E6 does so
by interacting with a cellular protein E6-AP to form a ubiquitin ligase that targets p53
for degradation via the proteosome (Scheffner et al. 1993). Another major function
of E6 important for immortalization is to activate telomerase via up regulation of
hTERT expression (McMurray and McCance 2003). Furthermore, the high-risk E6
protein have been shown to interact with a number of other cellular proteins, such as
the focal adhesion protein paxillin, p300/CBP, BAK12, IRF-3, and PDZ domain
proteins such as hDLG, MUPP-1, MAGI-1, and hScrib. These interactions contribute
to HPV-induced cellular transformation (Narisawa-Saito and Kiyono 2007). E7
binds to members of the Rb tumor suppressor protein family and inhibit their ability
to modulate the function of E2F transcription factors, leading to constitutive activation
of S phase genes (Dyson et al. 1989). The HPV-16 E7 protein has been shown to
interact with HDACs, and this interaction occurs through an intermediary protein
called Mi2. This binding is independent of E7s interaction with Rb and has been
found to be important for the maintenance of HPV episomes in undifferentiated keratinocytes (Brehm et al. 1999). The E6 and E7 protein derived from low-risk types
have been shown to have impaired ability to interact with cellular proteins thus
partially explaining their non-transforming nature (Heck et al. 1992).
The productive life-cycle of HPV is intimately tied to epithelial cell differentiation
(Longworth and Laimins 2004). On primary infection, the virus maintains its
genome as a low copy episomal DNA within the nuclei of the infected basal cells.
834
P. Kumar et al.
As basal cells divide, daughter cells migrate to the upper layers of the epithelium
and differentiate. Normally the cells exit the cell cycle as differentiation progresses;
however, HPV infected cells fail to exit the cell cycle and continue to support DNA
synthesis. The contribution of viral oncogenes E6 and E7 in creating a cellular environment favorable for viral DNA replication has briefly been discussed before.
During carcinogenic progression of cervical lesions, HPV DNA often integrates
into cellular chromosomes; an event that has been shown to positively correlate with
the severity of the lesions (Jeon et al. 1995). Whether the integration event is essential for carcinogenesis is a matter of debate. However, it has been noticed that viral
genome integration occurs downstream of the early genes E6 and E7, often resulting
in the disruption of E2 gene, a transcription factor that regulates viral transcription.
Loss of E2 is thought to relieve repression of early promoter resulting in increased
expression of transforming E6 and E7 proteins (Pett and Coleman 2007). HPV integration sites are randomly distributed over the whole genome with preferences for
relatively few loci (Wentzensen et al. 2004). There is no evidence to support the
targeted disruption of a critical cellular oncogene by integrated viral genome.
Although cervical cancer has been listed as an AIDS-defining condition by CDC
since 1993, numerous studies have failed to consistently demonstrate high incidence
of cervical cancer among HIV-infected women. The incidence of cervical cancer
never reached epidemic proportions in the pre-HAART era like other AIDS-associated
malignancies (KS and NHL) in women with AIDS, even though both HIV and HPV
share the same epidemiological risk factors (being sexually transmitted infections).
The association between HIV and cervical cancer varies considerably from country
to country. Studies conducted in Africa, where both HIV and cervical cancer are
endemic, have found a weaker association between the two than the data reported
from Europe or America (La Ruche et al. 1998; Mbulaiteye et al. 2006b; Sitas et al.
1997). It is likely that progression of HPV infection to cervical cancer, which can
take years, might exceed the mean survival time in developing countries. Also
patients in developing countries have higher background rates of cervical cancer in
general population as they usually do not have access to routine screening and
timely treatment thus making an inconclusive link between cervical cancer in HIVpositive and HIV-negative patients.
Data from several AIDS-cancer registries match studies in the United States have
reported fivefold or greater risk of cervical cancer in patients with AIDS compared to
the general population (Frisch et al. 2000, 2001). However, it is not entirely clear
whether HIV increases the oncogenicity of HPV types or how HIV alters the natural
history of HPV infection. It seems plausible that diminished HPV-specific immune
response due to HIV infection may allow for viral persistence and subsequent
progression to cervical cancer. The contribution of HIV-induced immunosuppression
to the increased risk of HPV infection and CIN is poorly understood. Recent studies
by Bigger et al. and Chaturvedi et al. have reported that the relative risk for cervical
cancer was not significantly associated with low CD4 T-cell count at the time of AIDS
onset and did not increase with increasing time after AIDS onset suggesting that there
is no relationship between the incidence of cervical cancer and HIV-induced immunosuppression (Biggar et al. 2007; Chaturvedi et al. 2009). Although increasing risk for
835
in situ carcinoma, a precursor for cervical cancer was found to be associated with
increasing time after AIDS onset. This is consistent with several studies that have
documented that HIV-seropositive women are at increased risk of low-grade cytological
abnormalities e.g., CIN1 (Harris et al. 2005; Strickler et al. 2005). It has been suggested
that lower CD4 count (reflective of immunosuppression) levels may promote early
stages of cervical dysplasia through poorly controlled HPV infection while later
events that are involved in the progression of malignant state seem not to be related to
immune status (Biggar et al. 2007). Strickler et al. (2003) investigated the prevalence
of high-risk HPV16 in HIV-seropositive women and found that its detection was not
greatly affected by lower CD4 counts suggesting that HPV16 may be more efficient
at evading immune surveillance than other HPV types, contributing to its strong
association with cervical cancer (Strickler et al. 2003).
Some studies have addressed the possibility of a direct interaction between HIV
and HPV which in theory could result in enhanced HPV-induced pathology. The
HIV-encoded tat protein has been shown to directly increase gene expression of
HPV 16 E6 and E7 genes in infected keratinocytes (Vernon et al. 1993). Similar
experiments showed that tat protein could increase HPV-18 E1 and L1 protein
expression (Dolei et al. 1999).There is little knowledge about the immune factors
that influence the progression of CIN to cancer in HIV-infected women. Nicol et al.
analyzed the local cytokines milieu from cervical biopsies of HIV/HPV co-infected
women and found that there was overexpression of the cytokine, RANTES, member
of the interleukin-8 super family of cytokines suggesting that cytokines may have an
important role in the pathogenesis of HIV/HPV co-infection (Nicol et al. 2008).
HIV Pathogenesis and Immune Suppression

Depletion of CD4+ T cells represents a fundamental event in HIV pathogenesis and is
generally associated with HIV-induced immunodeficiency. However, in recent years,
it has been increasingly recognized that HIV-associated immune suppression is not
primarily due to inactivation of the immune system, but rather due to chronic activation, rapid turnover, and activation-induced cell death. At first, immune activation
may seem like a compensatory mechanism to achieve T cell homeostasis after virusmediated CD4+ T cell destruction; however, several studies demonstrate that immune
activation is directly responsible for increased proliferation and death of CD4+ and
CD8+ T cells (Hazenberg et al. 2000; Sousa et al. 2002). In order to understand how
immune activation leads to defects in immune surveillance and the reactivation of
other co-infecting oncogenic viruses, one must first have a better understanding of the
natural history of HIV infection and the underlying immunopathology.
The natural history of HIV infection has been well studied and reviewed extensively (Cadogan and Dalgleish 2008; McMichael et al. 2010; Mogensen et al. 2010)
(Fig. 32.1). Exposure to infected bodily fluids through sexual contact or sharing
needles is the primary route HIV enters the body. The initial exposure of the virus
is followed by an acute phase of infection, which is defined as the period from the
836
P. Kumar et al.
Acute HIV syndrome
Dissemination of virus
to secondary lymphoid
organs
Clinical latency
Development of AIDS
associated malignancies
and
opportunistic infections
Immune activation
Circulating CD4+ T cells
HIV load
Immune
response
Mucosal CD4+ T cells
Acute
(5-10 Wks)
Chronic
(1-15 years)
AIDS
Fig. 32.1 The course of natural HIV-1 infection and kinetics of immune activation
initial detection of viral RNA until the presence of HIV-specific antibodies. Acute
phase usually range from days to several weeks after infection. Prior to the acute
phase and shortly after initial exposure of the virus via sexual route, there is a brief
eclipse phase where HIV first replicates locally in the vaginal or rectal mucosa
before any viral RNA can be detected in the plasma. It has been demonstrated in the
SIV model that the cells initially infected are resident memory T cells in the mucosa.
As a result of innate immune response, granulocytes, macrophages, and lymphocytes
are recruited to the initial site of infection. Macrophages are among the first cells
infected by HIV. In contrast to T cells, they are more resistant to the cytopathic
effects of HIV and thus are believed to be the principal reservoirs for long-term
infection (Salazar-Gonzalez et al. 2009; Veazey et al. 2003). Dendritic cells (DC)
located beneath the mucosal surfaces are also involved in HIV transmission. HIV
exploits the physiological migration properties of DCs to gain access to CD4+ T
cells in lymph nodes. Some subsets of mucosal DCs expressing DC-SIGN lectin
receptor bind gp120, the viral envelope, with high affinity. These DCs facilitate the
initial establishment and spread by carrying the virus from mucosal sites of exposure
to the lymphoid tissues and transferring infection to CD4+ T cells in the lymph node.
This further allows the virus to replicate and disseminate to secondary lymphoid
tissues such as the gut-associated lymphoid tissue (GALT).
Acute phase of infection is followed by a massive depletion of CD4+ memory
T cells in the mucosa, the lymph nodes, and the GALT within a few days after infection, accompanied by production of high levels of circulating virus. However, acute
infection does not seem to affect the nave and resting central memory T cells that
837
do not express CCR5 co-receptor, enabling these cells to replenish the T-cell population after the acute phase of infection. As these cells are short-lived, they only
partially substitute for the CD4+ effector memory T cells deleted during the acute
phase of infection (Grossman et al. 2006). Active viral replication in the infected
individuals subsequently leads to viremia, which usually peaks, between 2 and 3
weeks after initial infection. During this period, patients suffer from influenza-like
symptoms like fever, sore throat, and lymphadenopathy. T cell levels in the peripheral
blood gradually return close to normal but T cells in the GALT remain severely
suppressed. There is an establishment of latent viral reservoirs in resting memory
CD4+ T cells, which are generated when activated CD4+ T cells return to a quiescent
state. Latent HIV persists as a stably integrated but transcriptionally silent provirus.
In this state, the virus is unaffected by immune responses or antiretroviral drugs.
Eventually, the viremia levels decrease as the initial infection is partially contained
by the immune response and reach a steady viral set point, which marks the initiation
of the chronic phase of infection.
The chronic phase of infection can last for years even in the absence of any antiretroviral treatment but vary from individual to individual. During the chronic phase,
there is continuous immune activation and an accelerated cell turnover even though
the circulating peripheral CD4+ T cells are still near the normal level (Ford et al.
2009) HIV-infected individuals display elevated markers of activation (e.g. CD38,
Ki67) and apoptosis of CD8+ and CD4+ T-cells. Although the precise origin of
generalized immune activation is still not fully understood but several mechanisms
may be involved, including antigenic stimulation by the virus itself or its encoded
protein like envelope protein gp120 that has been shown to activate B cells via the
stimulation of various cytokines (He et al. 2006). The accessory protein Nef can
also promote lymphocyte activation either directly (Wang et al. 2000) or through
infection of macrophages (Swingler et al. 1999). Immune activation may also be
induced by other viruses, like EBV, KSHV or CMV that reactivate as a result of
suboptimal immune control. Last but not the least, substantial damages in the
mucosal barriers occurred during both the acute and chronic phase of infection,
allow the translocation of bacterial products such as lipopolysaccharide (LPS) into
the circulation which may lead to systemic activation of immune system (McMichael
et al. 2010). Immune activation causes considerable cellular turnover, senescence,
and apoptosis. Repeated stimulation gradually drives HIV-specific T cells toward an
irreversible exhaustion of their replicative capacities. In other words, immune
activation results in consumption of nave and memory cell pools with a concomitant
expansion of short lived effector CD4+ cells which ultimately become the new targets
for viral replication. Over time, this vicious cycle of immune activation and HIV
replication results in a constant decline in CD+ T cell counts, a gradual increase in
viral loads, followed by the development of opportunistic infections and AIDSassociated malignancies.
There are a number of possible mechanisms that can contribute to tumorigenesis
in HIV-infected individuals. The most discernible one being the depletion of CD4+
T cells during the course of infection, which subsequently leads to a lack of immune
surveillance against various co-infecting oncogenic viruses like EBV, HPV, and
838
P. Kumar et al.
KSHV. These viruses can be reactivated during HIV-induced immunosuppression.

The role and mechanism of how these viruses can lead to oncogenic transformation
has been described earlier. Another possible mechanism for the development of
AIDS-associated malignancies, especially AIDS-associated B cell lymphomas is
the chronic activation of B cells. AIDS-associated B cell lymphomas are often characterized by the presence of genetic abnormalities like the chromosomal translocation
of c-Myc proto-oncogene next to the immunoglobulin heavy chain gene locus, a
seminal event in the genesis of BL, or mutations involving other oncogenes, such as
BCL2 or BCL6, commonly observed in DLBCL. These chromosomal alterations
may occur due to defects in either of the two germinal center reactions following
B-cell activation namely somatic hypermutation or class switch recombination.
Chronic activation of B cells increases the likelihood of chromosomal translocations
and somatic mutations in cellular oncogenes. One factor that can potentially contribute toward defects in germinal center reactions following chronic B cell hyper
activation is an aberrant expression of activation-induced cytidine deaminase (AID),
the DNA-mutating enzyme that plays a central role in somatic hypermutation and
class switch recombination. A recent study found that indeed AID expression was
markedly elevated in those patients who developed AIDS-NHL, when compared to
AIDS and HIV-negative controls (Epeldegui et al. 2007).
Dysregulation of the cytokine network may be another independent risk factor
for the development of B-cell malignancies in HIV patients. Cytokines play important
roles in B-cell activation, proliferation, and apoptosis. In fact, numerous cytokines
associated with B cell lymphomagenesis have been found to be elevated during HIV
infection. Notably levels of IL-10 (Sasson et al. 2010), IL-6, TNF alpha, and
CXCL13, which is a homeostatic B cell chemokine (Patke and Shearer 2000); were
found to be elevated prior to the development of AIDS-NHL (Widney et al. 2005).
It is noteworthy that IL-6 and IL-10 are potent B cell-stimulatory cytokines. Earlier
studies proposed that HIV induced a TH1 to TH2 shift which would result in an
inability to mount a successful cell-mediated immune response (Clerici and Shearer
1993) but later studies did not support this idea (Graziosi et al. 1994). We do not
fully understand whether cytokine dysregulation is the cause or outcome of
lymphomagenesis. However, it is important to understand the role of cytokines
during HIV-induced immunosuppression as it may allow new therapeutic approaches
based on the use of either recombinant cytokines or specific antagonists.
Evolving Spectrum of AIDS Associated Malignancies

in Post-HAART Era
The introduction of highly active antiretroviral therapy (HAART) in the mid-nineties
significantly changed the natural history of AIDS, its prognosis and the mortality
rates for people infected with HIV. Since then, many studies have been conducted to
determine the effect of HAART on the incidence of AIDS defining and non-AIDS
defining malignancies. The results of these studies are not very consistent and some
KS and NHLs
San Francisco City Clinic

cohort, USA
North America, Europe,

and Australia
52 European HIV clinics
Chelsea and Westminster

Cohort, Great Britain
Buchbinder et al. (1999)
International collaboration on HIV and

cancer (2000)
Mocroft et al. (2000)
Matthews et al. (2000)
NHLs
NHLs
KS, NHLs, Cervical cancer

and 20 other cancers
19921994 vs. 19971998
KS and NHLs
19881999
19941998
19771999
19931996
19881997
NHLs
17 Western European
countries
Ledergerber et al. (1999) Swiss HIV cohort study
Franceschi et al. (1999)
Table 32.1 Studies reporting the impact of HAART on the incidence of malignancies
Author/group
Study site/cohort
Malignancies studied
Study period
Jacobson et al. (1999)
Multicenter AIDS cohort
KS and NHLs
19841997
study, USA
(continued)
Major findings
Decline in KS (25.67.5 per 1,000
person-years)
Increase in NHLs (21% per year)
but recent possible decrease
Increase in NHLs (3.6% in 1994
to 4.9% in 1997)
Decline in KS (hazard ratio 0.08)
No change in NHL (hazard
ratio 0.61)
Decline in KS incidence (3.50
per 100 person-years)
No change in NHL incidence
(remained between 1.41.8)
Decline in KS and NHL incidence
(IRR, 0.32 and 0.58,
respectively)
No change in other cancers (IRR,
0.96)
Percentage of NHLs as ADI have
increased (from 4 to 16%)
The incidence of AIDS-related
lymphomas has not changed
over time (P = .933), but
contributes to a greater
percentage of first ADI in the
HAART era (P < .0001).

839
Australia
London, Great Britain
Euro-SIDA study, Europe
29 centers in France
SEER program, USA
Australia
France
Univ of Alabama at
Birmingham
HIV clinic
Ives et al. (2001)
Kirk et al. (2001)
Besson et al. (2001)
Eltom et al. (2002)
Dore et al. (2002)
Herida et al. (2003)
Bedimo et al. (2004)
Levine et al. (2000)
Grulich et al. (2001)
Study site/cohort
Los Angeles, USA

Author/group

AIDS defining and nonAIDS defining
Non-AIDS defining
NHLs
KS and NHLs
Non-AIDS defining
NHLs
KS and NHLs
KS and NHLs
Lymphomas
Study period
19892002
19921999
19932000
19731998
19931994 vs. 19971998
19941995 vs. 19971999
19901998
19801998
19821998
Decrease in prevalence of small

noncleaved lymphoma
(P = 0.005)
Increase in diffuse large cell
lymphoma (P < 0.0001)
(P trend 0.045)
34% decline in KS, non-significant
increase (51%) in NHLs
Significant decline in NHLs
(1.990.30 cases/100 person
years of follow-up)
Decline in incidence of AIDSrelated lymphomas (86.042.9
per 10,000 person-years)
Increase in non-AIDS defining
cancers
Percentage of NHLs as ADI have
increased (from 4.4 to 6.3%)
No change in overall incidence,
Lung cancer increased
Decrease in AIDS-defining
malignancies (IRR, 0.31)
malignances (IRR, 10.87)
Major findings
840
P. Kumar et al.
Millitary Clinics,
Multicenter study, USA
San Diego County, USA
Mocroft et al. (2004)
Burgi et al. (2005)
Tri-service AIDS Clinical

Consortium and
Multicenter AIDS
Cohort Study
Swiss HIV cohort study
Shiels et al. (2008)

19902006
19842006
KS
19912002
19882000
19882003
19942003
Study period
AIDS defining
AIDS defining and AIDS

associated
NHLs
Non-AIDS defining
KS
Annual reduction of 39% in KS
incidence
Decline in non-AIDS defining
cancers (OR 0.21)
Decline in NHL incidence
(29.66.5 per 1,000 person-years)
Decline in KS (SIR 2,800790 per
100,000 person years) and
NHL incidence (SIR 9.86.5 per
100,000 person years)
cancers (from 31.4 to 58% of
cancers)
Decrease in AIDS-defining
malignancies (IRR, 0.29)
Major findings
Franceschi et al.
(2008)
Powles et al. (2009)
Decline in KS incidence (33.35.1

per 1,000 person-years)
Chelsea and Westminster
Non-AIDS defining
19852007
Cohort, Great Britain
cancers (SIR, 1.96)
Millitary beneficiaries,
AIDS defining and non19842006
Decrease in AIDS-defining in late
Crum-Cianflone et al.
AIDS defining
HAART era (P < 0.0001)
(2009)
cancers (P < 0.0004)
Seaberg et al. (2010)
AIDS defining and non19842007
KS and NHL declined (IRR, 0.13
AIDS defining
and 0.23, respectively)
Anal cancer increased (IRR, 5.84)
No chance in HL (IRR, 0.75)
NHLs Non-Hodgkins lymphomas, ADI AIDS-defining illnesses, KS Kaposis sarcoma, IRR Incidence rate ratio, SIR Standardized incidence ratios,
OR Odds ratio
Colorado, Florida, and

New Jersey
Engels et al. (2008)
Diamond et al. (2006)
Study site/cohort
Euro-SIDA study, Europe
Author/group

841
842
P. Kumar et al.
recent and prominent studies have been summarized in Table 32.1 (Levine et al.
2000; Engels et al. 2008; International Collaboration on HIV and Cancer 2000;
Bedimo et al. 2004; Besson et al. 2001; Buchbinder et al. 1999; Burgi et al. 2005;
Crum-Cianflone et al. 2009; Diamond et al. 2006; Dore et al. 2002; Eltom et al.
2002; Franceschi et al. 1999, 2008; Grulich et al. 2001; Herida et al. 2003; Ives et al.
2001; Jacobson et al. 1999; Kirk et al. 2001; Ledergerber et al. 1999; Matthews
et al. 2000; Mocroft et al. 2000, 2004; Powles et al. 2009; Seaberg et al. 2010; Shiels
et al. 2008). In recent years, the longer survival rates associated with HAART have
led to a steady increase in diseases with a longer latency period, especially non-AIDSdefining malignancies. Decreased incidence for certain malignancies may be due to
a better immune surveillance for malignancies.
These studies were mostly conducted in HIV-infected individuals in Western
countries, where one-third of deaths in these individuals are due to cancer. One point
that needs to be emphasized from this table is the lack of any report from sub-Saharan
Africa, despite this region bearing the brunt of the HIV epidemic. One of the reasons
has been the slower roll out of HAART in this region and poor availability of registries
and records in order to evaluate a change in incidence. Incidence of cancer in the
pre- and post-HAART era in Africa has also been difficult to measure due to the
considerably shorter life-span of people living with HIV as compared to Western
countries. Another limitation of many of these studies is that the length of time the
participants have been on anti-HIV therapy has been short. Considerably longer
follow-up is required to gather more comprehensive data on this topic.
As evident from Table 32.1, the incidence of non-AIDS-defining malignancies
have increased in the post-HAART era. In contrast, most studies conclude that KS
and NHLs have declined in incidence. The data on the impact of anti-HIV therapy
on the development and persistence of HPV-related disease is conflicting. Some
studies have found a regression in HPV-related disease, while others have found
limited or no regression (De Vuyst et al. 2008). More recent studies are now evaluating
the factors associated with the risk of developing malignancies including the use of
specific antiretroviral agents, ART drug class, the duration of ART, and the level
of immunosuppression.
Concluding Remarks
The incidence of AIDS-related KS and NHL has decreased dramatically since the
introduction of HAART, especially in the developed countries where HAART is
widely available and drug failures are being monitored. Similarly, in the developing
countries with the recent scale up of the treatment, it is expected that these AIDSdefining cancers will also experience a decrease. However, higher risk in developing
other non-AIDS-associated malignancies in HIV-infected and treated individuals
are already being observed. The impact of HAART on these cancers remains unclear
but it is expected there will be an increase in the overall risk in developing nonAIDS-defining cancers, and that there will a shift in the spectrum of these malignancies because of the increasing size of the HIV-infected and treated population.
32
Viral Malignancies in HIV-Associated Immune Deficiency
843
The development of these cancers could be due to a number of reasons but the exact
mechanisms are not known. It could be due to chronic immune suppression or due
to co-infection or reactivation of other viruses associated with cancers such as EBV
or KSHV. For HIV-infected individuals in developing countries, there could be other
additional factors which include poor nutritional status, other co-morbid diseases,
drugdrug interactions, and cytotoxicity of HAART therapy. In conclusion, the
evolving spectrum of AIDS-defining malignancies along with increasing incidence
of non-AIDS associated cancers remains a challenge for the diagnosis, care, and
treatment of HIV-infected patients and warrants further research.
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activates lytic cycle replication of Kaposis sarcoma-associated herpesvirus: role of JAK/STAT
signaling. J Virol 81:24012417
Index
A
Acute-transforming retroviruses, 758
Adenovirus type 12 E1A, tumorigenesis
E1A and E1B genes, 489
MHC class I
COUP-TFII/HDAC recruiting,
transcription repression, 495496
CTL avoidance, shutoff, 490491
transcription level, shutoff, 492, 493
neuronal and tumor-related genes,
expression of
E1A12 oncoprotein, 500
Spacer region, 499, 500
neuronal gene induction
NRSF degradation, 502504
NRSF, schematic representation of,
501, 502
NF-kB binding
and COUP-TFII/HDAC binding,
MHC class I repression,
496497
p50 and p65 phosphorylation, blocking
of, 492494
NK cell lysis
activating ligands, reducing expression
of, 497499
resistance to, 497
Adult T-cell leukemia (ATL), 613
genomic instability, 621623
vs. HTLV1-transformed cells, 623624
oligoclonal expansion, 620621
telomerase activity, 625
tumor suppressors, 621
AEV. See Avian erythroblastosis virus (AEV)
AIDS-associated lymphoma (ARL)
Burkitts lymphoma (BL), 820822
diffuse large B-cell lymphomas (DLBCL),

822825
Hodgkins lymphoma, 825827
primary effusion lymphoma (PEL),
827829
Alpha fetoprotein (AFP), 522
ALV. See Avian leukosis virus (ALV)
Antiviral therapy and HCC, 572573
ARL. See AIDS-associated lymphoma (ARL)
Ataxia telangiectasia mutated (ATM), 473, 474
ATL. See Adult T-cell leukemia (ATL)
Avian erythroblastosis virus (AEV), 692
Avian leukosis virus (ALV), 83, 681684
Avian reticuloendotheliosis virus T (REV-T),
691
B
BAC. See Bronchioloalviolar carcinoma
(BAC)
BART miRNA, 803
BHRF1 miRNA, 803
Bittner agent. See Mouse mammary tumor
virus (MMTV)
BK polyomavirus and transformation
cytopathic disease, 422423
discovery, 419
genome and life cycle
agnoprotein, 422
infection, replication/transformation, 422
large (LTag) and small tumor antigen
(sTag), 421
noncoding control region (NCCR), 421
primary host cell specificity, 420
regions, 420
secondary host cell specificity, 421

DOI 10.1007/978-1-4614-0016-5, Springer Science+Business Media, LLC 2012
853
854
BK polyomavirus and transformation (cont.)
in human cancers
kidney-pancreas transplant patient, 425
prostate cancer, 426, 427
urothelial cancers, 426
oncogenic transformation, in experimental
systems, 424
BL. See Burkitts lymphoma (BL)
Bone marrow-derived nonhematopoietic cells
transformation, JC virus, 441442
Bone marrow reconstitution, 591
Bronchioloalviolar carcinoma (BAC), 755
Burkitts lymphoma (BL), 820822
C
Cervical cancer
HIV-associated immune deficiency
AIDS-defining condition, 834
HIV/HPV co-infection, 835
HPV infections, 832833
immunosuppression, 834835
and HPV
integration of, 470471
oncogenes, contribution of, 471472
c-onc genes, 679
Consensus degenerate hybrid oligonucleotide
primers (CODEHOP), 254
CTL epitopes, T-antigen, 394396
Cytochrome P450 (CYP) pathway, 590591
D
Decoy cells, 426
Diffuse large B-cell lymphomas (DLBCL),
822825
DNA viruses
human cytomegalovirus, 120
PI3K-Akt-TSC-mTOR signaling pathway
cap-dependent translation, 122124
4E-BP1 phosphorylation, 122
eIF4F translation initiation complex, 120
intersections, 124, 125
oncoprotein, 121
PKB, 121
RAFT1/FRAP, 121
SV40 large T (SVLT), 123
TSC1 and TSC2, 122
replication, 119
tumor, oncogenic viruses and cancer
transmission, 106
Warburg effect
citrate diversion, 127
glucose and glutamin metabolism, 126
Index
HCMV-infected cells, 126
tricarboxylic acid cycle, 125
E
EBER. See EBV-encoded RNA (EBER)
EBV. See Epstein-Barr virus (EBV)
EBV-encoded RNA (EBER)
B cell transformation and
lymphomagenesis, 798800
cellular gene expression, 797798
lymphomagenesis and apoptosis
protection, 796797
protein translation shutoff prevention,
795796
structure and expression, 793794
EBV nuclear antigen 2 (EBNA2), 89
EBV nuclear antigen leader protein
(EBNALP), 180181
Endogenous retroviruses (ERV), 693694
env gene, 756
Enzootic nasal tumor virus (ENTV), 782783
Epidermodysplasia verruciformis (EV), 111
Epstein-Barr virus (EBV). See also
Lymphocryptovirus (LCV)
animal models, 142
antiviral agents, 190
associated tumors, oncogenic viruses and
cancer transmission, 110111
BILF1, 55
biology, 134137
and Burkitts lymphoma, 137139
EBER (see EBV-encoded RNA (EBER))
EBNA1, 59
EBNA3C, 60
ENBA5, 61
encoded oncogenes, 8990
encoded proteins, 174175
gastric carcinoma, 141
genome, 172
HHV8 and Kaposis sarcoma, 142143
HIV1, coinfection with, 596597
and Hodgkins disease, 140
immunotherapy, 188189
infection diagnosis and malignancies, 188
KS lesions, 144145
LMP1, 59, 6465
LMP2A, 50
lymphomas, immunosuppression, 141
lytic and latent genes, 173
microRNA
host cellular genes targeting, 804809
structure, 802804
and nasopharyngeal carcinoma, 139140
Index
855
NK-cell lymphomas, 141142

and notch signaling, 6668
structure, 172173
T-cell lymphomas, 141142
tumorigenesis
cell-cycle progression, 185188
cell signaling, 182183
chromatin remodeling, 183185
vaccination, 190191
virus-mediated cell proliferation, 4648
and Wnt Signaling, 70
F
Flexner, S., 1315
FoxN1, 696
Fv1, 696
G
Gag gene, 756
g 1-herpesvirus. See Lymphocryptovirus (LCV)
H
HAART. See Highly active antiretroviral
therapy (HAART)
HCV. See Hepatitis C virus (HCV)
Head and neck cancer (HNC), HPV role in,
475476
Hepadnaviruses and hepatocellular carcinoma
chronic HBV infection
biology of, 533536
hepatocyte evolution and liver cancer,
544553
virus and liver, changes in, 541543
ground squirrel models, 531, 532
integration of, 532
origin of, 536537
orthohepadnavirus and avihepadnavirus, 531
persistent injury, to liver, 532
progression of, 533
replication
double-strand, linear virus DNA (DSL
DNA), structure of, 540
HBeAg and HBx, 537, 538
HBV life cycle, in hepatocytes, 538,
539
HBV, rcDNA genome of, 537, 538
polymerase/pregenome complex, 538
therapy, 554
woodchuck models, 531, 532, 553
Hepatitis B virus (HBV). See also Viruses
and cancer
biology of
endothelial cell layer and hepatocytes,
534, 535
immune response, 535, 536
perinatal infection, stages of, 533, 534
conjectures, 3739
discovery of, 2831
DNA integration, into host chromosomes,
513
genetics of, 3637
and hepatocellular carcinoma, 110
hepatocyte evolution and liver cancer
HBx and HCC, 547550
oncogenes, 544
regeneration and HCC, 545547
transgenic mice, 544
viral envelope proteins and HCC,
550553
life cycle, in hepatocytes, 538, 539
pre-S2/S proteins and large envelope
protein, 514
protein-primed reverse transcription, 512
rcDNA genome of, 537
viral load, 513
virus and liver, changes in
genetic basis, 542
host immune response, 541542
resistance to infection, 542, 543
titer production of, 541
X protein, 513
Hepatitis C virus (HCV)
clinical perspective, and HCC
antiviral therapy, 572573
epidemiological link, 571572
etiology of, 573
core protein, 515516
genome, 514515
NS5A protein, 516
replication, 515
steatosis and oxidative stress, 516
virological perspective, and HCC
experimental systems, 574
genome and polyprotein product, 575,
576
molecular biology of, 575578
NS34A enzyme complex, 577
NS5A phosphoprotein, 578
NS5B RNA polymerase, 578
proteins and carcinogenesis, 575576
tropism, 574575
856
Hepatitis viruses
causes, of cancer death, 509, 510
epidemiology, 511512
HBV
DNA integration, into host
chromosomes, 513
pre-S2/S proteins and large envelope
protein, 514
protein-primed reverse transcription, 512
viral load, 513
X protein, 513
HCC (see Hepatocellular carcinoma
(HCC))
HCV
genome, 514515
NS5A protein, 516
replication, 515
steatosis and oxidative stress, 516
Hepatocellular carcinoma (HCC)
age, incidence distribution, 511, 518
detection and management of
alpha fetoprotein (AFP), 522
chronic hepatitis virus effect, 523
molecular and biomarker information,
subclassification, 523
poor prognosis and, 521, 522
serum markers for, 524
etiologies of, 509, 510
HBV and HCV, 509, 511
HBx and
DDB1, 547, 549
indirect effect, 550
nucleotide excision repair, 549
reported activities of, 547, 548
transformed state, of tumor cells, 549,
550
HCV (see Hepatitis C virus (HCV))
hepatocyte regeneration and
cirrhotic nodules and non-cirrhotic
livers, 546
clonal hepatocyte repopulation, 545, 546
molecular pathogenesis of
chronic hepatitis virus, immune
response, 519
chronic infection, molecular and
cellular events, 520
clinical conditions, progression of, 518
inherited and acquired mutations,
519521
microRNA role, in
hepaocarcinogenesis, 521
molecular targets and pathways, 520
pathways of, 517519
Index
origin of, 536537
viral envelope proteins and
ground glass hepatocytes (GGH), 551
HBx, 553
hepatocyte death, 551
as oncogenes, 551
PreS2 mutation, 552
S, M, and L protein, 550
spheres and rods, 550551
Hepatocyte
evolution and liver cancer
HBx and HCC, 547550
oncogenes, 544
regeneration and HCC, 545547
transgenic mice, 544
viral envelope proteins and HCC,
550553
regeneration and
cirrhotic nodules and non-cirrhotic
livers, 546
clonal hepatocyte repopulation, 545,
546
Herpesviruses
EBV (see also Epstein-Barr virus (EBV))
animal models, 142
biology, 134137
and Burkitts lymphoma, 137139
gastric carcinoma, 141
HHV8 and Kaposis sarcoma,
142143
and Hodgkins disease, 140
KS lesions, 144145
lymphomas, immunosuppression, 141
and nasopharyngeal carcinoma, 139140
NK-cell lymphomas, 141142
T-cell lymphomas, 141142
KS (see Kaposis sarcoma (KS))
KSVH (see also Kaposis sarcoma herpes
virus (KSVH))
animal models, 155156
biology, 146
latent proteins and oncogenesis, 146151
multicentric Castlemans disease,
145146
primary effusion lymphoma, 145
MDV, 156157 (see also Mareks disease
virus (MDV))
Herpesvirus-mediated cancers
EBV EBER RNAs
B cell transformation and
lymphomagenesis, 798800
cellular gene expression, 797798
lymphomagenesis and apoptosis
protection, 796797
Index
857
protein translation shutoff prevention,

795796
structure and expression, 793794
HSUR, 800801
microRNA
biogenesis and function, 802
EBV-encoded, 802803
identifying targets and functions, 804
KSHV-encoded, 803804
viral genes targeting, 809
Herpesvirus saimiri (HVS)
classification, 203
HSURs, 800801
Saimiri sciureus, 202
Saimiri transformation-associated protein
(Stp), 203
tyrosine kinase-interacting protein (Tip), 204
HHV8. See Human herpesvirus 8 (HHV8)
Highly active antiretroviral therapy (HAART)
drug interactions demand management,
590591
HIV1/HTLV2 coinfection, 660, 661
HIV1, treatment, 589
with chemotherapy, 590
HIV1. See Human immunodeficiency virus
type 1 (HIV1)
AIDS-associated lymphoma
Burkitts lymphoma, 820822
diffuse large B-cell lymphomas,
822825
cervical cancer
AIDS-defining condition, 834
HIV/HPV co-infection, 835
HPV infections, 832833
Kaposis sarcoma
AIDS-associated KS, 830
classic KS, 829
endemic KS, 829
histological features, 829, 830
iatrogenic KS, 830
immune suppression, 831832
KSHV replication, 831
pathogenesis and immune suppression
acute phase, 836837
CD4+ T cell destruction, 835
chronic phase, 837
infection and immune activation, 836
macrophage, 836
tumorigenesis, 837838
viral entry, 835836
post-HAART era, AIDS, 838842
HPV. See Human papillomavirus (HPV)
HSUR, 800801
HTLV1. See Human T-lymphotropic virus
type 1 (HTLV1)
HTLV2. See Human T-cell leukemia virus
type 2 (HTLV2)
HTLV1-associated myelopathy/tropical spastic
paraparesis (HAM/TSP), 613
HTLV1 uveitis, 613
Human breast cancer, 748749
Human cytomegalovirus (HCMV), 120
Human herpesvirus 8 (HHV8)
epidemiology, 592
Kaposis sarcoma, coinfection, 592595
Human herpes viruses (HHV), 4648. See also
Herpesviruses
Human immunodeficiency virus type 1
(HIV1)
associated malignancies, 586588
immunodepletion heightens, 588589
Kaposis sarcoma, 592595
treatment
bone marrow reconstitution, 591
drug interactions demand management,
590591
HAART with chemotherapy, 590
highly active antiretroviral therapy, 589
viral coinfection, malignancy
anti-EBV cytotoxic T lymphocytes, 598
EBV, 596597
HCV, 600
HHV8, 592595
HPV, 598599
HTLV1, 600601
IL10 role, 597
multicentric Castlemans disease, 596
rare lymphoma, 595596
Human infections, polyomavirus SV40
contemporary human infections
predictions, 401403
evidence of, 403404
SV40 association, human cancer, 405408
SV40-contaminated vaccines, 400401
Human papillomaviruses (HPVs)
abortive viral infection, 467
cervical cancer, 832834
in head and neck cancer (HNC), 475476
high-risk, 463
858
Human papillomaviruses (HPVs) (cont.)
molecular events, malignant progression
integration and cervical cancers,
470471
oncogenes contribution, to human
cervical cancers, 471472
oncogenes repression, E2 protein, 471
oncogenic activities, host DNA damage
response, and genomic instability,
473475
persistent latent infection
bovine papillomavirus type 1 (BPV1),
468
Brd4, 468, 469
E6 and E7 oncoproteins, 468
mitotic chromosomes, tethering
mechanism, 469
productive viral life cycle
basal cells, 465
E4 protein, 467
E5 protein, 466, 467
E2 proteins, 465, 466
HPV16 genome organization and
functions, of gene products, 464
suprabasal cells, 466
therapeutic approaches, 477
Human T-cell leukemia virus type 2 (HTLV2)
accessory genes
Aph2 antisense gene, 655
ORF I, II, and V, 654655
culture and animal models, 664
discovery, 647648
genetic variability, 656
genome structure, 649, 650
hematopoiesis, 658659
HIV coinfection, 659661
neurologic abnormality, 657658
receptor and infectivity, 656657
regulatory genes
Rex gene, 652654
Tax gene, 651652
structural and enzymatic genes
envelope (env) gene, 651
gag gene, 649650
polymerase (pol) gene, 651
protease (Pro) gene, 650
Tax oncoprotein mechanisms
apoptosis, 664
cell cycle dysregulation, 663
NFkB interaction, 663
posttranscriptional effects, 662
viral transcription, 661662
virion structure, 648649
Human T-cell lymphotropic virus (HTLV), 45
Index
Human T-lymphotropic virus type 1
(HTLV1)
animal models, 625
ATL cells (see Adult T-cell leukemia
(ATL))
cell-associated transmission, 619620
cell transformation, oncoproteins, 586
epidemiological studies, 613
genome and replication, 614615
genome structure, 586
HBZ functions, 619, 630632
history of, 620
p12I, p13II, and p30II functions, 618
Rex functions, 618
Tax functions
IKK/NF-kB, 616618
viral mRNA transcription, 615616
viral origins, human cancers, 27
in vitro T-cell transformation
vs. ATL cells, 623624
signaling pathways, 624
telomerase activity, ATL cells, 625
I
Interferon treatment, HCC, 572573
International Committee on Taxonomy of
Viruses (ICTV), 449
J
Jaagsiekte sheep retrovirus (JSRV)
endogenous
enJSRV, 781782
ERVs, 780781
molecular biology
betaretrovirus, 762764
disease determinants, 767
Hyal2, 769
LTR binding sites, 766767
molecular clones, 764
Orf-X, 768769
Rej, 768
sequence deduction and endogenous
identification, 762
transcription specificity, 764766
oncogenesis mechanisms
cell transformation, 770
Env, 770771
transformation, env domains, 771772
pathogenesis
disease latency, 779780
Index
859
immune responses, 780

OPA, flocks, 778779
transformation, signal
EGFR inhibitors, 775
env transformation, 776777
Hyal2/RON pathway, 777778
PI3K-Akt-mTor pathway, 772774
Ras-Raf-MEK-MAPK pathway, 774775
Src inhibitors, 775
JC virus (JCV)
bone marrow-derived nonhematopoietic
cells transformation, T-Ag, 441442
cell culture and animal models, of
and human cancer, association of, 433,
434, 436438
latency, reactivation, and cellular tropism,
434435
neural stem cells transformation, 443, 444
and oncogenic potential, molecular
mechanism studies, 435
pathogenesis of
considerations, 443
and dissemination, from bone marrow,
440, 441
stem cells in, 439, 440
T-Ag-induced, 440
PML-type configuration, 442, 443
and stem cells, brain and bone marrow
hematopoietic cells, 438
nonhematopoietic cells, 439
T-Ag expression, 442
JSRV. See Jaagsiekte sheep retrovirus (JSRV)
K
Kaposis sarcoma (KS)
classical, 219
clinical features and treatment, 221222
co-factors, 220221
cytokines and angiogenic factors, 151155
endemic, 219
epidemic/AIDS, 220
epidemiology, 251
etiological agent, 252253
HIV1 and HHV8 coinfection, 592595
AIDS-associated KS, 830
classic KS, 829
endemic KS, 829
histological features, 829, 830
iatrogenic KS, 830
immune suppression, 831832
KSHV replication, 831
iatrogenic, 220
KSHV
latency, 253
molecular characteristics, 252253
morphology, 251252
proliferative disorder, 223
Kaposis sarcoma herpes virus (KSHV). See
also Rhadinoviruses
animal models, 155156
associated diseases, 216
biology, 146
epidemiology, 252
etiological agent, KS, 252253
gene expression, 252253
genomic conservation, 255257
host, 258
K1 gene, 51
K15 gene, 51
LANA1, 62
latency-associated nuclear antigen (LANA)
functions, 253
vs. RFHV LANA sequence analysis,
256257
latent proteins and oncogenesis, 146151
microRNA
structure, 802804
modulator of immune recognition, 256
molecular biology, 252
multicentric Castlemans disease, 145146
multicentric Castlemans disease (MCD)
IL6 role, 227
incidence, 226227
and notch signaling, 68
origin and evolution, 215216
primary effusion lymphoma (PEL)
clinical features and therapy, 224225
KSHV-associated lymphomas, 223224
pathogenesis, role of, 225226
solid, 224
primary infection, 216218
vs. RFHV genome, 255257
vs. rhadinoviruses genome, 258260
RRV, 208, 209, 211
transmission and risk factors, 216
tumorigenesis
mechanisms, 228233
viral proteins involvement, 233236
vCyclin, 61
vGPCR, 5354
vIL6, 56
860
Kaposis sarcoma herpes virus (KSHV). See
also Rhadinoviruses (cont.)
viral chemokines, 53
vIRFs, 6263
virus-mediated cell proliferation, 4748
and Wnt signaling, 70
Kidney transplantation, BK polyomavirus, 425
KS. See Kaposis sarcoma (KS)
KSHV. See Kaposis sarcoma herpes virus
(KSHV)
L
Latency-associated nuclear antigen1 (LANA)
Kaposi sarcoma, 222
viral protein involvement, tumorigenesis,
233
Liver, digestive system, 517
Lung alveoli, epithelial cell, 760
Lymphocryptovirus (LCV)
BARTs, 181182
and cancer, 170171
chemotherapy, 190
cytotoxic T cells use
EBV-specific T cells, 189
unmanipulated donor T cells, 189
EBERs, 181
EBNA1, 177178
EBNA2, 178179
EBNA3 family, 179180
EBNALP, 180181
EBV
antiviral agents, 190
encoded proteins, 174175
genome, 172
immunotherapy, 188189
infection diagnosis and malignancies,
188
structure, 172173
vaccination, 190191
in human, 171
LMP1, 175
LMP 2A and 2B, 176177
radiation therapy and surgery, 190
targeting B-cells, antibody therapy,
189190
viral species, 170
Lymphomagenesis, MDV
initiating events, 325326
lymphoma progression, 326327
Lymphoproliferative disorder (LPD), 208
Index
M
Macaca nemestrina rhadinovirus 2 (MneRV2),
258
Major histocompatibility complex (MHC)
class I, adenovirus type 12 E1A
COUP-TFII/HDAC recruiting,
transcription repression, 495496
NF-kB and COUP-TFII/HDAC binding,
repression of, 496497
shutoff
CTL avoidance, 490491
transcription level, 492, 493
Mardivirus. See Mareks disease virus (MDV)
Mareks disease virus (MDV)
gene products, oncogenesis
MDV-encoded microRNAs, 320324
phosphoprotein 14 and RLORF9,
319320
RLORF4, 319
herpesviruses, 156157
history of, 308
lymphomagenesis
initiating events, 325326
lymphoma progression, 326327
Meq
binding proteins, 313314
deletion mutant, phenotype, 316317
dimerization partners, 312313
localization and dynamics, 311312
splice variants, 315316
target genes, 315
viral interleukin 8, 317318
viral telomerase RNA, 317
viral ubiquitin-specific protease, 318319
pathogenesis, 308309
RNA association, 799
MDV. See Mareks disease virus (MDV)
Meq, MDV
Binding Proteins, 313314
deletion mutant, phenotype, 316317
dimerization partners, 312313
localization and dynamics, 311312
splice variants, 315316
target genes, 315
viral interleukin 8, 317318
viral telomerase RNA, 317
viral ubiquitin-specific protease, 318319
Merkel cell polyomavirus (MCV)
genome, appearance and structure of, 450
Merkel cell carcinoma (MCC) and viral
detection
digital transcriptome subtraction
(DTS), 451, 452
Index
861
in immunosuppressed populations, 451

integration of, 452, 453
origin of, 451
prevalence of, 452
viral carcinogenesis of, 453, 454
polyomaviruses, 339
small and large T antigens
conserved region 1 (CR1), 456
DnaJ domain, 456
domains, 455
indirect evidence for, 458
p53, 456458
PP2A, 458
replication, 453
retinoblastoma (RB) family-binding
motif LXCXE, 456
T antigens, 456458
transformation of, 455
MicroRNAs (miRNAs)
role, in hepaocarcinogenesis, 521
EpsteinBarr virus
biogenesis and function, 802
identifying targets and functions, 804
and KSHV-encoded, 802804
viral genes targeting, 809
mLANA, 278279
MLV. See Murine leukemia virus (MLV)
Mouse mammary tumor virus (MMTV)
genome transformation, 740
germline transmission, 740741
human breast cancer, 748749
replication, 741742
transcriptional control
mammary gland differentiation and
transformation, 743
proviral expression, 742
regulatory elements, 742
virus production, 742
tumor induction
common integration sites,
744745
Env protein, 747
oncogene integration, 745746
oncogenes activation, 747
pregnancy state, 746
proviral integrations, 744
retroviral insertion, 744
wild-type and transgenic mice, 745
Mtv loci, 740
Multicentric Castlemans disease (MCD), 596
Murine gammaherpesvirus 68 (MHV68). See
also Murine gammaherpesvirusassociated tumorigenesis
immune response
antibody response, 289293
malignant disease control, 293296
immunomodulation
M1, 286287
M2, 282284
mK3, 285286
Murine gammaherpesvirus-associated
tumorigenesis
immune system role, 287
implications and application, 296297
MHV68 immune response
antibody response, 289293
malignant disease control, 293296
MHV68 immunomodulation
M1, 286287
M2, 282284
mK3, 285286
mLANA, 278279
prooncogenic alterations, cell signaling
NF-kB, 279280
PI3K/Akt, 280
twist/snail, 281
v-bcl2
characteristics, 274
gammaherpesvirus Bcl2 orthologs,
275
oncogene, 276
v-cyclin
classification, 271
effects, 273
oncogene, 272
role, 272
and tumorigenesis, 273274
vGPCR, 276277
Murine leukemia virus (MLV), 685686
N
Natural killer (NK) cell lysis, adenovirus type
12 E1A
NKG2D activating ligands, reducing
expression of, 497499
resistance to, 497
Neural stem cells transformation, JC virus,
443, 444
Neuronal gene induction, adenovirus type 12
E1A, 501504
Neuron-restrictive silencer factor (NRSF)
Ad5-and Ad12-, 502, 503
degradation, 502504
schematic representation of, 501, 502
ubiquitination, 501502
862
NF-kB
binding, adenovirus type 12 E1A
and COUP-TFII/HDAC binding, MHC
class I repression, 496497
p50 and p65 phosphorylation, blocking
of, 492494
murine gammaherpesvirus-associated
NHL. See Non-Hodgkin lymphoma (NHL)
Non-acute retroviruses, 758
Non-Hodgkin lymphoma (NHL)
anti-EBV cytotoxic T lymphocytes, 598
IL10 role, 597
lymphocryptoviruses, 170
Nonhuman primate gamma-herpesviruses
herpesvirus saimiri (HVS)
classification, 203
Saimiri sciureus, 202
Saimiri transformation-associated
protein (Stp), 203
tyrosine kinase-interacting protein
(Tip), 204
RhLCV
Cercopithecine herpesvirus 15, 204
EBV infection, 206
miRNAs, 207
retroperitoneal fibromatosis (RF), 205
RRV
G protein-coupled receptor (GPCR), 209
KSHV, 208, 209, 211
lymphoproliferative disorder (LPD), 208
vIL6 detection, 210
Nonobese diabetic/severe combined
immunodeficient (NOD/SCID), 625
NS34A enzyme complex, HCV, 577
NS5A phosphoprotein, HCV, 578
NS5B RNA polymerase, HCV, 578
O
Oncogenesis mechanisms
endogenous murine retroviruses, 693694
Env signaling
friend virus and env gene-mediated,
694695
gene cooperativity, 692693
host factors influences, 695696
insertional mutagenesis
enhancer-mediated, 687688
gene function disruption and tumor
suppressor genes, 689690
gene therapy, 688689
microRNA activation, 690691
promoter insertion, 685686
regulatory sequence disruption, 686687
Index
oncogene capture
ALV and RSV, 681684
v-onc-containing viruses generation,
680681
retroviral classification, 679
Oncogenic viruses and cancer transmission
control and prevention, 105106
epidermodysplasia verruciformis (EV), 111
EpsteinBarr virus-associated tumors,
110111
hepatitis B virus and hepatocellular
carcinoma, 110
infection and human cancer, 101104
in molecular biology, 104105
rumor viruses, 112113
transmissible cancer cells, 113114
transmission and virulence, 108109
transmission dynamics
DNA tumor viruses, 106
iatrogenic infections, 107108
retroviruses, 106107
viral cancers, multifactorial facets,
109110
OPA. See Ovine pulmonary adenocarcinoma
(OPA)
Open reading frame (ORF), 255256
Orthoretrovirinae, 679
Ovine pulmonary adenocarcinoma (OPA)
clinical signs, 759
gross pathology, 759760
histological feature
acinar growth, 760
mitotic index, 761
papillary form, 760
retrovirus transmission, 761762
transformed cells, 761
history, 755
Oxidative stress, HCV, 516
P
p53
in human cervical cancers, 472
Merkel cell polyomavirus, 456458
Papillomaviruses (PVs). See Human
papillomaviruses (HPVs)
PEL. See Primary effusion lymphoma (PEL)
Phosphoinositide 3 kinase-like kinase (PIKK),
473
PI3K-Akt-TSC-mTOR signaling pathway
cap-dependent translation, 122124
4E-BP1 phosphorylation, 122
eIF4F translation initiation complex, 120
intersections, 124, 125
oncoprotein, 121
Index
863
PKB, 121
RAFT1/FRAP, 121
SV40 large T (SVLT), 123
TSC1 and TSC2, 122
pol gene, 756
Polyomaviruses (PyV). See also Merkel cell
polyomavirus
BK virus, 419
cell cycle progression, LT, 345
DNA damage responses, 359360
genome destabilization, 357359
genome organization, 340343
history and discovery, 338339
icosahedral capsid and MCV genome,
appearance and structure of, 450
MPyV middle T antigen, 352355
non-neoplastic disease association,
345346
phylogenetic comparison, 339340
properties, 344
small T antigen, 355356
species of, 449
transformation assays
immortalization, 346
non-SV40 LT proteins, 351352
SV40 large T antigen, 347351
transformation, 347
tumors, in animals, 356357
viral life cycle, 343344
Polyomavirus SV40
hamster model, infection and oncogenesis,
390393
human infections
contemporary human infections
predictions, 401403
evidence of, 403404
SV40 association, human cancer,
405408
SV40-contaminated vaccines,
400401
large T-antigen early protein
cell cycle dysregulation, 382
cytoplasmic T-antigen, 382385
transformation, maintenance, 382
mouse model, cellular immune responses
CTL epitopes, T-antigen, 394396
SV40 microRNA and CTL evasion, 396
SV40 T-antigen, cellular proteins
DnaJ domain and hsc70, 387388
p53 tumor suppressor protein, 388389
T-ag, 389390
tumor suppressor proteins, Rb family,
388
T-antigen variable domain, 385387
transgenic mouse models

loss of dependence, T-antigen, 398
Rb and p53 Pathways, 397398
T/t-antigen gene signature, 399
viral genome and virus-encoded proteins,
377379
viral late proteins, 380381
viral regulatory region, 379380
Posttransplant lymphoproliferative disorder
(PTLD), 171
Primary effusion lymphoma (PEL), 827829
Progressive multifocal leukoencephalopathy
(PML), 433435, 437
Prostate cancer, BK polyomavirus, 426
Protein kinase A catalytic subunit (PKAc), 494
Protooncogene, 721
R
Retroperitoneal fibromatosis-associated
herpesvirus (RFHV)
etiological assessment, RF tumors
immunohistochemical analysis,
262263
spindle tumor cells detection, 262
viral load, 260261
genomic conservation, 255257
host, 258
vs. KSHV genome, 255257
LANA vs. KSHV LANA sequence
analysis, 256257
prevalence, macaque, 260
Retroperitoneal fibromatosis (RF), macaques
epidemiology, 253
etiology, 254
phylogenetic analysis, 254
Retroviral transformation, 739
Retroviruses
cell transformation, oncoproteins, 586
cellular proto-oncogenes activation, 585
genome structure, 756
lifecycle, 757758
oncogenesis mechanisms (see Oncogenesis
mechanisms)
oncogenic properties, 758759
oncogenic types, 679
oncogenic viruses and cancer transmission,
106107
structure, 757
viral-encoded genes and cancer, 8283
RF. See Retroperitoneal fibromatosis (RF),
macaques
RFHV. See Retroperitoneal fibromatosisassociated herpesvirus (RFHV)
864
Rhadinoviruses (RV)
associated diseases, 216
discovery, 258
Kaposi sarcoma
classical, 219
clinical features and treatment,
221222
co-factors, 220221
endemic, 219
epidemic/AIDS, 220
iatrogenic, 220
proliferative disorder, 223
molecular characteristics, 258260
multicentric Castlemans disease (MCD)
IL6 role, 227
incidence, 226227
origin and evolution, 215216
primary effusion lymphoma (PEL)
clinical features and therapy, 224225
KSHV-associated lymphomas, 223224
pathogenesis, role of, 225226
solid, 224
primary infection, 216218
transmission and risk factors, 216
tumorigenesis
mechanisms, 228233
Rhesus macaque lymphocryptovirus (RhLCV)
Cercopithecine herpesvirus 15, 204
EBV infection, 206
miRNAs, 207
retroperitoneal fibromatosis (RF), 205
Rhesus macaque rhadinovirus (RRV)
G protein-coupled receptor (GPCR), 209
KSHV, 208, 209, 211
lymphoproliferative disorder (LPD), 208
vIL6 detection, 210
Rhesus rhadinovirus (RRV)
discovery, 258
prevalence, macaque, 260
RLORF4, 319
RNA-dependent DNA polymerase, 757
Rous, P., 1, 2, 912, 1519
Rous sarcoma virus (RSV)
in biomedical sciences, 728
c-src confirmation in vertebrates
mRNA splicing, 719
protooncogene, 721
provirus insertion, 719
role, human cancers, 723
transduction mechanism, 720
virion recombination, 719, 721
DNA discovery and replication, 709712
Index
filterable agent discovery, 705706
genome organization, 714715
pathogenicity, birds and mammals, 706707
reverse transcription
circular DNA formation, 712714
DNA synthesis, 712
integration, host chromosome, 713714
src viral oncogene
identification, 715717
protein product isolation, 722723
transformation biology, 707709
virion
structure, 708, 724
virion assembly
attachment, 724
Gag protein, 727
maturation, 724
replication, 725726
transformation, 724725
viral RNA genome arrangement, 724
virogene-oncogene hypothesis, 717718
Rumor viruses, 112113
RV. See Rhadinoviruses (RV)
S
Schmorl, G., 69
SFFV. See Spleen focus forming virus (SFFV)
Sheep pulmonary adenocarcinoma. See Ovine
pulmonary adenocarcinoma (OPA)
Simian vacuolating virus 40 (SV40), 449
Solid tumors, JC virus, 436
Spindle cells, 251
Spleen focus forming virus (SFFV), 694695
Squamous cell carcinomas (SCCs), 475
Steatosis and oxidative stress, HCV, 516
Stem cells and JC virus (JCV), brain and bone
marrow, 438439
SV40 T-antigen, cellular proteins
DnaJ domain and hsc70, 387388
p53 tumor suppressor protein, 388389
T-ag, 389390
tumor suppressor proteins, Rb family, 388
T
T-antigen variable domain, 385387
Tax (transactivator encoded by the X region),
HTLV1
cellular senescence
p21Cip1/Waf1 and p27Kip1 upregulation, 630
precancerous condition, 629630
oncogenic activities
cell cycle progression, 626
Index
865
chromosome instability, 628629

DNA damage, 628
double-stranded DNA breaks, 628
genetic/chromosome instability, 627
NF-kB activation and cell
transformation, 626
PDZ domain-containing proteins, 627
in transgenic mice, 627
Telomerase reverse transcriptase (TERT), 688
Thromobospondin 1 (THBS1), 804
Transferrin receptor 1 (TfR1), 741
Transgenic mouse models, polyomavirus
SV40
loss of dependence, T-antigen, 398
Rb and p53 Pathways, 397398
T/t-antigen gene signature, 399
Tumorigenesis. See also Murine
gammaherpesvirus-associated
tumorigenesis
EBV
rhadinoviruses
mechanisms, 228233
viral proteins involvement
K1/VIP, 235236
LANA, 233
miRNAs, 234
vFLIP, 234
vGPCR, 235
vIL6, 234235
viral cyclin, 233234
V
v-bcl2
gammaherpesvirus Bcl2 orthologs, 275
oncogene, 276
vGPCR, 276277
Viral cyclin (vcyclin)
classification, 271
effects, 273
oncogene, 272
role, 272
and tumorigenesis, 273274
viral proteins, in tumorigenesis, 233234
Viral-encoded genes and cancer
human cancer
EBV-encoded oncogenes, 8990
HBV-encoded oncogenes, 88
HCV-encoded oncogenes, 88
HPV-encoded oncogenes, 88
HTLV1-encoded oncogenes, 87
KSHV-encoded oncogenes, 9092
large DNA tumor viruses, 92
retroviruses, 8283
small DNA tumor viruses, 8485
tumor viruses, 8182
Virogene-oncogene hypothesis
Rous sarcoma virus, 717718
Viruses and cancer
first cancer vaccine, HBV, 3536 (see also
Hepatitis B virus (HBV))
global vaccination programs, 3435
origins of, 2728
prevention, 26
secondary antiviral prevention, 36
vaccine field trials, 3233
vaccine invention, 3132
Virus-mediated cell proliferation
cell cycle machinery modulation
EBV EBNA1, 59
EBV EBNA5, 61
EBV EBNA3C, 60
EBV ENBA5, 61
EBV LMP1, 59
KSHV LANA1, 62
KSHV vCyclin, 6162
KSHV vIRFs, 6263
p53-mediated cell cycle arrest, 5859
Rb-mediated cell cycle arrest, 5758
cellular signaling pathways
EBV and notch signaling, 6668
EBV and Wnt Signaling, 70
EBV LMP1, 6465
KSHV and notch signaling, 68
KSHV and Wnt signaling, 70
KSHV K13 (vFLIP), 6566
NF-kB signaling pathway, 6364
notch signaling pathway, 66
wingless-type (Wnt) signaling pathway,
6870
chemokine/cytokine system, modulation
biological effects, 52
EBV BILF1, 5556
KSHV vGPCR, 5354
KSHV vIL6, 56
KSHV viral chemokines, 53
signaling pathways, 5455
oncogenic human herpesviruses, 4648
self-proliferation, viral strategy, 48
viral proteins mimicking growth receptor,
4852
v-onc genes, 679
866
W
Warburg effect
citrate diversion, 127
glucose and glutamin metabolism, 126
HCMV-infected cells, 126
tricarboxylic acid cycle, 125
Warthin, A.S., 46
Welch, W.H., 34
Index
Wistar-King-Aptekman-Hokudai (WKAH),
625
Wnt, 744
X
X-linked severe combined immunodeficiency
(SCID-X1), 688

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Current Cancer Research

For further volumes:

Cancer Associated Viruses

Baruch S. Blumberg, M.D., D.Phil.

The Hepadnaviruses have also been addressed in the compendium, where we

Peyton Rous: A Centennial Tribute to the Founding

Viruses and Cancer: A Historical Perspective HBV

Virus-Mediated Cell Proliferation .........................................................

Viral-Encoded Genes and Cancer .........................................................

Oncogenic Viruses and Cancer Transmission ...................................... 101

DNA Viruses and Cancer: Taking a Broader Look ............................. 119

Herpesviruses and Cancer ..................................................................... 133

Lymphocryptoviruses: EBV and Its Role in Human Cancer ............. 169

Nonhuman Primate Gamma-herpesviruses and Their Role

Rhadinoviruses: KSHV and Associated Malignancies ........................ 215

Retroperitoneal Fibromatosis Herpesvirus and Kaposis

Murine Gammaherpesvirus-Associated Tumorigenesis...................... 267

Mareks Disease Virus-Induced T-Cell Lymphomas ........................... 307

Polyomaviruses and Cancer ................................................................... 337

Polyomavirus SV40: Model Infectious Agent of Cancer ..................... 377

BK Polyomavirus and Transformation ................................................. 419

Polyomavirus JC and Human Cancer: Possible Role

Merkel Cell Polyomavirus ...................................................................... 449

Human Papillomaviruses and Cancer................................................... 463

Tumorigenesis by Adenovirus Type 12 E1A ......................................... 489

Overview of Hepatitis Viruses and Cancer ........................................... 509

Hepadnaviruses and Hepatocellular Carcinoma ................................. 531

Hepatitis C Virus and Hepatocellular Carcinoma ............................... 571

Human and Animal Retroviruses: HIV-1 Infection

HTLV-1 and Oncogenesis ....................................................................... 613

Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology

Mechanisms of Oncogenesis by Avian and Murine

Rous Sarcoma Virus: Contributions of a Chicken Virus

Mouse Mammary Tumor Virus and Cancer ........................................ 739

Jaagsiekte Sheep Retrovirus and Lung Cancer ................................... 755

Small RNAs and Their Role in Herpesvirus-Mediated Cancers ........ 793

Viral Malignancies in HIV-Associated Immune Deficiency ................ 819

Index ................................................................................................................. 853

James C. Alwine Department of Cancer Biology,

Janet S. Butel Department of Molecular Virology and Microbiology,

Jennifer Gordon Department of Neuroscience and Center for Neurovirology,

Kamel Khalili Department of Neuroscience and Center for Neurovirology,

Leslie J. Parent Department of Medicine, Penn State College of Medicine,

Susann Santag Institute of Virology, Hannover Medical School,

Peyton Rous: A Centennial Tribute

UnitedVRG - Medical Release Group

V. Wunderlich and P. Kunze

Fig. 1.1 Photograph

A Fresh Age of Medical Endeavor in America:

Fig. 1.2 Photograph

UnitedVRG - Medical Release Group

V. Wunderlich and P. Kunze

Aldred Scott Warthin: A Consummate Pathologist

Fig. 1.3 Photograph

Warthins research on the familial incidence of cancer had a particularly lasting

UnitedVRG - Medical Release Group

V. Wunderlich and P. Kunze

In the summer of 1898, Warthin was a guest of Schmorl at Dresden Friedrichstadt

Georg Schmorl: A Scientist with Most Infectious Enthusiasm

UnitedVRG - Medical Release Group

Fig. 1.6 Portrait of Christian

Peyton Rous in Dresden

UnitedVRG - Medical Release Group