Theory and Technology of Transgene

Applied in Biomedicine

December 2005
 Theory and Technology of Transgene
Applied in Biomedicine

Class Description

Introduction of Virology
Transduction of Transgene into Animals
Viral Vectors
Adenovirus (AdV), Adeno-associated virus (AAV)
Retrovirus (RV) and Lentivirus (LV)
Non-viral Vectors
Transduction by Proteins
Physical and Chemical Methods
Regulatable Systems and Cell Specific Promoters
Gene Therapy
 Class Organization

4 Sessions

Two Parts in each session

Part 1. The general background in the session
(will be offered by the instructor)
Part 2. Four research papers which will be
presented by the participants
I. Introduction of Virology
Dimensions
 Linnaean System of Classification
Swedish Carolus Linnaeus, 18th century
Classification Human as an example
Kingdom Animalia
Phylum Chondata
Subphylum Vertebrata
Class Mammalia
Order Primates
Family
Genus Homo
Species Sapiens
http://anthro.palomar.edu/animal/default.htm
 Five Domains of Life

Kingdom Types of Organisms

Monera bacteria, blue-green algae, and spirochetes

Protista protozoans and algae of various types
Fungi funguses, molds, mushrooms, yeasts, mildews, and
smuts
Plantae mosses, ferns, woody and non-woody flowering
(Plants) plants
Animalia sponges, worms, insects, fish, amphibians, reptiles,
(Animals) birds, and mammals
In biology, a kingdom or regnum is the top-level, or nearly
the top-level, taxon of organisms in scientific classification.
Originally two kingdoms were distinguished: the Animalia
for animals, and the Vegetabilia or Plantae for plants. Early
authors also treated minerals in a third kingdom Mineralia.

Viruses, which Kingdom?

UIUC microbiologist Carl Woese, Archaea

(Crafoord Prize in Biosciences. The Royal Swedish Academy of Sciences ),
The Archaea are a major group of prokaryotes. Their classification among
living things is difficult because they are similar to bacteria in some respects but
similar to eukaryotes in others.
 Classification of Virus

International Committee on Nomenclature of Viruses (ICNV), 1966,

Moscow
International Congress of Microbiology
1st report in 1970
6th report in 1995 The Classification and Nomenclature of Viruses
Nucleic acid type, capsid symmetry, envelope, virion characteristics

Virology, Bernard N. Fields etc. 3rd Edition, LW&W, 1996, Philadelphia, PA, USA
 Classification of Virus-cont

1 order, Mononegavirals, 3 families

71 families
The family Flaviviridae, Yellow fever, Dengue,
HPC, West Nile virus
The family Picornaviridae, HPA, HPB
The family Retroviridae, HTLV, Lentiviruses
The family Adenoviridae, AdV
The family Parvoviridae, AAV
11 subfamilies
164 genera, including many floating genera
>4,000 member viruses
 Virus

DNA Viruses
ssDNA Viruses, AAV
dsDNA Viruses, AdV
RNA Viruses
ssRNA Viruses, HIV
dsRNA Viruses, Rice dwarf phytoreovirus (RDV)
Enveloped Viruses, HIV
Non-enveloped Viruses, AAV, AdV, Herpes viruses
 HIV (MoMuLV)--Models of Viruses

Retrovirus, Moloney murine leukaemia virus, MoMuLV

Lentivirus, Human immunodeficiency virus, HIV-1
 HIV and AIDS, the History and Now

After AIDS was recognized in 1981 by Gottlieb and coworkers as a new

human disease, several papers were published by Gallo and his
associates during 1983-4, invoking the oncovirus responsible for adult T-
cell leukaemia as the cause of AIDS. In 1983 the French scientist Barre-
Sinoussi and her colleagues succeeded in isolating a new agent in the
disease, a lentivirus, which they named LAV. The French immunologist
Klatzmann and his colleagues discovered that LAV killed CD4+ T-cells,
furnishing an explanation for the pathogenesis of AIDS and providing a
mechanism for how AIDS developed.
Karpas A. Biol Rev Camb Philos Soc. 2004 Nov;79(4):911-33.

Dr. Robert Gallo of Institute of Human Virology, University of Maryland and

Dr. Luc Montagnier of Institut Pasteur, France, two well-known competing
researchers with highly publicized rivalry, have buried their hatchets and
formed a partnership to jointly develop and conduct clinical trials on AIDS
vaccines. Discovery Medicine, Feb. 2002.
HIV Genome
 Important Genes in HIV

I. Gag
The major structural proteins. The precursor for the virus
core. It encodes four structural proteins
MA (matrix)
CA (capsid)
NC (nucleocapsid)
p6

II. Pol
The major enzymes. Three main enzymatic components
PR (protease)
RT (reverse transcriptase)
IN (integrase)
 Important Genes in HIV

III. Env (gp160)

The envelope protein. The partner to be recognized by T
cell.
Among one of the most extensively studied HIV proteins
SU (surface or gp120), outer membrane envelope, binds
to CD4 and coreceptor on T cells
TM (transmembrane or gp41)

IV. Rev
Crucial to viral replication and nuclear export
RRE, Rev-response element, a multistem-loop RNA
element to which Rev binds and expose the NES (nuclear
export signal
 Important Genes in HIV

V. Tat
Transactivating protein for transcription
Play important role in HIV life-cycle
Promoter is located in the 5 LTR (long-terminal repeat)
Transcription is enhanced 100- to 500-fold with in the
presence of Tat
HIV Life Cycle
 HIV Life Cycle

Virus Entry
Reverse Transcription
Nuclear Import
Integration
Gene Expression
RNA Export
Viral Particle Production
Budding
Maturation
 Virus Entry-1

The players in the virus entry

HIV-1 T cells
Envelope proteins (Env) CD4
gp160 Coreceptors
gp120 CXCR4
gp41 (TM) CCR5
(processed by a cellular protease)
 Virus Entry-2
The structure of Env

gp120, 5 conserved regions, C1-C5, C3 and C4 are

responsible for CD4 interaction. 5 variable regions V1-V5.
gp41, 3 regions, ectodomain, transmembrane sequence and
cytoplasmic tail
 Virus Entry-3
CD4 binding
1980s, CD4 molecule the primary cell-surface receptor for
SIV/HIV
CD4/Env interaction in CD4, a small segment of the N-
terminal extracellular domain
CD4/Env interaction in gp120, primarily C3 and C4

{Pseudotyped viruses (such as VSV-G and others) bind and

enter the target cells in a CD4-independent manner}
 Virus Entry-4
The structure of a complex of gp120/CD4 and hAb

Nature 393, 648 - 659 (18 June 1998)

 Virus Entry-5
Coreceptor interactions
G-protein coupled 7-transmembrane receptors
Macrophage-tropic (or M-tropic), CCR5, b-chemokine
receptor. The found of CCR5 gene, Craig Venter
T-cell-line tropic (T or TCL-tropic), CXCR4 (fusin), a-
chemokine receptor
HIV tropism, V3 region
Virus nomenclature
X4, generally T-tropic
R5, generally M-tropic
R5X4, dual-tropic strains that use both CCR5 and CXC4
 Virus Entry-6
Membrane fusion

Primary function of gp120

Bind to the target cell
Interact with coreceptor
Induce conformational changes in gp41
The ectodomain of gp41 is at the heart of the fusion
reaction
The ectodomain: a highly hydrophobic N-terminal fusion
peptide and 2 heptad repeat motifs, the N-helix and the C-
helixa 6-helix bundle
 Virus Entry-7
Steps leading to membrane fusion induced by HIV-1 Env
 Post-Entry Events
The viral core enters the host cytoplasm
Uncoating
The formation of the reverse transcription complex (RTC)
The formation of the preintegration complex (PIC)
 Reverse Transcription-1
Reverse transcriptase

HIV-1, a heterodimer of two subunits, p66 and p51.

p51 is formed when the c-terminal 15kD RNase H of p66 is removed by PR
Different conformation of p66 and p51
Reverse Transcription-2
 Reverse Transcription-3

1. A primer (tRNA) is bound to the primer binding site (PBS), DNA systhesis
proceeds to the 5 end of the RNA molecule, RNA/DNA hybrid.
2. RNA degraded by RNase H, generating the minus-strand strong stop DNA.
3. First strand transfer. The minus-strand strong stop DNA jumps from 5 to 3.
4. Minus-strand systhesis.
5. Plus-strand systhesis using the RNA remaining from minus-strand as primer.
Primary priming sites, polypurine tract (PPT)
6. tRNA bound to the PBS is removed by RNase H.
7. Plus-strand systhesis proceeds to the end of the minus-strand

High mutation rate of HIV-1 RT (3X10-5 per cycle of replication) because of the
jumping and loose interaction between RT/template. One of the reasons that
HIV can rapidly evade host immune response and develop resistance to
antiviral drugs.
Reverse Transcription-4
 Reverse Transcription-5

RT as an early target to develop antiviral drugs

Two general classes

The nucleoside analogs, AZT (zidovudine), ddI
(didanosine), ddC (zalcitabine) and 3TC
(lamivudine)

The nonnucleoside inhibitors, nevirapine and

delavirdine.
 Nuclear Import
There is still a lot of debate which proteins are involved in this
process.
Players: Vpr, IN and central DNA flap
 Integration-1

Integrase (IN) catalyzes the insertion of the linear,

double-stranded viral DNA into the host cell
chromosome to form the provirus. Integration is an
essential step in retrovirus replication.
 Integration-2
The structure of the integrase

Three domains
N-terminal domain that contains a zinc-finger
a central core sequence, active site
a C-terminal domain

IN functions as a multimer
 Integration-3
Integration steps

1. 3-end processing. IN clips off several nucleotides

from the 3 termini of both strands to generate a
molecule of dsDNA with 3-recessed ends.
2. IN makes a staggered cleavage in the cellular
target DNA.
3. Strand transfer. The 3-recessed viral DNA ends
are joined to the ends of the cellular DNA.
Cellular repair enzymes fill in the gaps.
 Gene Expression-1

HIV-1 LTR, site of transcriptional initiation, U3/R junction

LTR, U3 (unique, 3-end), R (repeat), and U5 (unique, 5-end)
RNA polymerase II
Gene Expression-2
Structure of HIV-1 LTR
 Gene Expression-3
Function of Tat protein

The existence of Tat protein (101 aa, an transcriptional

transactivator) greatly increases the RNA synthesis
(over 2-logs).
1. Tat acts on an RNA element, the transactivation
response region (TAR), a stem-loop structure.
2. Tat binds to the TAR bulge region
3. Tat needs some cellular proteins for function to
phosphorylate the C-terminal domain of RNAP II.
Those proteins are cyclin T1, cyclin-dependent
kinase CDK9, and positive-transcriptional elongation
factor b.
 RNA Export-1
Elements required for RNA export
 RNA Export-2
RNA species generated from HIV provirus:
1. Unspliced mRNAs-Gag, GagPol polyproteins.
2. Partially spliced mRNA-around 5 kn, Env, Vif, Vpu and Vpr
3. Small (1.7-2.0 kn) mRNA-Rev, Tat and Nef

Challenge, the unspliced and partially spliced RNA need

to be exported to cytoplasm

Rev (regulator of expression of viral proteins), a 19 kd

(116aa) phosphoprotein

RRE (Rev responsive element, 250 nu), a cis-acting

RNA elements
 RNA Export-3
REV/RNA complex
Rev, 2 functional domains
Arg-rich motif required for RNA binding and nuclear localization
Leu-rich motif mediated nuclear export
RRE, a large, highly structured RNA element, folds into a series SLs
A Rev monomer initially binds to SL-2, and promotes a cooperative
protein-protein and protein-RNA interactions to form multimer
of Rev
Finally form of complex of ~ 8Rev:1RRE
Rev can recycle back to nuclei by using its nuclear localization signal
(NLS) after exporting the RNA into cytoplasm.
HIV Virus Particle Assembly-Plasma Membrane
The Viral Particle Production-Gag proteins
The Viral Particle Assembly-1
 The Viral Particle Assembly-2

The NC domain of Gag directs genome packaging

Chimeric hHIV-1 with MoMuLV NC that preferentially package

MoMuLV

MoMuLV chimeras containing the HIV-1 NC domain preferentially

package the HIV-1 genome

MoMuLV chimeras with MoMuLV NC only pack the full-length

HIV-1 genome and ignore spliced HIV-1 mRNA
 The Viral Particle Assembly-3

Zinc knuckles of NC domain are essential for genome

packaging

The NC domains of all retroviruses, except the

apumaretroviruses, contain 1 or 2 conserved CCHC arrays that bind Zn++
with high affinity

C-X2-C-X4-H-X4-C
C, Cys; H, His; X, variable amino acid

Mutations that inhibit zinc binding, the treatment that blocks zinc
from binding, result in non-infectious viral particles that are unable to
replicate
The Viral Particle Assembly-4

Structure of the HIV-1 NC (p7) protein

 The Viral Particle Assembly-5

RNA elements that promote genome packaging

The most efficient packaging is generally achieved for RNAs that

contain relatively large portions of the 5-UTR.
The 5-UTR also contains
- U5 control regions that recruit cellular proteins required to initiate
ribosomal translation;
- A tRNA-primer binding site (PBS) that is required for reverse
transcription;
- The 5 splice donor (SD) site that participates in the generation of spliced
viral transcripts;
- Elements that promote RNA dimerization.
 The Viral Particle Assembly-6
RNA elements that promote genome packaging (MoMuLV)

DIS, dimerization initiation site; SD, splice donor site; SL, stem loop
 The Viral Particle Assembly-7
Potential dimerization mechanism for DIS-2 (278-309) (MoMuLV)

NC-binding sequence UAUCUG is highlighted by red

 The Viral Particle Assembly-8
Potential 2nd structure 204-380 (DIC-1 and DIC-2) of the dimeric 5-UTR (MoMuLV)
 The Viral Particle Assembly-9
Proposed model for specific packaging of a dimeric genome
 The Viral Particle Assembly-10
Model for dimeric genome recognition and the early stage of retrovirus assembly
 The Viral Particle Assembly-11
HIV genome structure, splicing pattern and location of the packaging site

DIS, dimerization initiation site; SD, splice donor site; SL, stem loop
 The Viral Particle Assembly-12
Structures of the complexes of HIV-1 NC protein and stem-loops
 The Viral Particle Assembly-13
Predicted 2nd structure of the intact HIV-1 5-UTR

PBS, primer-binding site; CAP, 5 cap; LTR, long terminal repeat; TAR, trans-
acting responsive element
 Env Synthesis, Transportation and
Incorporation

The HIV-1 Env precursor protein gp160, rough ER

The Env gp160 is transported to the cell surface via the secretary pathway
The Env gp160 is cleaved by a host protease, furin or furin-like enzyme to
generate gp120 (SU) and gp41 (TM)
The gp120 domain is heavily glycosylated
The functional multimeric form of Env appears to be trimer
The Env is recruited into virion through the interaction between the gp41
cytoplasmic tail and the MA
 Budding

Retroviruses encode specific sequences that promote viral particle release-

late or L domain
HIV-1, the L domain is present in p6. PT/SAP, which interacts with WW
family of cellular proteins
 Maturation

During or shortly after virus release from the plasma membrane,

the viral PR cleaves the Gag and GagPol polyprotein
precursors to generate the mature Gag and Pol proteins
PR-mediated Gag and GagPol processing sets in motion a series
of structural rearrangements, which is considered as a 2nd
assembly or re-assembly reaction
The absolute requirement for PR-mediated virion maturation has
been applied to the treatment of HIV infection using PR
inhibitors
HAART, highly active antiretroviral therapy (cocktail treatment),
combines 3 different drugs (anti-RT inhibitors, PR inhibitors
and others)
Maturation
 Role of the HIV Accessory Proteins

Accessory (auxiliary) proteins, not required in culture for viral replication

Vpu
Vpr
Vif
Nef
 Role of the HIV Accessory Proteins-Vpu

Vpu, viral protein u, an 81 aa integral membrane phosphoprotein

Unique in HIV-1
Two major functions during HIV-1 replication
It enhances the release of virus particles
It promotes the degradation of CD4
 Role of the HIV Accessory Proteins-Vpr

Vpu, viral protein r, a 14 kd, 96 aa protein

Incorporated efficiently into virion, interaction with a Leu-rich motif
in the C-terminal of p6
Major functions
Weakly stimulates gene expression from HIV LTR
Induces the arrest of Vpr-expressing cells in the G2 phase, which
facilitates transport of the viral PIC to the nucleus
 Role of the HIV Accessory Proteins-Vif

Vif, viral infectivity factor, highly conserved among LV

Vif mutation can cause profound defects in virus infectivity
 Role of the HIV Accessory Proteins-Nef

Nef, negative factor, a 27 kDa membrane-associated phosphoprotein

through its membrane binding myristic acid moiety
Nef plays an important POSITIVE role in LV pathogenesis
HIV Life Cycle
Major references for this session
1. HIV replication, Freed 2001
2. How retroviruses select their genome
Papers will be discussed in this session
1. HIV-1 gp41 as a target for viral entry inhibitor.
2. A hard way to the nucleus.
3. The molecular basis of HIV capsid assembly.
4. The packaging and maturation of the HIV pol protein