Viral infection and cancer
Summary
Infection with specific viruses has a role in the
pathogenesis of some cancers in human beings. However,
the incidence of such cancers is much lower than the
frequency of virus infection, suggesting either that
infection alone does not result in cancer and that cellular
events in addition to the presence of the virus must occur,
or that cancer occurs only if viral proteins are expressed in
an inappropriate cell type or in an immunocompromised
host. Molecular analysis of viruses found in association
with cancer has revealed that they function, at least in
part, by encoding proteins which can associate with and
subvert the function of host cell-encoded tumour
suppressor proteins which regulate pathways of growth
arrest and apoptosis. Better understanding of the
mechanisms underlying this association will have
diagnostic, prognostic, and therapeutic implications in the
near future.
King’s College School of Medicine and Dentistry, Rayne Institute,
123 Coldharbour Lane, London SE5 9NU, UK (J D H Morris PhD,
A L W F Eddleston FRCP, T Crook PhD)
Correspondence to: Dr J D H Morris
754
Introduction
The possibility that infectious agents are involved in the
aetiology of cancer in human beings, or perhaps even have
a direct causative role, has excited investigators since
Rous demonstrated at the start of this century that
chicken sarcomas could be transmitted by cell-free
filtrates. This observation, together with the later
discovery of other oncogenic animal viruses such as
polyoma virus, prompted the hope that many human
cancers would prove to have an infectious aetiology. In the
context of these expectations, which have persisted for
almost a century, what is currently known about the role
of viral infection in the pathogenesis of cancer?
Carcinogenesis requires multiple steps
It is now clear that acquisition of a malignant phenotype
usually requires accumulation of multiple genetic changes
by the cell.’ Fundamental amongst these changes are the
activation by genetic mutation of growth-promoting
properties of cellular oncogenes such as the ras family,
and the inactivation of tumour suppressor genes such as
p53, causing loss of the negative growth-regulatory
functions. Many other changes also occur in tumour cells,
the phenotype effects of which include abnormal
expression of proteins regulating angiogenesis, and of
enzymes such as the matrix metalloproteinases and
cathepsins which are involved in destruction of the
cellular basement membrane and facilitate dissemination
of tumour cells. It is becoming evident that, whereas the
frequency of activation of the growth-promoting
properties of the oncogenes varies widely between types of
tumour, subversion of the negative regulatory effects of
tumour-suppressor-gene products may be an invariable
requirement for carcinogenesis. Some of the biochemical
pathways of these products are the targets of some cancer
viruses.
Viruses and cancer
Viruses are the most important class of infectious agents
associated with human cancer, in terms both of the
number of cancers which have an established aetiological
link with viruses, and the numbers of cases. Viruses which
contribute to the development of human cancers are most
readily described in groups rather than in relation to the
specific cancers with which they are linked. However,
before looking at these groups, some general observations
may be useful.
Prevalence of infection is always much higher than the
incidence of the associated tumour. For example, Epstein
Barr Virus (EBV), a ubiquitous infectious agent with a
long evolutionary relationship with its human host, causes
cancer only under conditions of host immunosuppression
or when specific viral proteins are expressed in an unusual
cell type.
 The presence of the virus (and expression of viral
proteins) is not in itself sufficient for the development of
the associated tumour. Additional factors-genetic,
epigenetic, hormonal, immunological-are necessary for
malignant transformation. In some instances, an
oncogenic virus initiates a multi-stage process of
carcinogenesis by expressing proteins which associate with
host-cell-encoded tumour-suppressor proteins, thereby
abrogating their cell-cycle check-point functions and
predisposing the cell to genetic instability.
* Development of cancer often (but not invariably) occurs
many years after infection. This latency period may be a
reflection of the requirement for accumulation of the
additional genetic changes mediating malignant
progression in a virally infected cell.
Retroviruses
In the involvement of retroviruses in animal
contrast to
cancers, only two have been implicated in human cancer:
human T-cell leukaemia virus (HTLV-1) and human
immunodeficiency virus (HIV).
HTLV-1-HTLV-1has been established by serological
and molecular analyses as the cause of adult T-cell
leukaemia (ATL), a disease characterised by malignant
growth of CD4+ T-lymphocytes, with presenting
symptoms of hypercalcaemia, lymphadenopathy, and
immunodeficiency.The prognosis for patients with acute
ATL is bad, with average survival after diagnosis of only a
few months. Only 1-4% of individuals infected with
HTLV-1develop ATL, with a latency period of several
decades after infection. Infection with HTLV-1is also
linked to the neurological disorder tropical spastic
paraparesis.3 A notable characteristic of leukaemic cells
isolated from patients with ATL is the absence of
detectable viral-gene expression, suggesting that continual
expression of viral gene products may not be necessary for
maintenance of malignant phenotype. Unlike other
known animal retroviruses, HTLV-1encodes a putative
oncogene Tax, which has no known cellular homologue.
Little is known of the mechanism by which Tax exerts a
transforming function as it is an extremely difficult gene
to express stably in cells, perhaps as a result of its toxicity.
Nevertheless, Tax has been implicated in growth
deregulation via transcriptional upregulation of growth-
related genes such as fos and certain lymphokines.
HIV-1-The increased incidence of some tumours in HIV-
infected individuals4 is almost certainly due to the chronic
immunosuppression which occurs in the disease, and is
not a result of any direct oncogenic activity of HIV.
Studies of the types of tumour which arise in these
patients have provided unequivocal evidence of the role(s)
of viruses in cancer, and the propensity of virally-related
cancers to occur in situations where host immunity is
compromised (exemplified by the EBV-related
immunoblastic lymphomas referred to below). In addition
to increased incidence of EBV-related immunoblastic
lymphomas (together with other non-Hodgkin’s
lymphomas) and Kaposi’s sarcoma in HIV-related
disease, there is now increasing evidence that human
papillomavirus (HPV)-related anogenital cancers, notably
cervical cancer in and anal cancer in men, have a
women
greater incidence in patients with HIV, strengthening the
case for the involvement of viruses in these tumours.5
DNA and RNA tumour viruses
Several DNA and RNA tumour viruses are aetiologically
linked to human cancer, including HPV, hepatitis B and
C viruses (HBV, HCV), and the herpesviruses Epstein-
Barr and the newly described Kaposi’s sarcoma associated
herpesviruses (KSHV).
Papillomaviruses--HPVs constitute a large group of
small DNA viruses which infect epithelial tissue.6 Over 70
different types have been described, and more almost
certainly await identification. Although HPV infection is
common, this usually results in self-limiting benign
lesions such as skin warts due to infection with HPV types
1 and 2. However, there is now evidence that sexually-
transmitted infection of the genital epithelium with "high-
risk" HPV types (mainly 16 and 18) is causally linked to
development of high-grade pre-malignant lesions (cervical
intraepithelial neoplasia [CIN] III) and anogenital cancer,
principally of uterine cervix, but also of anus and vulva.
Epidemiological evidence associating specific HPV
types with cancer is convincing, despite earlier confusion
resulting from overestimation of HPV prevalence in
cytologically normal cervical smears.7 DNA sequences
from "high risk" HPVs are detectable in the majority of
cervical and anal tumours, and a lower proportion of
vulval cancers. An illustration of the importance of HPV
in CIN and anogenital cancer, is provided in the study by
Cuzick and co-workers,8 where a substantial number of
apparently normal cervical smears were shown by
polymerase chain reaction (PCR) to have high levels of
DNA sequences from "high risk" virus types, and
subsequently shown by colposcopy to have underlying
CIN II/III. This finding extends a previous study by the
same authors,9 and implies that routine testing for HPV
,
(by automated PCR for example) would provide a useful
adjunct to standard cytological testing.
Given, however, this clear association between HPV
and anogenital cancer, why do only a proportion of
women infected with "high risk" HPV types develop
cancer? It is likely that, in most cases, the infection is
cleared without inducing cytological change. In a small
proportion of women, however, the infection persists,
predisposing them to CIN and cancer. It is clear that
HPV infection alone is insufficient for carcinogenesis,’"
additional changes are required. Identification of these
changes would be expected to have a major impact on the
incidence of anogenital cancer, yet at present they remain
poorly defined. The ability of HPV-encoded proteins E6
and E7 to abrogate the apoptotic response of cells to
DNA damage is evidence that these proteins function in
tumourigenesis, at least in part, by nullifying the
mechanisms by which cells maintain genomic stability in
the face of genotoxic attack. These functions are mediated
through interaction with host-encoded tumour-
suppressor-gene products. The E7 protein binds to and
functionally inactivates proteins of the retinoblastoma
(RB) family, whilst E6 associates with, and promotes the
proteolytic degradation of p53. The E6 and E7 proteins
from "high-risk" virus types perform these functions more
efficiently than "low-risk" viruses.11,12
Herpesviruses: Epstein-Barr virus
Whereas infection with HPV is commonly relative to the
incidence of its associated tumours, this phenomenon is
even more pronounced in the case of EBV, a
lymphotropic herpes virus, endemic in all human
755
populations and transmitted orally to uninfected
individuals during the early years of life. In the majority of
such people the virus is then carried as a life-long latent
infection of lymphoid tissue and carries no cancer risk."
However, the virus has oncogenic potential, since a small
number of EBV-encoded proteins which are expressed in
viral latency have the ability to deregulate control of cell
growth. 14 The conservation of these proteins amongst
naturally-occurring EBV isolates implies that they
function normally as critical components of viral
persistence in latency, but when expressed in an
immunologically compromised host they may be involved
in tumourigenesis. Although EBV was first associated
with Burkitt’s lymphoma (BL) and undifferentiated
nasopharyngeal carcinoma (NPC), more recent evidence
(principally the detection of EBV-DNA sequences) has
suggested that the virus may be involved in the
pathogenesis of other tumours.15 In the
immunocompetent host, these include Hodgkin’s disease,
non-Hodgkin’s lymphoma, thymic lymphoepithelioma,
salivary gland carcinoma, and urogenital carcinoma; in
the immunocompromised host, immunoblastic
lymphomas are seen in AIDS, and lymphoproliferative
disease in graft recipients.
Immunoblastic lymphomas-Perhaps the most
convincing example of tumours in which EBV is involved
(and in which it may well be the sole agent responsible)
are the immunoblastic lymphomas seen in patients with
long-term immunosuppression (eg, bone marrow
recipients and renal allograft patients) or AIDS. The
common factor is profound suppression of T-lymphocyte
function, and the tumours which arise are invariably EBV
positive. 16 These tumours express a number of EBV-
encoded genes (the viral latent proteins) and retain a
lymphoblastoid phenotype. They are therefore
phenotypically similar to EBV-immortalised B-
lymphoblastoid cell lines. The characteristic absence of
karyotypic instability in these tumours is consistent with
the hypothesis that one or more EBV-encoded proteins
are responsible for the malignant phenotype of these
tumours, and implies that under conditions of impaired
T-cell function, EBV is not only necessary but also
sufficient for tumourigenesis.
Burkitt’s lymphoma-In contrast to immunoblastic
lymphomas, the natural history of other EBV-associated
tumours much closely follows the classical multi-stage
model of carcinogenesis. Moreover, the evidence linking
EBV to tumour development is less definitive. BL is a
high-grade B-cell lymphoma characterised by reciprocal
chromosome translocations between the c-myc locus on
chromosome 8 and one of the immunoglobulin loci on
chromosomes 2, 14, and 22. Three forms of BL are
recognised. The endemic form originally described by
Burkitt, is found in Africa and New Guinea, where it
occurs coincidentally with the malarial parasite
Plasmodium falciparum. In endemic BL, the majority of
tumours have EBV DNA sequences in all malignant cells,
whereas sporadic BL, whose worldwide incidence is
20-100 fold lower than the endemic form, occurs
independently of malaria and rarely (10-15%) in
association with EBV. In AIDS-related BL, EBV DNA is
detectable in 30-50% of patients. In BL the only virally-
encoded protein expressed is EBNA-1, a protein which
functions to maintain the episomal EBV genome.
756
Arguments that EBV has a direct causative role in BL are,
as expected, most readily applicable to the endemic form
of the disease, although the clonality of EBV in sporadic
BL is an observation consistent with an aetiological role,
rather than the view that the virus is a passenger-there
because of EBV infection of established BL cells.
Definitive evidence of a causative role for EBV in BL
awaits demonstration of convincing mechanisms by which
the virus contributes to malignant transformation, as well
as explanations for cases of BL which lack EBV
Nasopharyngeal carcinoma-In contrast to BL, EBV
DNA sequences are invariably present in undifferentiated
NPC, an epithelial tumour which shows wide
geographical variation in incidence. The basis for these
variations remains obscure, but may reflect differences in
genetic predisposition between racial groups or the effect
of environmental (including dietary) factors. In NPC,
EBV latent gene expression is different from BL, with
expression not only of EBNA 1 but of two latent
membrane proteins, LMPl and 2. The aetiological
association of EBV with NPC is somewhat stronger than
with BL and is consistent with the ability of LMP1 to
inhibit terminal differentiation of epithelial cells in
culture.
Hodgkin’s disease-Almost 50% of cases of HD in the
northern hemisphere are positive for EBV DNA
sequences, with a higher frequency in more aggressive
mixed cellularity forms of the disease than in other forms.
By in-situ hybridisation, the viral genome has been
localised to the malignant Reed-Sternberg cells in the
tumour, further supporting a specific role for EBV
Herpesvirus: Kaposi’s sarcoma-associated human
herpesvirus
The cloning from KS tissue of a virus with DNA
sequence homology to y-herpesviruses has added another
candidate to the list of potential human tumour viruses."
The observation that this organism occurs in both AIDS-
related and unrelated forms of the disease, strongly
18
supports an aetiological role for this herpesvirus.
Preliminary studies of the virus suggest that it has the
largest genome of any known herpesvirus.
Hepatitis Band C viruses
The lifetime risk of hepatocellular carcinoma (HCC) has
been estimated to increase by a factor of 30-100 in
chronic carriers of hepatitis B virus (HBV).19 It has also
been recognised for some time that cirrhosis of the liver
carries with it a high risk for development of HCC,
particularly in older men. It has been argued that all the
factors which can cause cirrhosis carry the same risk of
HCC, and that this is entirely a consequence of the
cirrhosis itself. "0 On the other hand, there are
undoubtedly cases, particularly in younger patients with
hepatitis B infection, where hepatocellular carcinoma has
arisen in a non-cirrhotic liver. This has encouraged the
search for specific virus-driven oncogenic events in
infected hepatocytes.21 HBV has a DNA genome of about
3000 base pairs encoding four open reading frames, of
which the most interesting from an oncogenic viewpoint is
the X antigen (HBx). This protein has a transactivating
function which may have a role in tumourigenesis by
regulating expression of other genes. Alternatively, the
propensity of the viral genome to integrate into the host
Figure: Functional inactivation of p53 by virally-encoded oncoproteins and its biological
consequences
DNA damage causes stabilisation of p53 protein in cells, activating the transcriptional regulatory functions of
p53 and resulting in Gl-S cell cycle arrest and/or apoptosis. The EBV-immortalised lymphoblastoid cell (B) does
not encode a protein which associates with p53, and therefore undergoes p53-dependent Gl-S arrest and/or
apoptosis. In contrast, neither Gl-S arrest nor apoptosis occur in the genital keratinocyte (K) expressing HPV E6
or the hepatocyte (H) expressing HBVx. since these viral proteins encode proteins which associate with, and
thereby inactivate the function of p53.

genome (and thus to generate gross chromosomal
abnormalities at the site of integration) raises the
possibility that insertional mutagenesis of host genes may
occur. Consistent with this possibility, there is good
evidence that integration is important for development of
HCC in the woodchuck. Woodchuck hepatitis virus
frequently integrates near the chromosomal location of
the myc gene family, resulting in increased expression of
the gene. Other evidence suggests that, as is the case with
HPV, viral-gene products which can interact with tumour-
suppressor genes may also be important in HBV-related
mechanisms to inactivate p53 and other tumour-
suppressor proteins. Studies of EBV-immortalised cells
suggest, however, that this may not invariably be the case,
since recent work shows that Gl-S checkpoint and
apoptosis-regulating function of p53 appears to be fully
intact in EBV-immortalised B lymphoblasts which express
the full range of EBV-encoded latent proteins.24 A directly
related question is why any virus should have followed an
evolutionary pathway which could result (by causing
cancer) in the death of its natural host. The probable
answer is that the potentially oncogenic activities of the

oncogenesis. HBx has been shown stably to associate with
p53 and inhibit p53-mediated transcriptional regulation
in vitro.22 In common with other virus-related cancers,
HCC frequently occurs after decades of persistent
infection, suggesting that additional molecular changes
must occur for tumour development to ensue. In this
context, HBx-mediated abrogation of p53-dependent cell
cycle checkpoint functions provides an obvious molecular
pathway for the breakdown of genomic stability in cells
chronically infected with HBV.
With respect to HCV, a single-strand RNA virus, very
little is known of possible mechanisms, although again the
occurrence of non-cirrhotic cases of HCC in association
with HCV infection raises the possibility of such a direct
oncogenic effect.23
Common pathways to virally-associated
tumours?
The observation that "high-risk" HPV types, in common
with adenovirus and SV40 virus, encode oncoproteins
which are able to bind to and functionally inactivate
cellular tumour suppressor proteins raises the possibility
that other human oncogenic viruses might encode
functionally homologous proteins. This possibility is
strengthened by the observation of a p53-neutralising
function for HBx22 (figure), suggesting that many
potentially oncogenic viruses have convergently evolved
viruses are side-effects of viral proteins whose function in
the life cycle of the virus is either to drive replication of
host cells (and thereby facilitate viral replication) or to
abrogate the apoptotic response of the host cell to viral
infection or replication. Different viruses have
convergently evolved their own mechanisms in order to
achieve this. In the case of HPV and HBV, the viral
proteins which abrogate the function of p53 (E6 and HBx
respectively) are expressed in, and clearly associated with,
tumourigenesis. In the case of EBV, the lytic cycle protein
BZLFl, rather than a latent protein, has been reported to
abrogate p53 function, consistent with the hypothesis that
viral replication (which occurs during lytic phase of EBV
infection) might induce p53 function and thence
apoptosis in host cells. This suggestion would also explain
why a p53-neutralising function is not required during
EBV latency, but in turn implies that loss of function of
p53 is not an obligatory requirement for development of
all virally-related tumours. Alternatively, perhaps genetic
changes allowing subversion of p53-mediated growth
control occur later in carcinogenesis in EBV-related
tumours. Studies of BL suggest that this may indeed be
the case.25
Future prospects
The recent increase in knowledge about how viruses
cause cancer has opened the door to a new era in the

757
application of molecular biology to clinical problems of
cancer. Cuzick and co-workers8 point the way forward for
the use of molecular techniques in diagnostics. Trials of
therapeutic vaccines against HPV, and prophylactic
vaccines against EBV are underway. Similarly, the impact
of vaccination programmes against HBV on the incidence
of HCC will become clearer in the years ahead.
From a therapeutic viewpoint, the ability of viral
oncoproteins to associate with cell-encoded proteins
presents a target for rational design of molecules which
can inhibit and/or disrupt these complexes, perhaps with
therapeutic potential. Of even more interest is the
possibility of exploiting the presence and expression of
virally-encoded proteins in tumours for cancer-cell-
specific gene therapy. An obvious example would be use
of the invariant expression of EBNA-1 in the tumour cells
of undifferentiated NPC to target these cells for cytotoxic
or immunological attack. The clinical years ahead will
undoubtedly witness the impact of a vast amount of basic
research in the clinic.
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