BB20023/0110: DNA & disease
2005-06
Oncogenic viruses
Outline
DNA viruses
 Herpesviridae (, HHV8/Kaposi’s sarcoma,
Epstein-Barr virus)
 Papovaviridae (human papilloma virus – HPV)
 Hepadnaviridae (hepatitis B virus-HBV)
RNA viruses
 Flavivridae (hepatitis C virus)
 Retroviridae ( Human T-cell
lymphotropic virus –HTLV type I)
Overview of viral replication
EBV -Epstein-Barr virus
Epstein–Barr virus is the most potent transforming agent, widespread in all human
populations, and is usually carried as an asymptomatic persistent infection. In a small
minority of infected individuals the virus is associated with the pathogenesis of certain types
of lymphoid and nonlymphoid cancers, including Burkitt lymphoma (BL), Hodgkin disease
and nasopharyngeal carcinoma (NPC). BL and NPC have geographically restricted
distributions, with BL found in young children in the central Africa, while NPC is found
predominantly in the southern coastal region of China. Hodgkin disease has been associated
with EBV infection without a clear aetiological role. EBV is also associated with a number of
lymphoproliferative diseases, which are mainly of B-cell origin.
Replication and Latency:
Virus infection involves two cellular compartments:
(1) B cells, where infection is predominantly latent and has the potential to induce growthtransformation of infected cells; and
(2) epithelial cells, where infection is predominantly replicative.
EBV immortalises B-cells, the main mediator of primary and persistent infection. Primary
EBV infection is usually followed by latency, but in immunodeficiency, the proliferation of
cells can proceed unchecked. Following primary infection of B cells, a chronic virus carrier
state is established in which the outgrowth of EBV-transformed B cells is controlled by an
EBV-specific cytotoxic T lymphocyte response re-activated from a pool of virus-specific
memory T cells. At certain sites, latently infected B cells can become permissive for lytic
EBV infection. Infectious virus released from these cells can be shed directly into the saliva
or might infect epithelial cells and other B cells. In this way a virus-carrier state is established
that is characterised by persistent, latent infection in circulating B cells and occasional EBV
replication in B cells and epithelial cells
Dr. MV Hejmadi
0023: DNA: making, breaking & disease
Transformation:
The virus enters B cells by interaction of the
major viral glycoprotein gp350/220 with the
complement
receptor
(CR2/CD21,
an
immunoglobulin superfamily member). This
complex mediates an interaction between EBV
and major histocompatibility complex (MHC)
class II molecules, which serve as a co-receptor
for virus entry into B cells. Once the viral
genome has been uncoated and transferred to
the nucleus, circularisation and transcription
from the Wp promoter begin a cascade of
events leading to expression of all the latent
genes. The EBV nuclear antigen (EBNA)
leader protein (EBNA-LP) and EBNA2 are the
first proteins to be expressed and these are
sufficient to advance the cells to early G1
phase of the cell cycle. The EBV-infected
(EBV+) cells begin to proliferate in a manner
that depends on high cell density and on the
autocrine production of cytokines that promote
B-cell growth. Later, the EBV+ cells evolve
into cells that grow more rapidly and are less
dependent on autocrine growth mechanisms.
Following infection in vivo, the virus
establishes a latent infection in memory B cells
and this is characterised by the limited
expression of a subset of virus latent genes.
Role of Epstein–Barr virus in the pathogenesis of nasopharyngeal carcinoma In both
nasopharyngeal carcinoma (NPC) and Epstein–Barr virus (EBV)-positive gastric carcinoma, the
tumour cells carry monoclonal viral genomes, which indicates that EBV infection must have occurred
prior to expansion
of the malignant
cell
clone..
Multiple
genetic
changes
have
been found in
NPC,
with
frequent deletion
of regions on
chromosomes 3p,
9p, 11q, 13q and
14q and promoter
hypermethylation
of specific genes
on chromosomes
3p
(RASSF1A
and retinoic-acid
receptor 2) and
9p (genes that
encode
INK4A,
INK4B, ARF and
death-associated
protein
kinase).
Deletions in both
3p and 9p in low-grade dysplastic lesions and in normal nasopharyngeal epithelium from individuals
who are at high risk of developing NPC in the absence of EBV infection, indicate that genetic events
occur early in the pathogenesis of NPC and that these might cause predisposition to subsequent EBV
Dr. MV Hejmadi
0023: DNA: making, breaking & disease
infection.The above scheme (see figure; images show stained epithelial sections) has been proposed,
in which loss of heterozygosity (LOH) occurs early in the pathogenesis of NPC, possibly as a result of
exposure to environmental cofactors such as dietary components (such as salted fish). This results in
low-grade pre-invasive lesions that, after additional genetic and epigenetic events, become
susceptible to EBV infection. Once cells have become infected, EBV latent genes provide growth and
survival benefits, resulting in the development of NPC. Additional genetic and epigenetic changes
occur after EBV infection. CIS, carcinoma in situ; EDNRB, endothelin receptor B; H/E, staining with
haematoxylin and eosin; TSLC1, tumour suppressor in lung cancer 1.
HPV human papilloma virus
These viruses cause lower genital tract cancers; most commonly cervical cancer. Since HPVs
replicate in stratified epithelial cells only, they are the aetiological agents of squamous cell
carcinomas (SCCs) at various sites. Some of the high risk HPVs in cancer are HPV16, 18, 31 & 33.
Genome: HPV has a small circular genome of approximately 8 kbp and the mRNA is transcribed off
one strand of the DNA by host cell RNA polymerase II. There are five early genes coding for
proteins, which are involved in DNA replication (E1 and E2) or activation of cell growth (E6, E7 and
E5), and three late genes, two of which code for the major and minor capsid proteins (L1 and L2,
respectively) and one of unknown function (E4). Like most of the tumour viruses, the cell/growthactivating proteins affect either the signal transduction pathways or cell cycle control.
Replication: During the malignant phase of the disease, when the lesion is confined to the epithelium,
the viral DNA is in an episomal form, and amplification of the genome and viral particle production
occurs in the upper parts of the epithelium. In malignant cells, the genome is integrated randomly in
over 70% of cases, while in the rest there are episomal copies. All malignant cells continue to express
E6 and E7 proteins, suggesting that these proteins are essential for maintenance of the malignant
phenotype.
Cancer transformation: The transforming activity of HPV16 is associated with mainly E6 and E7
proteins and some from E5. E6 and E7 are multifunctional proteins that can increase cell
proliferation and survival by several pathways. E.g. E6 binds to the E6-associated protein (E6-AP)
and p53 (tumour suppressor) in a heterotrimeric complex. The result of this binding is the
premature degradation of p53 through the ubiquitin pathway. E6-AP is in fact a ubiquitin protein
ligase. Since one of the functions of p53 is to control the passage of cells through the G1 phase of
the cell cycle, any abrogation of this activity could lead to uncontrolled cell cycle progression.
E7 binds to the retinoblastoma family of tumour suppressor proteins, RB, p107 and p130. Normally,
late in G1 RB is phosphorylated, and this releases transcription factors such as E2F, important for
DNA synthesis. E7 can cause this same release of factors in the absence of RB phosphorylation and
drive cells into unregulated S phase. E7 has also been shown to bind the AP-1 family of
transcription factors and bind/compete with histone deacetylase for binding to RB.
HBV- hepatitis B virus
HBV is one of the aetiological agents of hepatitis and hepatocellular carcinoma (HCC). There is a
higher incidence of HCC in regions endemic to HBV infection contracted neonatally with persistence.
In USA and northern Europe, infection usually occurs in adulthood and persistence occurs in
approximately 1–10% of infected individuals. There is now a successful vaccine consisting of the
surface antigen (HBsAg) of the virus, which protects against infection of HBV.
RNA VIRUSES
replicate via a DNA copy of their RNA genome. They may be either
 oncogenic (e.g Rous Sarcoma Virus, human T-cell leukaemia virus 1 & 2) or
 non oncogenic ( e.g. Human Immunodeficiency Virus, HIV-1 & HIV-2)
Retroviral Replication
 RNA genome serves directly as mRNA
 Reverse transcriptase uses viral RNA as template for making cDNA strand using tRNA
as a primer
 Rnase H partially digests primer
 Circularises by RNA-DNA base pairing
Dr. MV Hejmadi
0023: DNA: making, breaking & disease
 DNA synthesised and tRNA primer excised by RnaseH
 RNA strand displaced and DNA strand linearly recombines with host DNA
HCV hepatitis C virus (Flaviridae)
HCV infection leads to persistent infection in 50–80% of individuals, a much higher rate than seen for
HBV in adults. HCV is increasingly recognized as a cause of HCC, and this will presumably continue
as HBV immunization increases. Approximately 10–20% of persistent carriers will develop cirrhosis
of the liver, and, of these, 1.5% will go on to develop HCC. At present, no other cancer has been
associated with HCV infection
HTLV-1 Human T-cell lymphotropic virus types I (Retroviridae)
HTLV-I causes adult T-cell leukaemia/lymphoma, which is endemic in Japan, West and Central
Africa Caribbean population of African descent and parts of the south Pacific islands. Infection
typically precedes cancer by several decades, and there is a very tight association between the
development of cancer and HTLV-I seropositivity. However, the lifetime risk of an HTLV-Iseropositive carrier is estimated to be 2–4%, and most have acquired the infection as children.
Genomic organization
This is a positive-sense, single-stranded RNA virus, which, like all retroviruses, carries two copies of
the genome in the virion. It has a genome of 9 kb and is organized with terminal repeats at either end.
The genome codes for viral structural proteins (Gag-core protein, Pol-polymerase and Env-envelope
proteins) and the organization of these is similar to that of other retroviruses. In addition, located at
the 3′ end of the genome are at least four ORFs coding for regulatory proteins.
Replication: As part of their natural replication process, retroviruses integrate proviral DNA
sequences into the host cell chromosome, as they code for an RNA-dependent DNA polymerase
which will produce a double-stranded complementary DNA molecule with long terminal repeat
sequences which allow efficient integration in a sequence-independent manner. This keeps all the
coding capacity of the genome intact, and so progeny RNA is produced from the integrated
sequences.
Activation of cell growth :HTLV-I codes for a number of proteins with sequences near the 3′ end of
the genome, often called the pX region. Two of the best-characterized proteins are Tax and Rex. Tax
can transactivate the HTLV-I promoter through binding to the transcription factor CREB (cAMP
response element-binding protein) and the coactivator CBP (CREB-binding protein), mediating the
interaction of the latter with the basal transcription machinery. In fact, Tax will activate a number of
different cellular promoters through CREB binding and also binding NFκB transcription factors,
resulting in the expression of a number of cellular genes, including IL-2, TGF-β, GM-CSF, IL-2α
receptor and c-myc. IL-2 is an important growth factor for T cells, the host cells for HTLV-I.
Rex is a nuclear protein and regulates the balance between spliced and nonspliced mRNA, thus
favouring the transcription and translation of the gag, pol and env genes over the cell regulatory genes
expressed through multiply-spliced transcripts. Rex also appears to affect cellular processes, and is
thought to act with Tax to increase expression of IL-2α receptor, by an as yet unknown mechanism.
Human Immunodeficiency Virus (HIV-1)
HIV genome contains 2 copies of +ve strand RNA molecule (9kb long each).
Genome encodes 9 genes including
3 structural genes
 gag (group specific antigen)
encodes matrix, capsid,
nucleocapsid proteins
 pol (polymerase) encodes reverse
transcriptase, integrase, protease
 env (envelope) encodes surface &
transmembrane protein
6 regulatory genes
 rev (regulatory virus protein)
 tat (transactivator)
 nef (negative regulatory factor)
 vif, vpr, vpu
Dr. MV Hejmadi
0023: DNA: making, breaking & disease
The 3 functions of the pol gene include
Reverse transcriptase: polymerase activity enabling copying of RNA into dsDNA
Integrase: splice HIV DNA permanently into host chromosome
Protease: cleaves polyprotein transcripts into packageable forms.
Course of HIV infection:
The virus is usually transmitted through sexual intercourse, direct exposure to contaminated blood,
or transmission from a mother to her fetus
or suckling infant.
HIV invades certain cells of the immune
system--including CD4, or helper T
lymphocytes or certain macrophages -replicates inside them and spreads to other
cells. HIV infections start with a dramatic
drop in CD4 cells (acute phase) within 3-6
weeks, followed by a steady state of viral
replication (set point) at about 6 months.
CD4 T cell concentrations gradually fall
over a period of 8 – 10 years (chronic
phase) to below 200 cells / mm3 of blood (
onset of AIDS).
COURSE OF HIV INFECTION
Group1 – seroconversion-acquisition of virus; flu like symptoms
Group 2 – asymptomatic stage (can be 10yr)
Group 3 – generalised persistent lymphadenopathy (GPL) lasts ~ 3mth
Group 4 – symptomatic infection
Antiretroviral or anti HIV therapy
All approved anti-HIV drugs attempt to block viral replication within cells by inhibiting either
RT or HIV protease.
 Nucleoside analogues mimic HIV nucleosides preventing DNA strand completion e.g.
Zidovudine (AZT), ddI, ddC, Stavudine
 Non nucleoside RT inhibitors (NNRTI) e.g Delavirdine and Nevirapine
 Protease inhibitors block active, catalytic site of HIV protease
Multidrug therapy
HAART (highly active antiretroviral therapy) usually consists of triple therapy including 2
nucleoside analogues + 1 protease inhibitor
1 non nucleoside RT inhibitor + 1(2) prot. inhibitor
Reading:
Chapter 13: Mol & Cell Biol of Cancer by Knowles and Selby
Optional Reading
 Epstein-Barr virus: 40 years on Nature Reviews Cancer 4 (10)757-68 Oct 2004 Young LS,
Rickinson AB
 Immunology of hepatitis B virus and hepatitis C virus infection Nature Reviews Immunology 5,
215-229 (2005) Barbara Rehermann & Michelina Nascimbeni
Dr. MV Hejmadi