1. Oncogenic viruses Key Concepts
Normal cells infected with certain viruses can be transformed into cancer cells due to expression or activation of viral oncogenes
Transformation can result in integration of viral genes or genomes into the host genome

2. Is cancer infectious?
1909, Peyton Rous discovers sarcoma-inducing agent in chickens
Figure 3.1; 3.2 The Biology of Cancer (© Garland Science 2007)

3. Normal cells infected with certain viruses can be transformed
Figure 3.7a The Biology of Cancer (© Garland Science 2007)

4. Viral transformation can induce cellular changes including tumourigenicity
Table 3.2 The Biology of Cancer (© Garland Science 2007)

5. Approximately one in six human cancers is caused by a human tumour virus!!
DNA viruses
RNA viruses
Herpesviridae
Human Herpes Virus 8 (HHV8) a.k.a Kaposi’s sarcoma associated virus
Epstein-Barr virus (EBV)
Papovaviridae
human papilloma virus (HPV)
Hepadnaviridae
hepatitis B virus-(HBV)
Flaviviridae
(hepatitis C virus HCV)
Retroviridae
Human T-cell lymphotropic virus (HTLV type I)
Hepatitis B virus (HBV) causes most cancers of the liver (hepatocellular carcinomas) and the development and use of an effective vaccine is preventing new infections with HBV and eliminating the hepatocellular carcinomas it causes
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia . Epidemiological analyses have revealed that HTLV-1 is often passed to infants from their infected mothers’ milk. In Japan, where adult T-cell leukemia is endemic, mothers infected with HTLV-1 have been encouraged not to breastfeed their infants, 80% of whom are now uninfected by HTLV-1. Blocking this viral transmission will almost certainly eliminate those adult T-cell leukemias, which otherwise would have arisen 50–60 years from now.
Human papillomaviruses (HPV) cause the vast majority of cervical carcinomas. In developed nations, early detection and surgical intervention prevents most cervical carcinomas, and vaccines are being developed for the most prevalent strains of HPVs that are associated with cervical carcinomas, which are likely to prevent infection and the associated cancers in developing nations.
Not all cancers that are caused by human tumor viruses are amenable to being prevented. Some human tumor viruses, such as Epstein–Barr Virus (EBV), are ubiquitous but primarily cause cancers in the developing nations and are, therefore, unlikely to be the subject for successful vaccine development. An alternative strategy to cope with the viral tumors that cannot practically be prevented is to develop treatments targeted against the viral products that are required to maintain the infected cells as tumors. This strategy is feasible only if such products exist. Animal tumor viruses have previously been shown to encode genes that are required to maintain tumors. Recently, two human tumor viruses, HPV and EBV, have also been found to encode such genes.
Rous sarcoma virus (RSV) causes tumors rapidly following its inoculation into the wing webs of susceptible newborn chicks. Early genetic analyses identified a viral gene that was necessary for maintaining the transformation of infected cells, but not their infection. This gene, termed Src, was later shown to be the sole gene of RSV that was required to induce tumors. Such direct studies are not practical with human tumor viruses, however. Furthermore, human tumor viruses predominantly infect only humans, and animal hosts are often unavailable to model their oncogenesis. These complexities necessitate less direct approaches to identify any human tumor viral products that maintain tumor phenotypes.

6. Overview of viral replication

7. Genome replication
DNA viruses
RNA viruses

8. How does tumourigenicity occur?
Viral genomes show the presence of several human gene homologues (cellular proto-oncogenes)
Infective viruses ‘kidnap’ proto-oncogenes which are then transformed into oncogenes
e.g. c-src/v-src ; v-myc/c-myc ; vIL6/ IL6 (interleukin 6)

9. Insertion of viral sequences into host DNA carrying the proto-oncogene
E.g. Insertion of ALV into c-myc protooncogene
Figure 3.23a The Biology of Cancer (© Garland Science 2007)

10. v-myc and c-myc (myc oncogene)

11. Table 3.4 The Biology of Cancer (© Garland Science 2007)

12. DNA viruses Herpesviridae
Human Herpes Virus 8 (HHV8) a.k.a Kaposi’s sarcoma associated virus
Epstein-Barr virus (EBV)
Hepatitis B virus (HBV) causes most cancers of the liver (hepatocellular carcinomas) and the development and use of an effective vaccine is preventing new infections with HBV and eliminating the hepatocellular carcinomas it causes
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia . Epidemiological analyses have revealed that HTLV-1 is often passed to infants from their infected mothers’ milk. In Japan, where adult T-cell leukemia is endemic, mothers infected with HTLV-1 have been encouraged not to breastfeed their infants, 80% of whom are now uninfected by HTLV-1. Blocking this viral transmission will almost certainly eliminate those adult T-cell leukemias, which otherwise would have arisen 50–60 years from now.
Human papillomaviruses (HPV) cause the vast majority of cervical carcinomas. In developed nations, early detection and surgical intervention prevents most cervical carcinomas, and vaccines are being developed for the most prevalent strains of HPVs that are associated with cervical carcinomas, which are likely to prevent infection and the associated cancers in developing nations.
Not all cancers that are caused by human tumor viruses are amenable to being prevented. Some human tumor viruses, such as Epstein–Barr Virus (EBV), are ubiquitous but primarily cause cancers in the developing nations and are, therefore, unlikely to be the subject for successful vaccine development. An alternative strategy to cope with the viral tumors that cannot practically be prevented is to develop treatments targeted against the viral products that are required to maintain the infected cells as tumors. This strategy is feasible only if such products exist. Animal tumor viruses have previously been shown to encode genes that are required to maintain tumors. Recently, two human tumor viruses, HPV and EBV, have also been found to encode such genes.
Rous sarcoma virus (RSV) causes tumors rapidly following its inoculation into the wing webs of susceptible newborn chicks. Early genetic analyses identified a viral gene that was necessary for maintaining the transformation of infected cells, but not their infection. This gene, termed Src, was later shown to be the sole gene of RSV that was required to induce tumors. Such direct studies are not practical with human tumor viruses, however. Furthermore, human tumor viruses predominantly infect only humans, and animal hosts are often unavailable to model their oncogenesis. These complexities necessitate less direct approaches to identify any human tumor viral products that maintain tumor phenotypes.

13. EBV- Epstein Barr Virus
most potent transforming agent,
widespread in all human populations
usually carried as an asymptomatic persistent infection (latent).
virus sometimes associated with the pathogenesis of certain types of lymphoid and epithelial cancers, including
Burkitt lymphoma (BL),
Hodgkin disease and
nasopharyngeal carcinoma (NPC).
Virus infection involves two cellular compartments: (1) B cells, where infection is predominantly latent and has the potential to induce growth-transformation of infected cells; and
(2) epithelial cells, where infection is predominantly replicative. Although the exact mode of primary and persistent EBV infection and the relative contributions of B cells and epithelial cells are uncertain, recent data point to the B-cell compartment as the main mediator of primary as well as persistent infection. 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

14. Nasopharyngeal carcinoma
Burkitt’s lymphoma
Nasopharyngeal carcinoma
Hodgkin’s lymphoma
Burkitt's lymphoma is endemic in regions with mean minimum temperatures that exceed 15·5°C and yearly rainfall of higher than 50 mL, which stretch from about 10° north to 10° south of the equator. Epstein–Barr virus (EBV) is a prototype gamma herpes virus that infects the majority of the population worldwide. Infection with EBV occurs in early childhood and is usually asymptomatic, although infection during early adulthood is frequently associated with infectious mononucleosis, a benign self-limiting lymphoproliferative disorder. Seroepidemiological evidence suggests that EBV infection is widespread in China, with all Chinese children in Hong Kong testing positive for EBV before the age of 15 years
40-50% of patients are
EBV seropositive
NPC tissue stained for the presence of EBV late antigens.

15. EBV genome and host cell transformation
EBV-encoded nuclear antigen 2 (EBNA2)
latent membrane protein 1 (LMP1) mimics CD40 receptor LMP2 mimics the B cell receptor

16. in vivo interactions between EBV and host cells
Putative in vivo interactions between Epstein–Barr virus and host cells.   a | Primary infection. Incoming virus establishes a primary focus of lytic replication in the oropharynx (possibly in the mucosal epithelium), after which the virus spreads throughout the lymphoid tissues as a latent (latency III) growth-transforming infection of B cells. Many of these proliferating cells are removed by the emerging latent-antigen-specific primary-T-cell response, but some escape by downregulating antigen expression and establishing a stable reservoir of resting viral-genome-positive memory B cells, in which viral antigen expression is mostly suppressed (latency 0). Different views of these events are shown. One view is that naive B cells are the main targets of new EBV infections in vivo. In this scenario, viral transformation drives naive cells into memory by mimicking the physiological process of antigen-driven memory-cell development in lymphoid tissues, a process involving somatic immunoglobulin-gene hypermutation during transit through a germinal centre. However, this is difficult to reconcile with the finding that EBV-infected B cells in tonsils from patients with infectious mononucleosis (IM) localize to extrafollicular areas — not to germinal centres — and show no evidence of ongoing hypermutation within expanding clones. An alternative view therefore envisages infection of pre-existing memory cells as a direct route into memory; this is consistent with the above observations on IM tonsils, but still leaves unexplained the apparent disappearance of the infected naive cell population. b | Persistent infection. The reservoir of EBV-infected memory B cells becomes subject to the physiological controls governing memory-B-cell migration and differentiation as a whole. Occasionally, these EBV-infected cells might be recruited into germinal-centre reactions, entailing the activation of different latency programmes, after which they might either re-enter the reservoir as memory cells or commit to plasma-cell differentiation — possibly moving to mucosal sites in the oropharynx and, in the process, activating the viral lytic cycle. Virions produced at these sites might initiate foci of lytic replication in permissive epithelial cells, allowing low-level shedding of infectious virus in the oropharynx, and might also initiate new growth-transforming latency III infections of naive and/or memory B cells; these new infections might possibly replenish the B-cell reservoir, but are more likely to be efficiently removed by the now well-established memory-T-cell response.

17. Summary of EBV
aetiology of several different lymphoid and epithelial malignancies.
EBV-encoded latent genes induce B-cell transformation in vitro by altering cellular gene transcription and constitutively activating key cell-signalling pathways.
EBV exploits the physiology of normal B-cell differentiation to persist within the memory-B-cell pool of the immunocompetent host.

18. Human papilloma virus (HPV)
90% of cervical cancers contain HPV DNA.
4 types (HPV-16, HPV-18, HPV-31, and HPV-45) accounts for ~ 80% of HPV-positive cancers.
HPV-16 & 18 most common type of HPV found in ~70% of cervical carcinomas.
HPV-6,11 : common in genital warts
Copyright © David Reznik, D.D.S. All Rights Reserved

19. HPV life cycle
HPVs infect the stratified squamous epithelia of skin and mucous membranes, where they cause benign lesions, some of which have the potential to progress to invasive cancer. (2–4). HPVs are small, nonenveloped viruses whose approximately 8-kb circular genome encodes 2 structural proteins, L1 and L2, that form the viral capsid, plus several nonstructural proteins that are important for the virus life cycle but are not incorporated into virions.
To establish infection, the virus must infect basal epithelial cells that are long lived or have stem cell–like properties (by microtrauma ).
Viral genome maintains itself as an episome in basal cells, where the viral genes are poorly expressed.
Viral replication takes place in suprabasal layers and is tied to the epidermal differentiation process. The presence of the virus causes morphological abnormalities in the epithelium, including papillomatosis, parakeratosis, and koilocytosis. Progeny virus is released in desquamated cells.
HPV infections tend to last months or years because the viral genome successfully parasitizes these cells and because the virus evades the immune system by limiting most viral gene expression and viral replication to suprabasal cell layers. Most infections are self-limited, presumably because the host eventually mounts a successful immune response
Infection established in basal epithelial layers where viral genome maintained as an episome
Viral replication occurs in suprabasal layers
Infections are therefore long lasting

20. Integration into the host genome

21. HPV 16 produces only eight proteins
Function L1
Major capsid protein in the virus particle; by itself, L1 can assemble into capsomers and then form virus-like particles (VLPs) L2
Minor capsid protein in the virus particle; L2 binds to DNA E6
Destruction of p53 tumor suppressor protein E7
Inactivation of Retinoblastoma tumor suppressor protein (Rb) E1
Replication of viral DNA; maintenance of viral episome; essential for viral replication and control of gene transcription E2
Essential for viral replication;
repression of E6 and E7 E4
Forms filamentous cytoplasmic networks E5
Prevents acidification of endosomes;
interaction with Epidermal Growth Factor (EGF) /Platelet-Derived Growth Factor (PDGF)
LCR
Origin of DNA replication; regulation of HPV gene expression
8 kb circular genome
mRNA is transcribed off one strand of the DNA by host cell RNA polymerase II.
5 early genes coding for proteins, involved in
DNA replication (E1 and E2) or
activation of cell growth (E6, E7 and E5),
3 late genes, two of which code for the major and minor capsid proteins (L1 and L2, respectively) and one of unknown function (E4).
The regulation of viral gene expression occurs at both the initiation of transcription and through post-transcriptional mechanism (Fig. 3). On going studies are examining the transcription factors that regulate early gene expression in undifferentiated cells and late gene expression in differentiated cells. Gene expression is also regulated through post-transcriptional control of splicing and polyadenylation. A set of tandem polyadenylation signals allows the virus to discriminate between expression of early and late genes. Efforts are in progress to dissect the mechanisms regulating this switch in polyadenylation site usage.
The dsDNA circular genome of HPV type 31. Viral gene expression is regulated by a keratinocyte specific enhancer (KE) and an auxilary enhancer (AE) located within the upstream regulatory region (URR). Early transcripts initiate at the promoter P97 while late transcripts initiate at the promoter P742 which is activated upon keratinocyte differentiation. Early transcripts utilize a polyadenylation signal (poly(A)Early) downstream of the E5 open reading frame while late transcripts use the signal (poly(A)Late) downstream of the L1 open reading frame

22. Development of cancer

23. E6 and E7 proteins inactivate tumour suppressor proteins p53 and pRB
Transforming activity of HPV16 is associated with mainly E6 and E7proteins
E6 and E7 are multifunctional proteins that can increase cell proliferation and survival by interfering with tumour suppressor activity.
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

24. Inactivation of pRB by E7

25. quadrivalent recombinant vaccine against HPV types 6, 11, 16 and 18
Gardasil© (Merck):
quadrivalent recombinant vaccine against HPV types 6, 11, 16 and 18
To prevent cervical cancers in children aged 9–15 years and women from years
expected to prevent up to 70% of
nearly 100 percent effective in preventing precancerous cervical lesions, precancerous vaginal and vulvar lesions and genital warts caused by infection with the HPV types 6, 11, 16 or 18 in women between the ages of 16 and 26.
Four multinational studies were conducted

26. Chapter 3: Biology of Cancer by RA Weinberg Optional reading
References
Chapter 3: Biology of Cancer by RA Weinberg
Optional reading
Oncogenic viruses by Dennis J McCance
Epstein-Barr virus: 40 years on Nature Rev Cancer 4 (10) Oct 2004 Young LS, Rickinson AB
How will HPV vaccines affect cervical cancer? Roden R, Wu TC Nat Rev Cancer Oct;6(10):753-63

27. The following slides are for general interest only (since there is not enough time to cover all viruses in detail) Translated as THERE WILL BE NO SPECIFIC QUESTION ON RETROVIRUSES IN THE EXAM

28. RNA viruses Unstable RNA genome prone to mutations
Generates genetic diversity and escape antiviral therapy
Can be oncogenic (e.g.hepatitis C virus HCV)

29. Retroviral replication
Figure The Biology of Cancer (© Garland Science 2007)

30. Human Immunodeficiency Virus HIV

31. HIV life cycle
See animation at

32. HIV genome 3 structural genes
gag (group specific antigen) encodes matrix, capsid, nucleocapsid proteins
pol (polymerase) encodes reverse transcriptase, integrase, protease
env (envelope) encodes surface & transmembrane proteins
6 regulatory genes
rev (regulatory virus protein)
tat (transactivator)
nef (negative regulatory factor)
vif, vpr, vpu, env (envelope) encodes surface & transmembrane protein
The HIV genome, transcripts and proteins.   a | HIV transcripts. Integrated into the host chromosome, the 10-kb viral genome contains open reading frames for 16 proteins that are synthesized from at least ten transcripts. Black lines denote unspliced and spliced transcripts, above which coding sequences are given, with the start codons indicated. Of these transcripts, all singly spliced and unspliced transcripts shown above those encoding the transcriptional transactivator (Tat) require regulator of virion gene expression (Rev) for their export from the nucleus to the cytoplasm. The RNA target for Rev, the Rev response element (RRE), is contained in the gene encoding envelope protein (Env).

33. Course of HIV infection
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)

34. 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

35. hepatitis C virus HCV Affects 3% of global population
Infects primarily hepatocytes
50-80% of infected individuals go on to develop hepatocellular carcinoma (HCC)
At least 6 genotypes known
Putative life cycle of HBV and HCV.   b | After entry to the cell, hepatitis C virus (HCV) nucleocapsids are delivered to the cytoplasm, where the viral RNA functions directly as an mRNA for translation of a long polyprotein. Replication occurs within cytoplasmic, membrane-associated replication complexes in a perinuclear membranous web24. Genomic RNA-containing plasmids bud through intracellular membranes into cytoplasmic vesicles, which fuse with the plasma membrane. E, envelope protein; HBeAg, HBV e antigen; HBsAg, HBV surface antigen; HBx, HBV X protein; NS, non-structural protein; POL, polymerase
Background: Hepatocellular carcinoma (HCC) is a primary malignancy of the hepatocyte, generally leading to death within 6-20 months. HCC frequently arises in the setting of cirrhosis, appearing years following the initial insult to the liver. However, 25% of patients have no history or risk factors for the development of cirrhosis. The extent of hepatic dysfunction limits treatment options, and as many patients die of liver failure as from tumor progression. Although it is currently one of the most common worldwide causes of cancer death, a major impact on the incidence of HCC should be achieved through current vaccination strategies for hepatitis B virus (HBV) infection, screening and treatment for hepatitis C virus (HCV) infections, and from the reduction of alcoholic liver disease. However, because the latency period from hepatic damage to HCC development is very long, it may be many years until the incidence of HCC decreases as a result of these interventions.

36. What causes hepatocellular carcinoma?
HBV and HCV co-infection?
HBV integrates into genome and produces a protein Hbx, involved in HCC
HCV does not integrate into the genome but can interact with host proteins and cause an inflammatory response, which can transform cells
e.g. HCV proteins NS3 and NS5A can disrupt transcription factors leading to proliferation and inhibition of apoptosis

37. HCV life cycle
Putative life cycle of HBV and HCV.   b | After entry to the cell, hepatitis C virus (HCV) nucleocapsids are delivered to the cytoplasm, where the viral RNA functions directly as an mRNA for translation of a long polyprotein. Replication occurs within cytoplasmic, membrane-associated replication complexes in a perinuclear membranous web24. Genomic RNA-containing plasmids bud through intracellular membranes into cytoplasmic vesicles, which fuse with the plasma membrane. E, envelope protein; HBeAg, HBV e antigen; HBsAg, HBV surface antigen; HBx, HBV X protein; NS, non-structural protein; POL, polymerase

38. Human Herpes Virus 8 (HHV8) or Kaposi’s sarcoma associated virus KSHV
Herpes virus family
Type 1 - causes ‘cold sores’ on lips (~90% of population)
Type 2 - sexually transmitted disease that causes "cold sores" on the genitals (~ 25% of US adults).

39. Human Herpes Virus 8 (HHV8) a. k
Human Herpes Virus 8 (HHV8) a.k.a Kaposi’s sarcoma associated virus HHV8 endemic regions

40. Kaposi’s sarcoma
sarcoma is a cancer that develops in connective tissues such as cartilage, bone, fat, muscle, blood vessels, or fibrous tissues (related to tendons or ligaments). Kaposi sarcoma (KS) was named for Dr. Moritz Kaposi who first described it in 1872.
This disease typically causes tumors to develop in the tissues below the skin surface, or in the mucous membranes of the mouth, nose, or anus. These lesions (abnormal tissue areas) appear as raised blotches or lumps that may be purple, brown, or red. Sometimes the disease causes painful swelling, especially in the legs, groin area, or skin around the eyes. Although the skin lesions of KS may be disfiguring, they usually are not life threatening or disabling. In most cases, the lesions cause no symptoms. In some, the lesions may be painful, especially if they cause swelling of nearby unaffected skin. KS does become life threatening when it is in the lungs, liver, or gastrointestinal tract. This can cause major symptoms. KS in the gastrointestinal tract, for example, can produce bleeding, while tumors in the lungs may cause difficulty breathing.
Endemic (African)
Acquired (Transplants)
Epidemic (AIDS related)

41. HHV8 and transformation
Most people infected with HHV8 do not get KS
Immunosuppressed individuals are susceptible
Viral homologues of several human proteins (e.g. v-cyc, vIL6)
Studies have found infection rates ranging from 3.5% to 25% in reports from different parts of the country. In Africa, this number is over 50% in certain areas. Most people infected with this virus do not get KS. A very small number will get the endemic, low-grade KS. But people who are immunosuppressed, such as those with AIDS, develop KS much more readily if they are infected with this virus.