Oncogenic viruses

1. BY
DR.A.SIVARANJINI

2. SYNOPSIS
 INTRODUCTION
 EPIDEMIOLOGY
 MECHANISM OF ONCOGENECITY BY VIRUSES
 VIRUSES ASSOCIATED WITH HUMAN TUMORS
 DIAGNOSIS
 PREVENTION
 APPLICATIONS

3. INTRODUCTION
 ONCOGENIC VIRUSES
Viruses that produce tumours in their natural host /
experimental animals
or
which induce malignant transformation of cells on culture.
 Features of viral oncogenesis
- cause cancer in humans & animals
- long latency between viral infection and tumorigenesis
- modulate growth control pathways in cells
- viral markers are present in tumor cells

4. YEAR SIGNIFICANCE
1908
Ellerman & Bang reported that cell free filtrates from chicken with leukemia
could transmit disease to healthy birds
1911 Rous - first described association of viruses with malignancy
- Fowl sarcoma caused by virus
- Nobel Prize in 1966
1932 Shope – demonstrated viruses causing tumors in animals
- isolated Rabbit Fibroma virus (1932) & Pappiloma virus (1933)
1957 Stewart & Eddy – discovered Polyoma virus
1962 Trentin – demonstrated sarcoma in newborn mice by human Adenovirus
Discovery of tumorigenic potential of Simian virus 40
1965 Burkitt – identified Epstein Barr virus as causative for Burkitt’s lymphoma.
First human tumor virus
1975 Blumberg et al – linked chronic hepatitis B infection to hepatocellular
carcinoma
1980 Second-generation recombinant HBV surface antigen subunit vaccine (against
HBV & HCC)
1974 Harald zur Hausen – proposed HPV as etiologic agent of cervical cancer
- Nobel Prize in 2008
1981 Gallo et al – proposed causal role of HTLV 1 in adult T cell Leukemia
1989 Houghton et al – proposed association between chronic HCV infn and HCC
1994 Chang et al – isolated kaposi’s sarcoma virus (HHV8)

5. EPIDEMIOLOGY
 About 10-20% of human tumours are caused by viruses

6. MECHANISM OF ONCOGENECITY
Introduction of new Alteration of expression of
‘Transforming gene’ preexisting cellular gene
into the cell
Loss of normal growth regulation processes
Affection of DNA repair mechanisms
Genetic instability
Mutagenic phenotype
DIRECT ACTING
INDIRECT
ACTING

7.  Oncogenesis -An abnormal growth of tissue resulting from
uncontrolled,
progressive multiplication of cells and serving no physiological
function. Result of genetic changes that alter the expression or
function of proteins that play critical roles in the control of cell
growth and division
 Proto-oncogenes - normal (pre-mutation) (pre-diseased) genes
- present in normal cells
- conserved in their genomes
- code for proteins which regulate cell growth
&differentiation
 Oncogenes - mutated versions of proto-oncogenes
- contribute to cancer development by disrupting a cell's
ability to control its own growth.

8. CELLULAR ONCOGENES
 Present in cancer cells
 Contains introns
characteristic of eukaryotic
cells
 Encodes proteins triggering
transformation of normal
cells
VIRAL ONCOGENES
 Present in viruses
 Host cell origin
 Do not possesss introns
 Also called ‘cancer genes’
 Encodes proteins triggering
transformation of normal
cells into cancer cells
VIRAL
ONCOGENE
HUMAN
ONCOGENE
ORIGIN NATURE
V-src C-src Chicken Sarcoma
V-ras C-ras Rat Sarcoma
V-myc C-myc Chicken Leukemia
V-fes C-fes Feline Sarcoma
V-sis C-sis Simian Sarcoma
V-mos C-mos Mouse Sarcoma

9. Conversion of Proto-oncogene to Oncogene

10.  Tumor suppressor genes
 Directly inhibit uncontrolled proliferation (RB,P53,)
 Maintain integrity of the genome (BRCA 1)
 House keeping genes
 P53 gene
 Retinoblastoma (Rb) gene

11.  TRANSFORMATION
alteration in a cell’s properties that leads to immortalization and
different growth patterns that result from alteration in cell cycle.
 Acute tranforming viruses
- retrovirus(alpha ,gamma)
- replication defective (does not produce viral infection)
- contain viral oncogene( transform cells in culture )
 Non acute transforming viruses
- retrovirus (alpha,beta,gamma)
- replication competent (cause viral disease)
- lack viral oncogene (high level of infection months before tumor )

13. TRANFORMATION

14. PROPERTIES OF TRANSFORMED CELLS
NORMAL CELLS TRANSFORMED
CELLS
BEHAVIOUR OF
TRANSFORMED
CELLS
Tumorigenic Non - Tumorigenic Forms tumors on
inoculation into
susceptible hosts
Growth Finite life
span(Senescence)
Immortal Infinite serial passsages
Density dependent
growth
inhibition(contact
inhibition)
Loss of contact
inhibition
Focus (piled up
appearance of focally
proliferative cells )
Anchorage dependent Anchorage
independent
Colony formation in
semisolid medium
Growth factor
dependent
Growth factor
independent
Growth in reduced
conc.of growth factors
DNA synthesis despite
nutrient deprivation

15. NORMAL CELLS TRANSFORMED
CELLS
BEHAVIOUR OF
TRANSFORMED
CELLS
Biochemical Oxidative
respiration
Aerobic glycolysis
(WARBURG
EFFECT)
Acidification of
culture
medium(lactic acid)

16. INTERACTION BETWEEN HOST &
ONCOGENIC VIRUS
 PERSISTENT INFECTIONS
-chronicity of the infections modulate growth control
mechanisms
 LATENCY OF VIRAL GENOME
- episomal copies of viral genome are maintained in
transformed cells
- viral genome is incorporated into host cell genome
- tumorigenesis after latent period

17.  EVASION OF HOST IMMUNE RESPONSE
- restricted expression of viral genome (EBV )
- infection of sites inaccessible to immune
response(HPV)
- mutation of viral antigens (EBV)
 EXHIBITION OF MARKED TISSUE SPECIFICITY

18. ONCOGENIC VIRUSES
TAXONOMIC
GROUPING
EXAMPLES PRIMARY TUMOR
TYPES
RNA VIRUSES
1.Flaviviridae Hepatitis C virus Hepatocellular carcinoma
2.Retroviridae
• Alpharetroviruses Rous sarcoma virus(RSV) Sarcoma
Rous associated virus(RAV) Bcell
lymphoma,erythroleukemia
Avian myeloblastosis virus (AMV) Myeloid/erythroid leukemia
Avian erythroblastosis virus (AEV) Erythroid leukemia
Myelocytoma virus (MC29) Myeloid leukemia
•Betaretroviruses Mouse mammary tumor virus(MMTV) Mammary carcinoma
Jaagsiekte sheep retrovirus Lung carcinoma

19. Gammaretroviruses Murine leukemia virus(MuLV) Leukemia, lymphoma
Murine sarcoma virus(MuSV) Sarcoma
Feline leukemia virus Leukemia,lymphosarcoma
Feline sarcoma virus Sarcoma
Simian sarcoma virus Sarcoma
Koala retrovirus T cell leukemia
Deltaretroviruses Human T lymphotropic virus(HTLV) Adult T cell leukemia
Bovine Leukemia virus B cell leukemia
•Epsilonretroviruses Walleye dermal sarcoma virus Sarcoma

20. DNA VIRUSES
1.Adenoviridae Types 2,5,12 Various solid tumors
2. Hepadnavirus Hepatitis B virus (HBV) Hepatocellular carcinoma
3.Herpesviridae Epstein-Barr virus(EBV)
(HHV4)
Burkitt’s lymphoma,
nasopharyngeal carcinoma
Kaposi sarcoma Herpes
virus(KSHV) (HHV8)
Kaposi sarcoma
4.Polyomaviridae SV40,polyoma virus Various solid tumors
5.Papillomaviridae HPV 6,11,16,18
Bovine papilloma virus
Papilloma,carcinoma
6.Poxviridae Shope fibromavirus Myxoma,fibroma

21. TUMOR VIRUSES
DNA VIRUSES RNA VIRUSES
VIRAL
ONCOPROTEIN
EXPRESSION
VIRAL
ONCOGENE
PROTO
ONCOGENE
CONVERSION
PROVIRAL
INSERTION NEAR
CELLULAR
ONCOGENE
TRANSFORMING NON
TRANSFORMING
INACTIVATION OF
TUMOR
SUPPRESSOR
GENES

22.  Viral Oncoproteins
- virus – encoded non structural proteins
- target tumor suppressor proteins of host cell
 Acquisition of proto-oncogene
- usually mutated in the process
- viral cellular genes are lost
- transformation of target cell
 Proviral Insertion
- activate cellular proto-oncogene
- replication intact
- induce tumors after long latent periods

23. MECHANISM OF VIRAL ONCOGENESIS
 Impairment of the signal transduction pathway
- Growth factor expression
- Growth factor receptor activation
- Cytoplasmic or membrane-bound kinases
- Transcription factors
 Inactivation of Tumor-suppressor genes
- Uncontrolled proliferation (Rb gene & P53gene)
- Inhibition of Apoptosis (P53 gene)

24. MITOGENIC SIGNAL TRANSDUCTION
PATHWAY

25.  In normal cells,
Ras-GTP (active)
(GAP) GTPase
activating protein
Ras – GDP(inactive)
Stops abnormal cell
proliferation
 In transformed cells,
Point mutations block Ras in
Active state by blocking
GAPs
Ras – GTP(active)
GAP
Ras – GTP (active)
Uncontrolled proliferation of
cells

26. Rb GENE MEDIATED ONCOGENESIS

28. VIRUSES CAUSING HUMAN CANCERS
VIRUS FAMILY VIRUS HUMAN CANCER
Papillomaviridae Human Papilloma virus Genital tumors
Squamous cell carcinoma
Oropharyngeal carcinoma
Herpesviridae Epstein –Barr virus Nasopharyngeal carcinoma
Burkitt’s lymphoma
Hodgkin disease
B cell lymphoma
Human Herpes virus 8 Kaposi sarcoma
Hepadnaviridae Hepatitis B virus Hepatocellular carcinoma
Flaviviridae Hepatitis C virus Hepatocellular carcinoma
Retroviridae Human T cell lymphoma virus Adult T cell lymphoma
Human immunodeficiency virus AIDS related malignancies

29. RNA VIRUSES

30.  FLAVIVIRIDAE (HCV)
 RETROVIRIDAE (HTLV-1)
 XENOTROPIC MURINE LEUKEMIA VIRUS(XMRV)
 Viral oncogenes capture cellular oncogene(proto-oncogene)
(reverse transcriptase mediated in retrovirus)
proto-oncogene is mutated to cause cancer
 About 70 proto-oncogenes identified
code for key cell signaling proteins involved in the control of
cellular proliferation and apoptosis

31. RETROVIRUS
Properties
 Virion – spherical,helical
nucleoprotein within
icosahedral capsid
 Genome – 2copies of single
stranded RNA
 Possess reverse
transcriptase(RNA
dependent DNA polymerase)
 Enveloped virus
 Not cytolytic (except
lentivirus)

32. SUBFAMILY GENERA EXAMPLES
Oncovirinae- all
oncogenic viruses
Alpharetrovirus Avian leukemia v.
Avian sarcoma v.
Betaretrovirus Mouse mammary tumor v.
Gammaretrovirus Mammalian leukemia
Mammalian sarcoma v.
Deltaretrovirus Hu.T lymphotropic v.
Bovine leukemia v.
Epsilonretrovirus Fish viruses
Spumavirinae-foamy
degeneration, no
infection
Spumavirus
Lentivirinae –slow
infections
Lentivirus HIV

33.  Standard leukemia virus (alpha,gamma retrovirus) – no viral
oncogene
gag – encodes core proteins(group specific)
pro – encodes protease enzyme
pol – encodes reverse transcriptase
env – encodes envelope glycoproteins
 Influence proto-oncogene by insertional
mutagenesis
 Deltaretrovirus, Lentivirus
tax/tat – transactivating regulatory gene
 Transforming retrovirus
onc – encodes for oncogenetic potential

35. CELL CYCLE OF RETRO VIRUSES

36. HUMAN T- CELL LEUKEMIA VIRUS
 Epidemiology
-First retrovirus implicated in human disease.
-20 million infected worldwide.
-High endemic areas – Latin America,Caribbean , Africa ,Japan
- IV drug users - U.S & Europe
 Adult T-cell leukemia/lymphoma
 Oncoproteins
Tax
- Enhanced proliferative potential of T – lymphocytes
- interference of cell regulation pathways & DNA repair
mechanisms
HBZ (HTLV 1 Basic leucine Zipper factor)
- Late stage tumorigenesis

37.  Accessory protein p12
- alters MHC 1 & T-cell receptor (TCR) cascade activation
 Accessory protein p13II
- targets mitochondria
- affects cell proliferation , apoptosis , ROS production

38. HIV
 Accessory protein Tat
- interferes with DNA repair mechanisms
- interfere with Rb gene mediated growth regulation pathway
HCV
 Flaviviridae. Single stranded RNA.
 Over 170 million chronic carriers
 Hepatocellular carcinoma
 Non Hodgkins B cell lymphoma
 Mechanism- inflammation cirrhosis

39. DNA VIRUSES
Hepatitis B virus HBV
Human Papillomavirus
Adeno virus

40. DNA virus oncogenesis
 Inhibition of tumor suppressor genes
- Rb gene mutation
- inactivates p53 mediated growth regulation
 Lack viral oncogene
 Oncoproteins interact with specific targets on the host cell

41. DNA virus Oncoproteins and their major targets
VIRUS TARGETS
Adenovirus E1A Rb family members
Adenovirus E1B19K Bak ,Bax
Adenovirus E1B55K p53
Adenovirus E4 orf 6 p53
Epstein Barr virus EBNA2 Glycogen synthetase kinase,RBP-J kappa/CBF 1
Epstein Barr virus Lmp 1 PI3K ,TNF signalling components
Epstein Barr virus LMP 2 Src family members
Hepatitis B virus X protein p53
Human Papilloma virus E5 EGF receptor
Human Papilloma virus E6 P53, PDZ proteins,
Human Papilloma virus E7 Rb family members, p21,p27,p600
KSHV ORF 50 ,KSHV K-bZIP p53
KSHV vCyclin Cyclin dependent kinase 6

43. HPV
 Small
 Non-enveloped
 Virion –Icosahedral
 Genome – double stranded ,circular DNA (8000bp )
 Closely related to Polyomaviruses
 16 genera (5 – human infections)
 Nearly 140 types are identified
 Classified using molecular criteria
 Epidemiology
- HPV induced cervical cancer is 2nd most common cancer
worldwide
- 16% of all female cancers are linked to HPV
- Papilloma virus is found in 90% of women with cervical cancers

44. HUMAN PAPILLOMA
VIRUS TYPE
CLINICAL LESION SUSPECTED
ONCOGENIC POTENTIAL
1 Plantar warts Benign
2,4,27,57 Common skin warts Benign
3,10,28,49,60,76,78 Cutaneous lesions Low
5,8,9,12,17,20,36,47 Epidermodysplasia
verruciformis
Benign - malignancy
6,11,40,42,43,44,54,61,70,72,8
1
Anogenital condylomas,
Laryngeal papillomas,
Mucaosal dysplasia &
intraepithelial neoplasia
Low
7 Hand warts in butchers Low
16,18,30,31,33,35,39,45,51-
53,56,58,59,66,68,73,82
High grade dysplasia,genital
carcinoma ,laryngeal &
esophageal carcinomas
High (espl with cervical
cancer)

45. HPV REPLICATION
 High tropism for epithelial cells of skin & mucous membranes
 Stages in viral replication are dependent on sequential
differentiation states of epithelial cells .
 DNA synthesis is supported only in basal cells of the squamous
epithelium.
 Hence difficult to propagate in vitro
 Opening Reading Frames(ORF) – encode viral proteins .
Located on only one of he 2 viral DNA strands
 No viral DNA polymerase. Dependent on host cell replication
machinery for viral genome replication.

46. ORF FUNCTION
L1 L1 protein-major capsid protein
(VLP vaccine)
L2 L2 protein- minor capsid protein
E1 Initiation of viral DNA
replication,helicase,ATPase
E2 Trancriptional regulatory
protein, genomic maintenance
E4 Late protein. Disrupts
cytokeratins
E5 Membrane transforming protein,
interacts with specific growth
factor receptors
E6 Transformation. Degradation of
p53, telomerase activation
E7 Transformation.
Inactivation of pRb .

47. HPV pathogenesis
 Sexually transmitted
 Peak incidence – adolescents & young adults
 Second most common cause cancer in women worldwide.
Major cause of cancer deaths in developing countries.
 Episomal HPV DNA – in skin carcinomas , premalignant
lesions
 Integrated HPV DNA – in cervical cancer cells
 Oncogeneticity – transforming oncoproteins interacting with
tumor suppressors(p53, Rb )

48. HERPES VIRUS
 EPSTEIN BARR VIRUS (HHV 4)
 KAPOSI SARCOMA HERPES VIRUS (HHV 8 ) GAMMA
Properties
 Large viruses
 Genome – linear double stranded DNA
 Icosahedral capsid with lipid containing envelope
 Acute infection followed by latency
 Recurrence from latent infection
 Latency genes

49. EBV
 Infectious mononucleosis
 Burkitt’s lymphoma
 Nasopharyngeal carcinoma
 Non Hodgkin’s lymphoma
 Remain latent in lymphoblast cell lines -
 Epidemiology
- Ubiquitous
- Burkitt’s lymphoma – children in Central Africa
- Nasopharyngeal carcinoma – Cantonese China , Alaskan
Eskimos
Malaria - cofactor
Tumors contain integrated & episomal forms of viral DNA

50.  Oncoproteins
 LMP1(Latent MembraneProtein 1)
- membrane protein wuth 6 domains
- receptor -TNF alpha
TRAF TRADD
(TNF Recceptor ass.factor) (TNF receptor ass.death
RIP domain)
(Receptor interacting protein)
Stimulation of transcription factors for AntiApoptotic
proteins
LMP 1

51.  EPSTEIN BARR NUCLEAR ANTIGEN (EBNA)
 EBNA 1
 EBNA 2
 EBNA LP Conversion of EBV infected peripheral
 EBNA 3A blood cells to lymphoblastoid cell lines
 EBNA 3C
 P53 mutation

52. KAPOSI SARCOMA HERPES VIRUS
 HHV 8
 KAPOSI’S SARCOMA- affects multiple organs with
prominent vascular endothelial component
 PRIMARY EFFUSION LYMPHOMA
 MULTICENTRIC CASTLEMAN DISEASE
 Epidemiology
- Mediterranean & African countries
- Elderly men high preponderance

53.  ONCOPROTEINS
- Seven latent genes
- Latency Associated Nuclear Antigen(LANA)
Impairs p53 & Rb up-regulates Beta – catenin pathway
V - FLIP(Flice inhibitory protein)
inhibits caspase activity

54. HBV
 Hepadnaviridae
 Genome – circular ,ds DNA
 Epidemiology
- endemic in Africa ,China, South east Asia
- over 250 million persistently infected
 Primary infections in neonates & children-90% chronic
Hepatocellular carcinoma
 Hepatitis B vaccination has lowered incidence of
infection & HCC

55.  Oncoprotein
1. X gene
- encoded by ORF X
- affects cellular gene expression
- interferes with p53 function
2. Mutated proto-oncogene N-myc2
3. Deletion of p53
3. Aflatoxin
- co factor
- Africa &China
4. ROS
- generated by inflammatory cells of Chronic active hepatitis
- DNA damage & mutagenesis

56. POLYOMA VIRUS
 MERKEL CELL VIRUS
 Merkel cell carcinoma
- highly lethal skin cancer
- immunocompromised
 Oncoproteins
- Large T antigen (LT)
- Small T antigen (ST)
- inactivate Rb tumor suppressor pathway

57.  SIMIAN VIRUS 40
 Osteosarcoma, lung carcinoma,brain tumors
 EPIDEMIOLOGY
- primary infection in Asian macaques
- accidental exposure through contaminated poliovirus
vaccines b/w 1955 and 1963
- PCR assays

58. DETECTION
 DETECTION OF TRANSFORMATION – studied in
established cell lines that are immortal
 GENETIC MAPPING - to identify specific viral genes with
transforming activity
 GENE SEQUENCING
 PCR
- HTLV 1
- HBV , HCV
- EBV (LMP 1,2)
 SOUTHERN BLOTTING
- HBV
- EBV - EBNA1 Ag

59.  ANIMAL INOCULATION
 MICROARRAY
 IN SITU HYBRIDISATION
- HPV (E6,E7)
- KSHV

60. VACCINES
HPV
 Non infectious ,recombinant vaccines
- Virus Like Particles(VLP) composed of L1 proteins(major
capsid protein)
- VLPs generate high titre type specific neutralizing antisera
 GARDASIL
- FDA approved (Merck)
- Quadrivalent vaccines (particles derived from HPV
6,11,16,18)
- also used in men to prevent genital warts (approved in
2009)

61.  CERVARIX
- FDA approved (GlaxoSmithKline)
- Bivalent vaccines ( HPV 16,18)
- immunity for 5 years
- contraindicated in pregnancy.
 Immune response is Type specific
 No cross protection against other HPV types
 Expensive
 Heat labile
IN PIPELINE
 Using L2 epitopes
 Prophylaxis against broader spectrum of HPV types
 Inclusion pf Early gene products E2,E6 ,E7

62. HBV
 Subunit,recombinant vaccine
 HBsAg
 HCV
 Genome is highly variable
 No effective vaccine
 HTLV
 No vaccine
 Interferons

63. EBV
 Chemotherapy
 Vaccine – in pipeline
KSHV
 Gancyclovir – inhibits viral DNA polymerase
 Incidence lowered in AIDS patients treated with HAART
 No vaccine

64. USES
Discovery of Tumor suppressor genes (DNA tumor
viruses)
GENE THERAPY
 Retroviruses
- treatment of genetic disorders (SCID),Chronic
granulomatous disease
- Adverse reaction – viral vector induced malignancy
 Adenovirus
 High level of transgene expression

65. GENE THERAPY

67. SUMMARY

68. REFERENCE
 Daniel Di Maio,Hung Fan;’Viruses,Tranfornation and
Cancer’;David M.Knipe,Peter M.Howley;Field’s Virology;5th
edition(153-184)
 Peter M.Howley,Dan Ganem,Elliot Kioff;’DNA and RNA
Viruses’;De Vita,Kellman,Rosenberg’s Cancer Principles &
Oncology;(173-192)
 Oncogenic Viruses;Ananthanarayanan&Paniker’s Textbook of
Microbiology;9th edition;(564-569)
 Joan C.M.Macnab , David Onions;’Tumor Viruses’;Baron
S.;Medical Microbiology 4th edition

69.  Janet S.Butel;’Human Cancer
Viruses’;Jawetz,Melnick&Adelberg’s Medical
Microbiology;25th edition;(591-608)
 A review of human oncogenes ;The Lancet;Vol 30;April
2009(321-322)
 Melissa Pulitzer ;’Molecular Diagnosis of Infection-Related
Cancers in Dermatopathology’;Seminars in cutaneous medicine
&surgery;Frontline Medical Communications;2012;(247-257)
 Emma J.Crosbie and Henry C.Kitchener;Human papilloma in
cervical screening and vaccination;Clinical
Science;2006,110;(543-552)

70. THANK YOU