Oncogenic viruses

Oncogenic viruses

1. ONCOGENIC VIRUSES 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 SIG NIFICANCE 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. R esult 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 )

12. TRANFORMATION

13. 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

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

15. 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

16. 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

17. 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

18. 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

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

20. 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

21. 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

22. 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)

23. MITOGENIC SIGNAL TRANSDUCTION PATHWAY

24. 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

25. Rb GENE MEDIATED ONCOGENESIS

26. 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

27. RNA VIRUSES

28. 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

29. 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 )

30. 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

31. 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

32. CELL CYCLE OF RETRO VIRUSES

33. 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

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

35. 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

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

37. 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

38. 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

39. 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 2 nd most common cancer worldwide - 16% of all female cancers are linked to HPV - Papilloma virus is found in 90% of women with cervical cancers

40. 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, 40,42,43,44,54,61,70,72,81 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 )

41. 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.

42. 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 .

43. 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 )

44. 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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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

51. 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

52. 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

53. 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

54. 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

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

56. 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)

57. 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

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

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

60. 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

61. GENE THERAPY

62. SUMMARY

63. REFERENCE Daniel Di Maio,Hung Fan;’Viruses,Tranfornation and Cancer’;David M.Knipe,Peter M.Howley;Field’s Virology;5 th 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;9 th edition;(564-569) Joan C.M.Macnab , David Onions;’Tumor Viruses’;Baron S.;Medical Microbiology 4 th edition

64. Janet S.Butel;’Human Cancer Viruses’;Jawetz,Melnick&Adelberg’s Medical Microbiology;25 th 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)

65. THANK YOU