Bohomolets Microbiology Lecture #15

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Bohomolets Microbiology Lecture #15

  1. 1. Viral OncogenesisRetrovirusesAIDS
  2. 2. Oncogenic virusesDNA viruses RNA viruses Herpesvirus  Retrovirus Adenovirus  Flavivirus Hepadnavirus Papillomavirus Poliomavirus Poxvirus
  3. 3. Feature of malignant transformation (1)Feature DescriptionAltered Loss of differentiated shapemorphology Rounded as a result of disaggregation of actin filaments and decreased adhesion to surfaceAltered Loss of contact inhibition of growthgrowth Loss of contact inhibition of movementcontrol Reduced requirement for serum growth factors Increased ability to grow in suspension Increased ability to be cloned from a single cell Increased rapidity of multiplication Increased ability to continue growing (“immortalization”)
  4. 4. Feature of malignant transformation (2)Feature DescriptionAltered Induction of DNA synthesiscellular Chromosomal changesproperties Appearance of new antigensAltered Reduced level of cyclic AMPbiochemical Increased anaerobic glycolysisproperties Loss of fibronectin Changes in glycoproteins and glycolipids
  5. 5. The 2 major concepts of the way viral tumorigenesis (1) The provirus modes. The genes enter the cell at the time of infection by the tumor virus The oncogene model.
  6. 6. Only 2 viruses are considered to be human tumor viruses: Human T cell leukemia virus. There are 2 types: HTLV-I and HTLV-II Human papillomavirus. Especially serotypes 16, 18, 33, 35, 11 Most cervical, vulvar and penile cancers are ASSOCIATED with types 16 and 18 (70% of penile cancers)Several other candidate viruses are implicated byepidemiologic correlation, by serologic relationship,or by recovery of virus from tumor cells
  7. 7. Some retroviruses have an extra gene “typical retrovirus” R U5 GAG POL ENV U3 R Rous Sarcoma VirusR U5 GAG POL ENV SRC U3 R
  8. 8. Retroviral oncogenes Avian Myeloblastosis VirusR U5 GAG POL MYB U3 R Feline Sarcoma Virus (FSV) R U5 dGAG FMS dENV U3 R Avian Myelocytoma Virus (MC29) R U5 dGAG MYC dENV U3 R
  9. 9. Not all tumor viruses of the retrovirus family contain onc genes. How do these viruses cause malignant transformation?The DNA copy of the viral RNA integrates near acellular oncogene, causing a marked increase in itsexpression.Overexpression of the cellular oncogene may playa key role in malignant transformation by theseviruses.
  10. 10. The 2 major concepts of the way viral tumorigenesis (2) The provirus modes. The oncogene model. The genes for malignancy are already present in all cells of the body. These oncogenes encode proteins that encourage cell growth, eg, fibroblast growth factor. In this model, carcinogenes such as chemicals, radiation, and tumor viruses activate cellular oncogenes to overproduce these growth factors. This initiates inappropriate cell growth and malingant transformation.
  11. 11. What do oncogenes encode? Proteins that are involved in growth control and differentiation: Growth factors Growth factor receptors Signal transduction proteins Transcription factors
  12. 12. OncogenesViral Oncogene is named V-oncCellular Proto-oncogene is namedC-onc
  13. 13. Proto-oncogeneA cellular (host) gene that is homologouswith a similar gene that is found in atransforming virusA cellular oncogene can only inducetransformation after: mutation some other change in the cell’s genome
  14. 14. Cellular oncogenes Genes can be assigned to sites on specific chromosomes myb mos myc mos and myc : chromosome 8 fes: chromosome 15fe s
  15. 15. Evidence that cellular oncogenes (c-onc) can cause tumors (1)Evidence DescriptionMutation of DNA isolated from tumor cells can transformc-onc gene nirmal cells. This DNA has a c-onc gene with a mutation consisting of a single base changeTranslocati Movement of c-onc gene to a new site on aon of c-onc different chromosome results in malignancygene accompanied by increased expression on the gene
  16. 16. Cancers often result from gene translocationsBurkitt’s Lymphoma8:14 translocation myc
  17. 17. Evidence that cellular oncogenes (c-onc) can cause tumorsEvidence DescriptionAmplification of The numbers of copies of c-onc genes isc-onc gene increased, resulting in enhanced expression of their mRNA and proteinsInsertion of Proviral DNA inserts near c-onc gene, whichretrovirus near alters its expression and causes tumorsc-onc geneOverexpression Addition of an active promoter site enhancesof c-onc gene expression of the c-onc gene, and malignantby modification transformation occursin the laboratory
  18. 18. Both DNA and RNA tumor viruses cantransform cells Integration of viral genome into the hostchromosomes occurs (usually) Similar mechanisms of transformation byeach type of tumor virus
  19. 19. DNA Tumor Viruses DNA genome Host RNA polymerase II mRNA Host enzymes protein virus OR TRANSFORMATIONIn transformation usually only EARLY genes are expressed
  20. 20. DNA Tumor Viruses In Human Cancer• Can transform cells or have lytic life cycle• Often integrate into host genome• In transformation ONLY early genes aretranscribed• These are genes that are also necessary for a PRODUCTIVE infection
  21. 21. Human viruses that can cause tumor growth in humanEpstein-Barr virus (Herpesviridae). Burkitt’slymphoma, nasopharyngeal carcinomaHerpes simplex virus type 2 (Herpesviridae).Carcinoma of the cervixHepatitis B virus (Hepadnaviridae). Hepatoma –hepatocellular carcinomaHepatitis C virus (Flaviviridae). Hepatocellularcarcinoma
  22. 22. retroviruses HIV and AIDS
  23. 23. Family Retroviridae (have approximately 150 species)Subfamily: Oncovirinae Lentivirinae Spumavirinae
  24. 24. Retroviruses Groups of Retroviruses• Oncovirinae important Tumor viruses• Lentiviruses important Long latent period Progressive chronic disease Visna HIV
  25. 25. Retroviruses Enveloped viruses round shape Size – 80-130 nm Nucleocalsid with cubical type of symmetry Structural enzyme - REVERSE TRANSCRIPTASE Structural genes – pol, env, gag Possibility to integrate into the host chromosome
  26. 26. Retroviral genome RNA Diploid Capped and polyadenylated Positive sense (same as mRNA) Viral RNA cannot be read as mRNA New mRNA must be made Virus must make negative sense DNA beforeproteins are made Therefore virus must carry structuralREVERSE TRANSCRIPTASE into the cell
  27. 27. Retroviral structural genesGag: encodes internal proteinsEnv: encodes envelope glycoproteinsPol: encodes enzymesReverse transcriptaseIntegraseProtease
  28. 28. Structure of retroviruses
  29. 29. Retrovirus life cycle Bind to surface receptor Fusion of membranesRelease of nucleocapsid to cytoplasm Nucleus
  30. 30. Parental RNA Reverse transcriptaseRNA/DNA Hybrid Reverse transcriptaseLinear DNA/DNA duplexCircular Duplex DNA IntegraseIntegration Host DNA polymerase Replication (DNA genome in cell) Host RNA pol IITranscription Viral RNA genome mRNA protein
  31. 31. Some retroviruses have an oncogene instead of their regular genes
  32. 32. HIV and AIDSAcquired Immunodeficiency SyndromeDisease caused by an infectious agent:a retrovirus – Human Immunodeficiency Virus
  33. 33. History of HIV discovering 1980 – isolation of Human T cell lymphotropic virus (HTLV-І) – causative agent of T-cell leukemia virus 1982 – isolation of HTLV-ІІ – agent of hairy cell leukemia 1983 – isolation of HIV-1 1985 – isolation of HIV-2 Robert Gallo Luc Montagnier
  34. 34. Classification HIVFamily RetroviridaeSubfamily LentivirinaeGenus LentivirusType HIV-1 HIV-2Gropes M, N, O A, BSubtypes At least 10 (A, B, No(genotypes) C, D, E, F, and others)
  35. 35. AIDS Statistics• Approximately 44,000,000 people in the world are HIV-infected• Approximately 14,000 new HIV infections occur daily aroundthe world• Over 90% of these are in developing countries• 1000 are in children less than 15 years of age.• Of adult infections, 48% are in women and 15% in individuals15-25 years• As of December 2003, 929,985 Americans reported with AIDS.• At least 501,669 of them have died (2002 figures)• 5,315 children under 15
  36. 36. Characteristic HIV-infection and AIDS IMMUNOSUPPRESSION OPPORTUNISTIC INFECTIONS Also Lymphadenopathy Hodgkin’s Lymphoma
  37. 37. HIV-associated opportunistic infection, that usually are signs of AIDS Agent DiseasePneumocystis carinii PneumoniaMycobacterium TuberculosistuberculosisMycobacterium avium Lung infectionHerpesviruses 6. 7 Kaposi’s sarcoma, Hodgkin’s lymphomaCytomegalovirus CMV-infectionCabndida albicans Affection of skin and oral mucosaCryptococcus Skin affectionneoformansCryptosporidia species Acute diarrheaToxoplasma gondii Neurological pathology
  38. 38. HIV and AIDS The Cellular Picture Loss of one cell type throughout the course of the disease CD4+ T4 helper cells A fall in the CD4+ cells always precedes diseaseThe virus only grows on T4 cells that are proliferating inresponse to an immune stimulus In advanced disease: the loss of another cell type CD8+ cytotoxic killer cells
  39. 39. The Genome of HIV
  40. 40. HIVMembrane: host derived Three genes GAG – POL – ENV Three polyproteins
  41. 41. HIVENV gene Two glycoproteins: gp160 gp120 and gp41• gp120 – adherence to cellular CD4 receptors• gp41 – fusion of viral envelope with cell cytoplasmicmembrane
  42. 42. HIV GAG gene Polyprotein Group-Specific Antigensp17: inner surface - myristoylatedp24: nucleocapsidp9: nucleocapsid associated with RNA
  43. 43. HIV• POL gene Enzymes• Polymerase (reverse transcriptase –RNA dependent DNA polymerase)• Integrase• Protease (cuts polyproteins)
  44. 44. Structure of HIV gp 120 gp 41 Matrix proteins Envelope Nucleocapsid with RNA and enzymes
  45. 45. Reproduction of HIVBudding virion Adherence to CD4 Penetration andCell synthesis DNAmembranemodified by on the RNAviral proteins template New viral Cell nucleus RNA New viral Viral progeny RNA Viral DNA enter into the proteins leave the nucleus nucleus and integrate with chromosome
  46. 46. Budding virions
  47. 47. T-cells infected by HIV (releasing viruses)
  48. 48. HIV - Life History Latency Specific destruction of CD4+ cells
  49. 49. HIV - Life History •Syncytia formation Profound significance for AIDS progression and therapy: spread from cell to cell and as result escaping of antibodies. Humoral antibody will not stop spread – need cell- mediated response
  50. 50. HIV - Life History Latency – Cellular – The problem of memory T4 cellsOnly activated T4 cells can replicate virusMost infected T4 cells are rapidly lyzed but are replacedSome T4 cells revert to resting state as memory cells which are long-livedMemory T4 cells cannot replicate the virus unless they become activated Clinical Latency HIV infection is not manifested as disease for years During apparent clinical latency, virus is being replicated and cleared
  51. 51. Transmission of HIV By sexual contact (57%) By transfer of infected blood (13%) By injection (13%) Vertical transmission - from infected mother to neonate, either at birth or via breast milk (17%)
  52. 52. HIV and AIDSThe cellular and immunological picture - The course of the disease
  53. 53. HIV and AIDSThe cellular and immunological picture - The course of the disease
  54. 54. HIV and AIDS The cellular and immunological picture The course of the disease 1. Acute Infection High virus titer Mild symptoms Fall in CD4+ cells but recovers Rise in CD8+ cells but recovers A high virus titer (up to 10 million viruses per mlblood) Macrophages infected
  55. 55. HIV and AIDS 2. A strong immune response Virus almost disappears from circulation Good cytoxic T cell response Soluble antibodies appear later against bothsurface and internal proteins Most virus at this stage comes from recentlyactivated (dividing) and infected CD4+ cells CD4+ cell production compensates for loss dueto lysis of cells by virus production anddestruction of infected cells by CTLs
  56. 56. HIV and AIDS 3. A latent state Latency of virus and of symptoms Virus persists in extra-vasculartissues Lymph node dendritic cells Resting CD4+ memory cells (last avery long time - a very stablepopulation of cells) carry provirus
  57. 57. HIV and AIDS• 10 billion HIV particles per day• Virus half life 5.7 hours• 100-10 million virions per ml blood (set point)• Small minority of T4 cells are infected• Virus found in lymph nodes
  58. 58. HIV and AIDS 4. The beginning of diseaseMassive loss of CD4+ cells CD4+ cells are the targets of the virus Cells that proliferate to respond to thevirus are killed by it Dendritic cells present antigen and virusto CD4 cells Epitope variation allows more and more HIV toescape from immune response just as response wanes Apoptosis of CD4+ cells HIV patients with high T4 cell countsdo not develop AIDS
  59. 59. HIV and AIDS5. Advanced disease - AIDS CD8+ cells destroy more CD4+ cells CD4 cell loss means virus and infectedcells no longer controlled As CD4+ cells fall below 200 per cu mmvirus titer rises rapidly and remainingimmune response collapses CD8+ cell number collapses Opportunistic infections Death in ~2 years without intervention
  60. 60. Virus destroys the cell as a result of budding Why do all T4 cells1. PUNCTURED MEMBRANE disappear?
  61. 61. Why do all T4 cells disappear? - 2 Most T4 cells are not HIV+Infected CD4 Could “sweep cell Cells Fuse up” uninfectedGp120 positive cells Uninfected Killing of CD4 cells CD4 cell 2. Syncytium Gp120 Formation negative
  62. 62. Why do all T4 cells disappear? Cytotoxi c T cell Killing of CD4 cells 3. Cytotoxic T cell-mediated lysisBUT: Most cellsare not infected
  63. 63. Killing of CD4+ cells4. Complement-mediated lysis Binding of free Gp120 toCD4 antigen makesuninfected T4 cell looklike an infected cell
  64. 64. 5. Apoptosis of T cells and macrophages
  65. 65. Laboratory diagnosis Detection of viral antigens in the patient blood by immuno-enzyme assay Detection of antiviral antibodies by immuno-enzyme assay ELISA Detection of viral RNA by PCR Detection of proviral DNA in infected cells by PCR
  66. 66. Strategies for drugs to treat AIDS (1)A prominent group of drugs (AZT, ddC) are molecularmimics called nucleoside analogs or reversetranscriptase inhibitors
  67. 67. Strategies for drugs to treat AIDS (2)Protease inhibitors plug into the active sites in HIVprotease
  68. 68. Strategies for drugs to treat AIDS (3)Ribozyme. The enzyme that effectively cleave the viralRNA in half
  69. 69. vaccine problem Population Polymorphism Retroviruses use host cell RNA polymerase II to replicate their genome Pol II has a high error rate 1:2,000-10,000 HIV genome 9749 nucleotides Therefore EVERY new virus has at least one mutation! Every possible single mutation arises daily 1% of all possible double mutations arise dailyThe HIV that infects a patient is very different from that seen by the time AIDS appears
  70. 70. Population Polymorphism• Variation in reverse transcriptase leads to resistance tonucleoside analogs drug problem• Variation in protease leads to resistance to proteaseinhibitors drug problem Polymorphism due to high mutation rate as a result of lack of proof-reading in reverse transcriptase and RNA pol II Sub-populations arise with altered cell tropism

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