Retroviruses and HIV

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Virology Report of Retroviruses / HIV
Biotechnology Class Course 37
Institute of Biotechnology Research and Development
Can Tho University

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Retroviruses and HIV

  1. 1. Retroviruses and Human Immunodeficiency Virus (HIV) Lecturer: Bùi Thị Minh Diệu
  2. 2. Group members • Trần Hoàng Đệ • Lâm Tấn Hào • Huỳnh Lê Bảo Ngọc • Trần Hạnh Phước • Hoàng Nguyễn Phương Trinh • Lý Hoàng Tuấn
  3. 3. Outline • Part 1: Retroviruses: – Discovery & Classification – Methods to study retroviruses – Retrovirus structure – Retrovirus cycle
  4. 4. Outline • Part 2: HIV-1: – History – Signs & symptoms of AIDS – AIDS transmission and epidemiology – HIV-1 structure & replication – Treatment and prevention of HIV/AIDS
  5. 5. Discovery • The finding of retrovirus (Rous, 1910) & reverse transcriptase (Temin & Baltimore, 1971)  revolution Figure 1 (from left to right) Peyton Rous, Howard Temin & David Baltimore
  6. 6. Classification Genus Examples Host Alpharetrovirus Rous sarcoma virus Chickens Betaretovirus Mouse mammary virus Mice Gammaretrovirus Murine leukemia virus Mice Deltaretrovirus Human T-cell leukemia virus type 1 Humans Epsilonretrovirus Walleye dermal sarcoma virus Fish Lentivirus Human immunodeficiency virus type 1 Simian immunodeficiency virus Feline immunodeficiency virus Humans Monkeys Cats Spumavirus Simian foamy virus Monkeys Table 1 7 genera of retroviruses
  7. 7. Methods • Apply many widely used methods in virology: – Purification of retroviral particles – Structural study with electron microscope
  8. 8. Purification of retroviral particles • On the basis of size and density • Achieved by centrifugation – Retroviral density ~ 1.16 g/ml  35% w/w sucrose • In retroviruses, physical/infectious particles > 100:1  flaws in properties measurement • Most purified retrovirus: AMV  early biochemical study of viral proteins
  9. 9. Electron microscope • Measure particle size • Define morphology • Study of retroviral structural proteins
  10. 10. Electron microscope • Use 2 techniques, each with its own drawbacks: – Negative staining: deformations in particles – Thin-section: • Require harsh fixation • Final appearance depends on plane of sectioning •  Particle size can also be measure by rate zonal sedimentation, but usually result in doubling in actual size
  11. 11. Cryo-electron microscope • Observe virus directly as an unstained particle • Drawback: low-contrast image  Apply computer-assisted program to generate 3D structure
  12. 12. Methods to study retroviruses • To sum up: – The exact arrangement of retroviral components remains uncertain – Models are necessary to represent the virion structure with suitable modification and prediction – A complete understanding of retrovirus may have to wait for the development of new techniques
  13. 13. Structure • Virion structure: – Roughly spherical, 100nm in diameter – Icosahedral or conical capsid – Packaging 2 identical copies of (+)strand RNA and viral enzymes (RT, PR, and IN) in enveloped virion Figure 2 A typical retrovirus virion
  14. 14. Structure • Genome structure: – The virus genome is a (+)strand RNA: 7–10kb Figure 3 Structure of retrovirus RNA
  15. 15. Retrovirus life cycle Early phase • Virus enters the cell  copies RNA genome  inserts the copy into the host cell genome. Late phase • Expression of viral RNA • Synthesis of viral proteins • Assembly of virions
  16. 16. Early phase • Retrovirus enters cell by the fusion pathways. • Viral RNA is converted into a double-stranded DNA copy by reverse transcription.  proviral DNA • A copy of proviral DNA is integrated into the cellular genome at a random site. Figure 4 Early phase of retrovirus life cycle
  17. 17. Attachment and Entry SU protein (virus) interacts with receptors (host) TM protein changes conformation, allowing virion membrane to fuse with host plasma membrane Figure 5 Retrovirus attachment and entry. Fusion of virion membrane and plasma membrane, the virion contents is modified to be the transcription complex
  18. 18. Reverse transcription • Reverse transcription: RNA  DNA – Enzyme: reverse transcriptase  2 activities: RNA/DNA dependent DNA polymerase, ribonuclease H – No proofreading  quasispecies – 9 steps
  19. 19. Figure 6 Reverse transcription. The first 6 steps
  20. 20. Figure 7 Reverse transcription. The last 3 steps
  21. 21. Integrase Integration sites: random (Cleave  ligate) Proviral DNA and host DNA can replicate together Figure 8 Integration of proviral DNA into host cell Integration
  22. 22. Late phase • Expression of viral RNA through transcription of proviral DNA • Synthesis of viral proteins through translation and post-translational modification • Assembly and budding of virions
  23. 23. Expression of viral RNA • 2 identical long terminal repeats LTRs: – Left LTRs: signaling the transcription initiation precisely at U3/R junction – Right LTRs: signaling cleavage and polyadenylation of the transcript Figure 9 Transcription of proviral DNA
  24. 24. Expression of viral RNA • Differential splicing generates multiple mRNAs • All retrovirus make at least 2 mRNAs: – Unspliced form  Gag & Gag/Pol proteins – Singly spliced form  Evn proteins
  25. 25. Synthesis of viral proteins • Gag: many structural proteins • Pol: fewer enzyme molecules •  2 mechanisms to ensure the Gag and Gag/Pol in a proper ratio (~ 95%:5%): – Suppression of translation termination – Ribosomal frameshifting
  26. 26. Suppression of translation termination • Correct recognition of stop codon UAG separating gag and pol reading frame  gag protein only • Gln-tRNAGln misreading UAG as CAG 1/20 time  translational readthrough  Gag/Pol polyprotein – Stimulated by pseudoknot Figure 10 Suppression of translation termination
  27. 27. Ribosomal frameshifting Figure 11 Ribosomal frameshifting A heptamer A 2nd structure
  28. 28. Figure 12 Retrovirus translation and post- translational modifications Evn is glycosylated and cleaved  SU & TM Gag, Gag/Pol is myristylated
  29. 29. Assembly of the virion Two different assembly pathways: Core assembly and then budding  “B-type”, “D-type” viruses Core assembly and budding simultaneously  “C-type” virus Figure 13 2 assembly pathways in retroviruses
  30. 30. Assembly of the virion • Only full-length RNA is encapsidated, thanks to psi () signal • As virion assembled and extrudes, protease cleaves Gag, Gag/Pol  mature & functional form  Virion become infectious
  31. 31. History • 1959: 1st case of HIV infection in human from Democratic Republic of the Congo (Africa) • 1981: A group of healthy young male in Los Angeles/San Francisco showed significant depletion of their immune system  suffered opportunistic infections (pneumonia)  The term acquire immunodeficiency syndrome (AIDS)
  32. 32. History • 1983: A retrovirus isolated from the blood of individuals with AIDS was characterized  human immunodeficiency virus type 1 (HIV-1) Figure 14 (from left to right) Luc Montagnier, Barré-Sinoussi, and Robert Gallo successfully isolated and characterized HIV-1 at the same time
  33. 33. AIDS Signs and Symptoms Acute infection • Last several weeks • Mononucleosis or influenza-like syndromes Clinical latency • Two weeks to >20 years depends on several factors • Few or no symptoms AIDS • Low CD4+ T cell level • Various opportunistic infections, cancers Figure 15 3 stages of HIV infection
  34. 34. HIV Transmissions • HIV-1 was probably transmitted to humans from chimpanzees infected with SIVcpz. • HIV can be transmitted from an infected person to another through: Blood, semen, vagina secretions, breast milk. • Activities that allow HIV transmission: – Unprotected sexual contact – Direct blood contact (injection drug needles, blood transfusions…) – Mother-to-Child transmission (Vertical transmission)
  35. 35. AIDS Epidemiology • HIV/AIDS is a global pandemic – approximately 35.3 million people living with HIV globally (2010), of which: 3.4 million children <15 • Sub-Saharan Africa and South East Asia are 2 regions most affected.
  36. 36. Sub-Saharan Africa • 12% world population <> contribute to 2/3 people infected with HIV • More women are infected than men • Reasons: • Widespread of sexually transmitted diseases • Unsafe blood transfusions • Poor state of hygiene and nutrition • Poor economic conditions • Lack of sex education Figure 16 Map of HIV prevalence in Africa in 2007
  37. 37. South & South East Asia • 4.2 – 4.7 M adults and children infected. – Largely concentrated in: injecting drug users, men who have sex with men (MSM), sex workers, and clients of sex workers and their immediate sexual partners
  38. 38. Vietnam • 220k people living with HIV (0.47% population) (2007): – 65% are injecting drug users (IDU): sharing needles • Women are more exposed to risk of contracting HIV – 90k (2007) – Reasons: from their partners – undisclosed IDU, from men having pre-marital or extra-marital sexual relationships
  39. 39. Structure • Specific features: – Cone-shaped capsid: Wide end: 40–60nm, narrow end: 20nm Figure 17 Diagram of HIV-1 virion structure (Only 1 mRNA molecule is shown covered with CA for clarity)
  40. 40. Structure • Genome structure is very complex • Splicing of HIV-1 primary transcript  >25 mRNAs, coding for Gag, Gag/Pol, Env and 6 additional proteins Figure 18 Genome structure and RNA splicing pattern of HIV-1
  41. 41. HIV Replication • Entry and attachment of HIV-1 • Functions of 6 additional proteins
  42. 42. gp120 (SU) (HIV-1) binds to CD4 receptor (cell surface) gp120 changes conformation  interact with chemokine receptors (CCR5/CXCR4)  gp41 (TM) change conformation Fusion and release of nucleocapsid HIV-1 Attachment and Entry Figure 19 Model of HIV-1 entry
  43. 43. 6 addition proteins • Virion protein R (Vpr) • Viral infectivity factor (Vif) • Virion protein unique to HIV-1 (Vpu) • Transactivator of transcription (Tat) • Regulator of expression of virion protein (Rev) • Negative effector (Nef)
  44. 44. Tat • Transactivator of transcription increases HIV-1 transcription by stimulating elongation by RNA pol II Figure 20 Mechanism of Tat function
  45. 45. Rev • Regulator of expression of virion protein mediates cytoplasmic transport of viral 9-kb (full- length) and 4-kb (singly spliced) mRNA Figure 21 Mechanism of Rev function
  46. 46. Virion protein R (Vpr) • Confer HIV-1 the special ability to infect non-dividing cell • Facilitate the packing of enzyme uracil DNA, glycolase in the virion  remove deoxyuridine, which blocks transcription • Arrest infected cell at G2 stage, at which transcription of HIV-1 is the most active  Enhance HIV-1 replication at multiple levels
  47. 47. Viral infectivity factor (Vif) • Structure: 139-amino acid protein • Found in cytoplasm of infected cells • Function: Vif prevents action of host protein APOBEC3G  viral DNA is not mutated by APOBEC3G  increases virion infectivity
  48. 48. Virion protein unique to HIV-1 (Vpu) • Structure: 81-amino acid protein • Found in Golgi apparatus, endosome compartment of infected cells • Function: enhance the release of progeny virions – Degradation of CD4 – Enhancement of virus release from the plasma membrane
  49. 49. Negative effector (Nef) • Structure: 210-amino acid protein • Found in the inner face of plasma membrane • Function: – Decrease in the expression of CD4 and MHC1 – Enhancement of virus infectivity – Modification of cell signaling  enhance release of progeny virion
  50. 50. AIDS Treatment • Chemotherapy: – 5 classes of antiretrovirals, each with its own mode of act (MOD) and drawbacks (D). • Immunotherapy • RNA interference
  51. 51. Chemotherapy • Nucleoside analog reverse-transcriptase inhibitors (NRTIs) • Non-nucleoside RT inhibitors (NNRTIs) • Protease inhibitors (PIs) • Entry inhibitors • Integrase inhibitors
  52. 52. Nucleoside analog reverse-transcriptase inhibitors (NRTIs) • MOD: Inhibit the viral reverse transcription • D: Has serious side-effects Figure 22 Azidothymidine (AZT) – the first NRTI to be used
  53. 53. Non-nucleoside RT inhibitors (NNRTIs) • Very varied chemical structure • MOD: Cause allosteric inhibition • D: – Side effects – Ineffective against HIV-1 and drug-resistant mutants Figure 23 Nevirapine (NVP) (left) and Efavirenz (EFV) (right) – 2 common NNRTIs
  54. 54. Protease inhibitors (PIs) • MOD: Compete with protease enzymes – Efficacy in triple therapy (used in combine with 2 NRTI) - Highly active antiretroviral therapy – HAART • D: Frequent and severe side- effects Figure 24 Ritonavir – the PI usually used in HAART
  55. 55. Entry inhibitors • MOD: Prevent membrane fusion by changing gp41 conformation • D: – Not cost-effective – Ineffective against drug-resistance strains
  56. 56. Integrase Inhibitors • MOD: Inhibit strand transfer • D: Low barrier for virus to easily overcome Figure 25 Raltegravir: (left) Structure and (right) Marketable form
  57. 57. Immunotherapy • No effective vaccine is available  Alternative treatment strategies are needed – Monoclonal antibodies are promising therapy – Using of immunogen need to be considerered
  58. 58. RNA Interference • Response to the double stranded RNA • Target both HIV genes and host cell receptors  Reduce the chance of synthesis of HIV proteins • Face numerous challenges for clinical application
  59. 59. HIV/AIDS Prevention • Mother-to-Child Transmission • Sexually Transmitted Infections – Microbicides • Post-exposure Prophylaxis • Pre-exposure Prophylaxis
  60. 60. Mother-to-Child Transmission • Treatment of the mother and infants with AZT – Combined with exclusive formula-feeding
  61. 61. Sexually Transmitted Infections • Safe sex message & practice – Important and cost-effective method
  62. 62. Microbicides • Microbicides: cellulose sulfate and PRO-2000, and non-ionic surfactants  applied topically to vagina/rectum  disrupt virus – cell interaction  appropriate for women • D: May cause inflammatory effect
  63. 63. Post-exposure Prophylaxis • Use antiretroviral drugs (ZDV, truvada & kaletra) immediately after possible exposure to HIV: – Occupational exposure – Sexual exposure – Pre/Post-natal treatment  Reduce the risk of HIV infection
  64. 64. Pre-exposure Prophylaxis (PREP) • The use of antiretroviral drugs (i.e. truvada) prior to exposure • Concerns: – Rapid development of resistance in cases of transmission – People undergo PREP may have some risky behavior
  65. 65. Conclusion • The discovery of retroviruses and reverse transcriptase is phenomenal • No available method provides a complete understanding of retroviruses • Retrovirus cycle consists of 2 phases • HIV-1 is a typical member of retroviruses • HIV has caused global pandemic – AIDS • Currently, there is no effective treatment or vaccine to completely eliminate HIV
  66. 66. Thank you for your attention! Figure 26 The red ribbon is a symbol for solidarity with HIV- positive people and those living with AIDS. World AIDS day: December 1st

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