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Retroviruses and HIV

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Virology Report of Retroviruses / HIV …

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

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  • 1. Retroviruses and Human Immunodeficiency Virus (HIV) Lecturer: Bùi Thị Minh Diệu
  • 2. Group members • Trần Hoàng Đệ • Lâm Tấn Hào • Huỳnh Lê Bảo Ngọc • Trần Hạnh Phước • Hoàng Nguyễn Phương Trinh • Lý Hoàng Tuấn
  • 3. Outline • Part 1: Retroviruses: – Discovery & Classification – Methods to study retroviruses – Retrovirus structure – Retrovirus cycle
  • 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. 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. 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. Methods • Apply many widely used methods in virology: – Purification of retroviral particles – Structural study with electron microscope
  • 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. Electron microscope • Measure particle size • Define morphology • Study of retroviral structural proteins
  • 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. Cryo-electron microscope • Observe virus directly as an unstained particle • Drawback: low-contrast image  Apply computer-assisted program to generate 3D structure
  • 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. 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. Structure • Genome structure: – The virus genome is a (+)strand RNA: 7–10kb Figure 3 Structure of retrovirus RNA
  • 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. 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. 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. Reverse transcription • Reverse transcription: RNA  DNA – Enzyme: reverse transcriptase  2 activities: RNA/DNA dependent DNA polymerase, ribonuclease H – No proofreading  quasispecies – 9 steps
  • 19. Figure 6 Reverse transcription. The first 6 steps
  • 20. Figure 7 Reverse transcription. The last 3 steps
  • 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. 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. 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. 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. 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. 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. Ribosomal frameshifting Figure 11 Ribosomal frameshifting A heptamer A 2nd structure
  • 28. Figure 12 Retrovirus translation and post- translational modifications Evn is glycosylated and cleaved  SU & TM Gag, Gag/Pol is myristylated
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. HIV Replication • Entry and attachment of HIV-1 • Functions of 6 additional proteins
  • 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. 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. Tat • Transactivator of transcription increases HIV-1 transcription by stimulating elongation by RNA pol II Figure 20 Mechanism of Tat function
  • 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. 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. 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. 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. 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. AIDS Treatment • Chemotherapy: – 5 classes of antiretrovirals, each with its own mode of act (MOD) and drawbacks (D). • Immunotherapy • RNA interference
  • 51. Chemotherapy • Nucleoside analog reverse-transcriptase inhibitors (NRTIs) • Non-nucleoside RT inhibitors (NNRTIs) • Protease inhibitors (PIs) • Entry inhibitors • Integrase inhibitors
  • 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. 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. 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. Entry inhibitors • MOD: Prevent membrane fusion by changing gp41 conformation • D: – Not cost-effective – Ineffective against drug-resistance strains
  • 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. Immunotherapy • No effective vaccine is available  Alternative treatment strategies are needed – Monoclonal antibodies are promising therapy – Using of immunogen need to be considerered
  • 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. HIV/AIDS Prevention • Mother-to-Child Transmission • Sexually Transmitted Infections – Microbicides • Post-exposure Prophylaxis • Pre-exposure Prophylaxis
  • 60. Mother-to-Child Transmission • Treatment of the mother and infants with AZT – Combined with exclusive formula-feeding
  • 61. Sexually Transmitted Infections • Safe sex message & practice – Important and cost-effective method
  • 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. 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. 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. 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. 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