Retroviruses and AIDS

1. Retroviruses and AIDS Dr Amanj Saeed MB.CH.B, MSc, PhD amanj.saeed@krg.org

2. Discovery of retroviruses • Retroviruses possess a unique enzyme known as RT (reverse transcriptase) • RT uses viral RNA as a template for making DNA copy which integrate in to the chromosome of the host cell and serves either as basis for viral replication or as oncogene. • Howard Temin and David Baltimore received Nobel Prize for discovery of RT enzyme.

3. Discovery of HIV • In 1981 new clinical syndrome characterized by profound immunodeficiency was recorded in male homosexual and termed AIDS. • Unusual prevalence of Pneumocystis carinii pneumonia in in a group of young previously healthy male homosexual. Kaposi’s sarcoma (rare cancer) in previously healthy male homosexual??

4. Discovery of HIV • First isolation of HIV-1 made by Luc Montagnier and Barre-Sinoussi at Pasteur institute in Paris in 1983. • This observation is confirmed by Robert Gallo in the USA.

5. Discovery of HIV • HIV-2 isolated from mildly immunosuppressed patient n west Africa. • 5000 cases of HIV-1 cases per Day? • 41 million people have been infected world wide. • HIV-2 account for 4.5% of HIV cases.

6. Retroviridae

7. Lentiviruses

8. Primates infected with lentiviruses > 30 species of African primates naturally infected with SIV SIV infections: natural acquired not known • Natural infections: • >50% of adults • nonpathogenic • Chimpanzee the only ape

9. Primate Lentiviruses HIV-2

10. Photograph by Karl Ammann

11. Photograph by Karl Ammann

13. Properties of HIV • Classification • The family Retroviridae is named for RT. • (Retro= Backwards) • Seven genera is now recognised (only two of them cause disease in human): • Lentivirus: containing HIV-1 and HIV-2, characterised by: • Cone shaped Nucleocapsid, absence of oncogenicity, and the lengthy and insidious onset of clinical signs.

14. Properties of HIV • BLV-HTLV retroviruses: contain HTLV-I and II: characterised by ability to cause tumours rather than immunosuppression. • Spumavirus : Causes characteristic foamy appearance in infected primate cell culture. (they are not pathogenic).

16. Global estimates for adults and children  2010 People living with HIV34.0 million [31.6 million – 35.2 million] New HIV infections in 2010 2.7 million [2.4 million – 2.9 million] Deaths due to AIDS in 2010 1.8 million [1.6 million – 1.9 million]

17. Adults and children estimated to be living with HIV  2010 Eastern Europe & Central Asia 1.5 million [1.3 million – 1.7 million] Western & Central Europe 840 000 [770 000 – 930 000] North America 1.3 million [1.0 million – 1.9 million] East Asia 790 000 [580 000 – 1.1 million] Middle East&North Africa 470 000 [350 000 – 570 000] Caribbean 200 000 [170 000 – 220 000] South & South-East Asia 4.0 million [3.6 million – 4.5 million] Sub-Saharan Africa 22.9 million [21.6 million – 24.1 million] Latin America 1.5 million [1.2 million – 1.7 million] Oceania 54 000 [48 000 – 62 000] Total: 34.0 million [31.6 million – 35.2 million]

18. Estimated number of adults and children newly infected with HIV  2010 Eastern Europe & Central Asia 160 000 [110 000 – 200 000] Western & Central Europe 30 000 [22 000 – 39 000] North America 58 000 [24 000 – 130 000] East Asia 88 000 [48 000 – 160 000] Middle East&North Africa 59 000 [40 000 – 73 000] Caribbean 12 000 [9400 – 17 000] South & South-East Asia 270 000 [230 000 – 340 000] Sub-Saharan Africa 1.9 million [1.7 million – 2.1 million] Latin America 100 000 [73 000 – 140 000] Oceania 3300 [2400 – 4200] Total: 2.7 million [2.4 million – 2.9 million]

19. Estimated adult and child deaths from AIDS  2010 Eastern Europe & Central Asia 90 000 [74 000 – 110 000] Western & Central Europe 9900 [8900 – 11 000] North America 20 000 [16 000 – 27 000] East Asia 56 000 [40 000 – 76 000] Middle East & North Africa 35 000 [25 000 – 42 000] Caribbean 9000 [6900 – 12 000] South & South-East Asia 250 000 [210 000 – 280 000] Sub-Saharan Africa 1.2 million [1.1 million – 1.4 million] Latin America 67 000 [45 000 – 92 000] Oceania 1600 [1200 – 2000] Total: 1.8 million [1.6 million – 1.9 million]

20. Children (<15 years) estimated to be living with HIV 2010 Eastern Europe & Central Asia 17 000 [14 000 – 23 000] Western & Central Europe 1400 [<1000 – 1800] North America 4500 [4000 – 5800] East Asia 16 000 [11 000 – 21 000] Middle East & North Africa 40 000 [27 000 – 52 000] Caribbean 16 000 [12 000 – 19 000] South & South-East Asia 160 000 [110 000 – 210 000] Sub-Saharan Africa 3.1 million [2.8 million – 3.5 million] Latin America 42 000 [30 000 – 54 000] Oceania 4600 [3600 – 5800] Total: 3.4 million [3.0 million – 3.8 million]

21. Estimated number of children (<15 years) newly infected with HIV  2010 Eastern Europe & Central Asia 2200 [1700 – 2900] Western & Central Europe <100 [<200] North America <100 [<200] East Asia 2100 [<1000 – 3800] Middle East & North Africa 6800 [4800 – 8800] Caribbean 1200 [<1000 – 1700] South & South-East Asia 20 000 [14 000 – 28 000] Sub-Saharan Africa 350 000 [300 000 – 410 000] Latin America 3500 [2100 – 5000] Oceania <1000 [<500 – <1000] Total: 390 000 [340 000 – 450 000]

22. Estimated deaths in children (<15 years) from AIDS  2010 Eastern Europe & Central Asia 1200 [<1000 – 1800] Western & Central Europe <100 [<200] North America <100 [<200] East Asia 1100 [<1000 – 1700] Middle East & North Africa 3900 [2700 – 5000] Caribbean 1000 [<1000 – 1300] South & South-East Asia 14 000 [8300 – 20 000] Sub-Saharan Africa 230 000 [200 000 – 260 000] Latin America 2400 [1300 – 3500] Oceania <500 [<500 – <500]

23. Morphology of HIV • HIV particle is 100-150 nm in diameter. • Outer envelope of lipid penetrated by 72 glycoprotein spike (the lipid envelope protein) • The envelope protein is composed of two subunits: the outer glycoprotein knob (gp120) and transmembrane protein (gp41) • The receptor binding site for CD4 is present on gp120 as well as very important antigen such as V3 loop.

24. Morphology of HIV • The inner surface of virus lipid envelope is lined by matrix protein (p17)?. • There is also abundant cellular proteins in the lipid envelope (MHC class I and II) antigens. • In HIV-1 the lipid envelope encloses an icosahedral shell of protein (p17), within which is a vase or cone shaped protein core (p24, p7, and p9) containing two molecules of positive sense ssRNA • The RNA genome is associated with several copies of RT, integrase, and protease.

26. HIV genome • Positive sense ssRNA genome • The genome is approximately 10kb in size • The genome contain control genes which can enhance viral replication: • rev: regulator of virus • tat: transactivation. • vif: viral infectivity • repressor genes: • nef: negative factor

27. HIV genome • The genome is flanked at each end by LTR • 3’ LTR has the polyadenylation signal and 5’LTR has the enhancer promotor sequence for viral transcription. • The pol gene code for RT, integrase and protease.

29. The HIV-1 genome rev vif nef tat 5’ LTR 3’ LTR vpu vpr gag pol env p17 matrix antigen p24 capsid antigen p6/7 nucleocapsid reverse transcriptase protease integrase envelope glycoprotein (gp120) transmembrane glycoprotein (gp41)

30. HIV genome • HIV binds to specific receptor on the surface of CD4+ T lymphocytes (T-helper cells) • It also infects: • B lymphocytes • Macrophages • dendritic cells • brain cells. • Second subsidiary receptor belongs to chemokine receptor family CXCR4 on the T-cells and CCR5 on the surface of macrophages.

32. Retroviruses

33. HIV lifecycle maturation virus binding fusion virion assembly and release ssRNA (+) reverse transcription translation of viral proteins dsDNA nuclear transport integration transcription nucleus cytoplasm

34. HIV life cycle • After attachment the virus penetrate the cell by fusion from without (Mediated by gp21 and gp41) • Synthesis of viral cDNA starts when the virion enters the cell cytoplasm. • The viral RT enzyme directs the synthesis of cDNA strand (the minus strand) using host positive RNA as a primer and the viral genomic RNA as a template. • Viral RNAse enzymatically remove the viral RNA while the RT synthesize the second DNA strand (plus strand).

35. HIV life cycle • Viral dsDNA will enter the Nucleolus of the host cell as a pre-integration complex (compose of viral protein M, Vpr, integrase, and dsDNA) • the integration of dsDNA to the host chromosome occurs (forming pro-viral DNA) • After integration viral and cellular factors are needed to activate HIV transcription. • Initial expression of viral RNA is stimulated by vpr and further stimulated by cellular transcription factors .

36. HIV life cycle • The primary RNA transcript is spliced to give 30 plus strand viral mRNAs. • Viral and cellular factors are required for early and late viral protein expression. • Early viral gene product include (tat, rev, and nef), accessory viral proteins (vif, vpr, and vpu) • Late viral gene products include (gap, pol and. env).

37. HIV life cycle • Assembly of new virion can begin by proteolytic cascade by viral proteases. • Different viral structural proteins begin to assemble with the p24 as a core and p7 enclosing viral RNA. • Viral genome assemble in the cytoplasm. • Retroviruses including HIV are release from the infected cells by budding from the infected cells. • The pro viral DNA may reside quietly in the chromosome for years.

38. Genetic Variability • RT has NO proof-reading mechanism therefore mutations (point point mutations and deletions/insertions) occur • Quasispecies = swarm of genetically distinct yet related viruses

39. Effects of Variability • Immune escape by changing/masking antigenic determinants • CTLs and Abs • Resistance to anti-retroviral drugs • Point mutations in enzymatic proteins • RT - resistance to nucleoside and non-nucleoside analogues • Protease - resistance to protease inhibitors • Altered cytopathogenicity • Env and particularly V3 mutations alter co-receptor usage • Different cell tropism, eg. Macrophages, T-cells, glial cells, langerhans cells etc. • Different tissue tropisms, e.g. brain

40. Integration • Double stranded cDNA (provirus) migrates to nucleus • Can exist extra-chromosomally as linear or circular form • Can integrate via the enzyme integrase

41. Activation • Once integrated the provirus responds to cellular nuclear factors e.g. SP1, NF-kB • Mediated through control regions in the 5’ LTR • Once active viral factors take over • Transactivation then control of RNA splicing events

42. Translation • Translation I.e.viral protein production • Virus release via budding on cell membrane • Morphological characteristics of budding virus is used for classification • type C, Type D morphology refers to morphology of budding/maturing virus

44. Summary • HIV member of the Retroviridae family (reverse transcriptase) • Entry mediated by CD4 plus co-receptor • Reverse transcription leads to errors • Virus can become integrated into chromosome (can be latent) • Transcription – short (spliced) then long RNAs • New virus buds at surface • Three main targets for therapy