Retroviruses and AIDS Dr Amanj Saeed MB.CH.B, MSc, PhD

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.

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??

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

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

Retroviridae

Lentiviruses

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

Primate Lentiviruses HIV-2

Photograph by Karl Ammann

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.

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).

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

Total: 34.0 million [31.6 million – 35.2 million] Western & Central Europe [ – ] Middle East & North Africa [ – ] Sub-Saharan Africa 22.9 million [21.6 million – 24.1 million] Eastern Europe & Central Asia 1.5 million [1.3 million – 1.7 million] South & South-East Asia 4.0 million [3.6 million – 4.5 million] Oceania [ – ] North America 1.3 million [1.0 million – 1.9 million] Latin America 1.5 million [1.2 million – 1.7 million] East Asia [ – 1.1 million] Caribbean [ – ] Adults and children estimated to be living with HIV  2010

Estimated number of adults and children newly infected with HIV  2010 Western & Central Europe [ – ] Middle East & North Africa [ – ] Sub-Saharan Africa 1.9 million [1.7 million – 2.1 million] Eastern Europe & Central Asia [ – ] South & South-East Asia [ – ] Oceania3300 [2400 – 4200] North America [ – ] Latin America [ – ] East Asia [ – ] Caribbean [9400 – ] Total: 2.7 million [2.4 million – 2.9 million]

Estimated adult and child deaths from AIDS  2010 Western & Central Europe 9900 [8900 – ] Middle East & North Africa [ – ] Sub-Saharan Africa 1.2 million [1.1 million – 1.4 million] Eastern Europe & Central Asia [ – ] South & South-East Asia [ – ] Oceania 1600 [1200 – 2000] North America [ – ] Latin America [ – ] East Asia [ – ] Caribbean 9000 [6900 – ] Total: 1.8 million [1.6 million – 1.9 million]

Western & Central Europe 1400 [<1000 – 1800] Middle East & North Africa [ – ] Sub-Saharan Africa 3.1 million [2.8 million – 3.5 million] Eastern Europe & Central Asia [ – ] South & South-East Asia [ – ] Oceania 4600 [3600 – 5800] North America 4500 [4000 – 5800] Latin America [ – ] East Asia [ – ] Caribbean [ – ] Children (<15 years) estimated to be living with HIV  2010 Total: 3.4 million [3.0 million – 3.8 million]

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

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

Morphology of HIV HIV particle is 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.

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.

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

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.

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

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.

Retroviruses

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

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).

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.

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).

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.

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

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

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

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

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

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