HIV molecular biology

1. HIV molecular biology BTY328: Virology wstafford@uwc.ac.za

2. Retroviruses • Oncoviruses: cancer causing eg. HTLV • Lentiviruses: slow virus diseases eg. HIV-1 and HIV-2 • Spumaviruses: not associated with human disease. • Retroviruses such as HIV are so named because they reverse the usual flow of genetic information within host cell in order to reproduce themselves (Central dogma: DNA-RNA-protein)‏

3. Reverse transcriptase and HIV integration into the host genome • Retroviral RNA is copied using the enzyme reverse transcriptase (RT) into a complementary DNA strand, this is copied into dsDNA, and this migrates to the host nucleus and becomes integrated into the host cell DNA as a provirus. • From this point on the process is irreversible as the viral genes are part of the cells genetic information (essentially an acquired genetic disease)‏

4. Active and latent HIV infection of CD4

5. HIV recognition of CD4 T-cell

6. Recognition, attachment HIV surface membrane has glycoproteins gp41 and gp120 􀁺 gp120 binds to CD4 receptor on CD4+ cell (T lymphocytes)‏ 􀁺 gp41 binds to a cytokine receptor, either CCR5 or CXCR4, fusing viral envelope with cell membrane and releases the capsid into the cell.

7. Reverse transcription and integration into the host genome Nucleocapsid contains two single stranded RNA molecules, as well as reverse transcriptase, which makes dsDNA from the RNA. 􀁺 dsDNA is transported to nucleus by viral protein R (VPR)‏ 􀁺 HIV integrase iserts viral dsDNA into host cell

8. HIV genome replication and protein production Incorporated viral DNA is transcribed onto two mRNAs, 􀁺 one strand contains information for HIV structural proteins 􀁺 other serves as genetic material of new virus Translated polypeptide is cleaved by protease, making functional HIV proteins

9. Assembly of HIV particles HIV Structural proteins spontaneously assemble around RNA and viral proteins, forming new virus 􀁺 Virus is encapsulated by cell membrane and buds off (exocytosis)‏ 􀁺 Through mechanisms not understood (adoptosis) host cells die.

11. Reverse transcriptase (RT) • Reverse Transcriptase: Heterodimer consisting of a full 66kDa RT monomer with RNaseH domain, and a 51kDa RT monomer from which the RNaseH domain has been cleaved. • HIV RT is a RNA-dependant DNA polymerase transcribing ssDNA from RNA template. • It has a subunit with RNaseH activity- hydrolysing the retroviral RNA from the RNA-DNA complex. • Also acts as a DNA-dependant polymerase to transcribe the second DNA strand complementary to the first DNA strand.

13. Integration in to the host genome • HIV integrase is a 32 kDaprotein produced from the C-terminal portion of the Pol gene product, and is an attractive target for new anti-HIV drugs. Integrase catalyzes two reactions; • 3'-end processing, in which two deoxynucleotides are removed from the 3' ends of the viral DNA. • the strand transfer reaction, in which the processed 3' ends of the viral DNA are covalently ligated to the host chromosomal DNA. • Integrase is a key step in allowing viral DNA to become a permanent member of the host genome.

14. Production of Viral RNA transcripts • Before HIVs provirus genes can be expressed, RNA copies that can be read by host cells protein-making machinery must be produced. • Proviral DNA produces new strands of RNA; some behave like mRNA to produce proteins essential for HIV production, others become encased with viral core proteins to become new viruses.

15. HIV genes • All retroviruses contain RNA sequences that code for same three genes. Flanking these genes is a sequence of similar nucleotides (LTRs).The LTR functions as a promoter. • 5'–LTR-- GAG POL ENV –LTR--3' • There are 9 HIV genes encoding proteins: • Structural proteins: GAG, POL, and ENV • Regulatory proteins: Tat and Rev • Accessory proteins: Nef, Vif, Vpu, Vpr

17. Structural Genes GAG- group specific antigens make up viral nucleocapsid- cylindrical core proteins (p24: a nucleoid shell protein with mol. wt. 24,000) and several internal proteins.When GAG is non-functional HIV loses ability to bud out of cell. POL codes for the following HIV enzymes: P10 protease, reverse transcriptase, endonuclease /integrase ENV codes for two major HIV surface proteins- gp120: located on external spikes of HIV and gp 41: transmembrane protein that attaches gp120 to surface of HIV.

18. Regulatory Genes TAT • A transactivator protein that exerts its effect on viral replication • Tat protein interacts with a short nucleotide sequence called TAR located within the 5'LTR region of HIV mRNA transcripts. • Once Tat protein binds to TAR sequence, transcription of provirus by cellular RNA polymerase II accelerates 1,000 times; in absence of Tat, viral transcription usually terminates prematurely.

19. REV Regulator of Expression of Viral proteins • Selectively increases synthesis of HIV structural proteins in latter stages of HIV disease, thereby maximising production of new virus. • Functions by transporting spliced & unspliced RNAs from nucleus to cytoplasm.

20. Accesory Genes Nef: N-myristylated and anchored to inner surface of plasma membrane, probably in a complex with a cellular serine kinase. Makes cell more capable of producing HIV progeny • Decreasing cell surface expression of CD4 (prevent host cell re-infection with HIV?)‏ • Decreases cell surface expression of MHC in infected lymphocytes and this reduces the cytotoxic T cells response to HIV. • Increases transcriptional activator Nf-kb thereby augmenting viral expression.

21. Summary