HIV lifecycle with more accurate depiction of budding

1. Translation of unspliced mRNA gives viral Gag, and occasionally, Gag-Pol proteins. These assemble to form immature particles. Proteolytic cleavage of Gag (by HIV protease) produces matrix (MA), capsid (CA), and nucleocapsid (NC), which rearrange to form the mature virus. HIV lifecycle with more accurate depiction of budding A Gag-Pol protein D’Souza & Summers, 2005, Nature Reviews 3: 643-655

2. Figure 9-15 Summary of HIV lifecycle See previous slide for correction.

3. Genomics and HIV Rapid sequencing of viral genomes Study transmission, mutation, drug resistance Genome wide association studies/genome sequencing of “elite controllers” (“long-term non-progressors”) Elite controllers: ~1 in 3000 Maintain 50 HIV/mL without anti-retroviral drugs (typically 104-106 HIV/mL before drugs)

4. Much of the human genome is “non-coding” DNA;Resolves the “C-value paradox” -- observation that genome size does not reflect complexity • Human genome: 1.5% protein-coding genes; 98.5% non-coding DNA. • Much of the non-coding DNA are transposable elements (transposons), sequences of DNA that can move to different positions with the genome; i.e., mobile genetic elements. • Discovered by Barbara McClintock in corn (1948). • Retrotransposons are one class of transposable element. They paste copies of themselves into genome in multiple places. • Retrotransposon DNA is first transcribed into RNA. • RNA copied into DNA by a reverse transcriptase (often encoded by the transposon itself). This should sound familiar…

5. Endogenous retroviruses in the human genome • 8-10% of human genome codes for retroviruses that are inherited along with other genes. Endogenous retroviruses are copied from a viral RNA genome and inserted as proviruses into the host genome. Intact retroviruses have LTRs (long terminal repeats) and coding sequences. Other retroelements can transpose but don’t contain functional genes. Everybody’s genome is littered with the remnants of ancient retroviral infections. The total burden of retroviral infection on the human genome is total to about one and a half average-sized chromosomes. There are more retroviruses in your DNA than there are genes. Paul Bieniasz, 2008

6. Producing viral particles • Retaining transpositional activity • Making proteins essential for host genome function • Decaying into “junk” DNAEndogenous retroviruses can move between species. Are xenotransplants safe?Need complete genomic sequences in many species to determine the number and location of endogenous retroviruses, their role in genome evolution, and their contributions to human disease. What is viral DNA doing in our genome?

7. Clicker question Is it likely that HIV could become an inherited part of the human genome? 1) YES 2) NO

8. Clicker question What is inside the core of HIV? 1) A lipid bilayer 2) Receptors 3) Viral enzymes and RNA 4) Carbohydrates 5) Mitochondria

9. Clicker question What is the typical course of an HIV infection? 1) AIDS-->flu-like symptoms-->symptomatic-->asymptomatic 2) Flu-like symptoms-->symptomatic-->asymptomatic-->AIDS 3) Symptomatic-->asymptomatic-->flu-like symptoms-->AIDS 4) Flu-like symptoms-->asymptomatic-->symptomatic-->AIDS

10. Clicker question How many genes does HIV have? 1) 1 2) ~10 3) ~100 ~1000 ~10000 ~100000

11. Clicker question HIV enters the host cell by 1) Budding 2) Receptor-mediated endocytosis 3) Fusion with the host membrane 4) Phagocytosis 5) Invitation

12. Clicker question Where does HIV persist in a latent phase? 1) Hides embedded in the genome 2) Hides in the Golgi apparatus 3) Hides in the Endoplasmic reticulum 4) Hides on the Y chromosome

13. Clicker question Where do the lipids on the HIV particles come from? 1) HIV has genes to make lipids for its particles 2) Lipids are acquired during processing in the Golgi Apparatus 3) A lipid bilayer is acquired from the host cell during budding 4) Lipids form spontaneously around the viral core because the core is hydrophobic

14. Clicker question HIV infects which of the following cell types: 1) Sperm cells 2) Red blood cells 3) B cells 4) T cells Macrophages Liver cells

15. Figure 11-23 HIV RNA is transcribed by viral reverse transcriptase into DNA that integrates into the host genome • Viral genome enters cell after fusion of viral and host cell membranes. • Viral reverse transcriptase protein packaged together with viral genome transcribes viral RNA into viral cDNA (complementary DNA). • Viral cDNA integrated into host cell genome by viral integrase. • Integrated cDNA is called the provirus. • Analogous to integrated prophage in a phage  lysogen Integrase enters cell along with viral genome. RT enters cell along with viral genome.

16. Consequences of integration of HIV genome into a host cell chromosome • HIV DNA that is integrated into a chromosome is duplicated when cell divides, therefore all progeny of the infected cell will contain HIV DNA. • Individuals with HIV in its latent, proviral form are healthy and show no signs of AIDS. • HIV can be passed among healthy individuals because HIV can residue as a provirus in T cells. Healthy individuals whose T cells contain integrated HIV DNA can transfer HIV in blood or semen, both of which contain T cells.

17. Figure 9-15 part 3 of 4 HIV lies dormant in resting T cells and replicates in activated T cells NFkB is normally supposed to be used by the cell to transcribe genes related to host defense -- it enters the nucleus to transcribe genes only under conditions of stress (e.g., when T cells are activated). Note that HIV is using the host cell’s normal defense mechanisms to transcribe its genes. Another example of evolution at work! Tat and Rev promote viral replication in activated T cells. NFkB is a host cell transcription factor that binds to the viral LTRs to initiate transcription by the host cell’s RNA polymerase.

18. Tat = transactivator protein • Tat protein binds to start of a new HIV RNA strand • Tat binds to TAR: Transactivator Active Region • Tat binds to TAR and activates transcription of HIV genes Tat is required for HIV-1 replication

19. Figure 9-15 part 4 of 4 Final steps in HIV assembly and budding from host cell HIV needs to export unspliced, singly spliced, and multiply spliced mRNAs from the nucleus to the cytoplasm in order to make all of its proteins. Eukaryotic cells normally prevent export of incompletely spliced mRNAs. Fully spliced Rev mRNA leaves the nucleus and gets translated in cytoplasm. Rev protein then enters the nucleus and binds to a specific site on the viral RNA and to a host transport protein to force export of unspliced viral mRNA. , Note there are some inaccuracies in this figure (maturation to a bullet-shaped capsid occurs AFTER budding).

20. Translation of unspliced mRNA gives viral Gag, and occasionally, Gag-Pol proteins. These assemble to form immature particles. Proteolytic cleavage of Gag (by HIV protease) produces matrix (MA), capsid (CA), and nucleocapsid (NC), which rearrange to form the mature virus. HIV lifecycle with more accurate depiction of budding A Gag-Pol protein D’Souza & Summers, 2005, Nature Reviews 3: 643-655

21. Figure 9-15 Summary of HIV lifecycle Not clear how cDNA enters nucleus with integrase. RT is primed by a host tRNA. Capsid might uncoat at a later stage. See previous slide for correction.

22. How does RT reverse transcribe only its own RNA? Host tRNALys3 packaged into virions along with HIV RNA. 3’ terminal 18 nucleotides of tRNALys3 anneals with 18 nucleotides at the 5’ of the HIV RNA (the primer binding site) tRNALys3 serves as a primer for RT. RT priming for in vitro reverse transcriptions done with oligo-dT or random primers

23. Genomics and HIV Rapid sequencing of viral genomes Study transmission, mutation, drug resistance Genome wide association studies/genome sequencing of “elite controllers” (“long-term non-progressors”) Elite controllers: ~1 in 3000 Maintain 50 HIV/mL without anti-retroviral drugs (typically 104-106 HIV/mL before drugs)

24. Much of the human genome is “non-coding” DNA;Resolves the “C-value paradox” -- observation that genome size does not reflect complexity • Human genome: 1.5% protein-coding genes; 98.5% non-coding DNA. • Much of the non-coding DNA are transposable elements (transposons), sequences of DNA that can move to different positions with the genome; i.e., mobile genetic elements. • Discovered by Barbara McClintock in corn (1948). • Retrotransposons are one class of transposable element. They paste copies of themselves into genome in multiple places. • Retrotransposon DNA is first transcribed into RNA. • RNA copied into DNA by a reverse transcriptase (often encoded by the transposon itself). This should sound familiar…

25. Endogenous retroviruses in the human genome • 8-10% of human genome codes for retroviruses that are inherited along with other genes. Endogenous retroviruses are copied from a viral RNA genome and inserted as proviruses into the host genome. Intact retroviruses have LTRs (long terminal repeats) and coding sequences. Other retroelements can transpose but don’t contain functional genes. Everybody’s genome is littered with the remnants of ancient retroviral infections. The total burden of retroviral infection on the human genome is total to about one and a half average-sized chromosomes. There are more retroviruses in your DNA than there are genes. Paul Bieniasz, 2008

26. Producing viral particles • Retaining transpositional activity • Making proteins essential for host genome function • Decaying into “junk” DNAEndogenous retroviruses can move between species. Are xenotransplants safe?Need complete genomic sequences in many species to determine the number and location of endogenous retroviruses, their role in genome evolution, and their contributions to human disease. What is viral DNA doing in our genome?

27. Clicker question Is it likely that HIV could become an inherited part of the human genome? 1) YES 2) NO

28. Summary of HIV lifecycle Bruce Walker, Harvard, HHMI holiday lectures

29. Clicker question What should you target to make an anti-viral drug? • An activity that is critical for viral function • An activity that is virally-encoded • An activity that is not similar to host activities • All of the above

30. Potential anti-HIV drugs might Block attachment to host cell Prevent fusion of viral and host membranes Inhibit reverse transcriptase Inhibit integrase Inhibit HIV protease

31. Attachment inhibition

32. D1 D2 D3 D4 PRO 542 -- the first two domains of CD4 fused to the constant (Fc) region of an antibody. CD4 The T cell co-receptor and HIV receptor. Recombinant soluble CD4 blocks gp120 binding to cell surface CD4 on T cells http://clinicaltrials.gov/ct2/show/NCT00055185 “The purpose of this study is to determine any adverse effects of PRO 542 after administration and to determine the anti-HIV effects of PRO 542 in the patient.” T cell plasma membrane

34. Could also block binding to CCR5 (HIV co-receptor) Progenics Pharmaceuticals http://www.progenics.com/prod_pro140.cfm

35. Pro 140 Pro 140 is designed to both block HIV and permit normal chemokine binding.

36. HIV fusion inhibition

37. HIV binding and fusion (movie from Dennis Burton, Scripps)

38. N-peptide C-peptide Heptad repeats form a-helices with one hydrophobic face; e.g., leucine zipper Trimer of coiled coils The gp41 ectodomain is a 6-helix bundle Structure thought to represent post-fusion state of gp41 Chan et al (1997) Cell 89:263

39. Excellent animation of fusion mediated by HIV gp41 http://www.molecularmovies.com/movies/gp41_092707.html