1. Retrovirus Biology Immunology/HIV Michael Para, MD 1

2. Learning Objectives  Describe the unique features and interrelationship of simple and complex Retroviridae  Identify the major structural components of HIV  Describe how HIV replicates  Describe and compare the mechanisms of action for the 5 antiretroviral drug classes  Describe how HIV becomes drug resistant and how this is prevented 2

4. GAG PRO POL R U5U3 R -AAA-3’5’-cap- Simple Retrovirus - ALV ENV GAGENV R U5 U3 R -AAA-3’5’-cap- Complex Retrovirus - HTLV POL TAX REX PRO Retroviridae are single stranded + sense RNA 4

5. 5 Schematic Diagram of the HIV-1 Viron

6. GAG PROENV R U5U3 R -AAA-3’5’-cap- POL GAGENV R U5 U3 R -AAA-3’5’-cap- Complex Retrovirus – HIV or HTLV (below) POL TAX REX PRO Simple Retrovirus – ALV with 4 genes 6 Spliced regulatory genes

7. oncoviruses (SIV) Evolutionary Relationship of Vertebrate Retroviridae 7

9. 9 Schematic Diagram of the HIV-1 Viron env

10. 10 Schematic Diagram of the HIV-1 Viron matrix nucleocapsid gag

11. Reverse Transcriptase env 11

12. 12 Reverse Transcriptase

13. HIV-1 RT/DNA Complex thumb 13

14. Mirror image dimer 14 HIV Protease with Inhibitor in Active Site

15. 15 Genes of HIV Structural and Regulatory - Spliced non-structural

16. CCR5 on macrophages or CXCR4 in T cells 1. gp120:CD4 binding 2. Conformational change of gp120 3. CCR5 Binding 4. gp41:membrane insertion 5. membrane-env fusion gp120 cytoplasm HIV virus CD4 gp41 CCR-5 16 HIV binding and entry into CD4 cell

17. 17 Replication of HIV Video

19. Live Cycle of HIV-1 Regulatory proteins Circularizes, Pre-intergration complex TAT, REV, NEF 19

20. Years CD4+ cell count 2 4 1012 8 1000 800 700 100 200 500 900 300 Death 400 600 AIDS PCP CMV MAC acute mono-like illness lymphadenopathy acne folliculitis tuberculosis shingles oral hairy leukoplakia thrush tinea bacterial pneumonia gingivitis sinusitis HIV RNA 10 7 10 7 10 2 10 2 10 3 10 3 10 4 10 4 10 5 10 5 Copies/ml 20 “Typical” Course of Untreated HIV

21. 21 Viral replication

22. 22 Zidovudine - AZT

23. RT with DNA Strands A closer view of the polymerase active site in the HIV-1 RT/DNA structure. The sites for the commonly occurring resistance mutations for NRTI drugs are indicated. Nucleoside analogue inserts here causing DNA chain termination 23

24. Mirror image dimer 24 HIV Protease with Inhibitor in Active Site

26. CCR5 on macrophages or CXCR4 in T cells 1. gp120:CD4 binding 2. Conformational change of gp120 3. CCR5 Binding 4. gp41:membrane insertion 5. membrane-env fusion gp120 cytoplasm HIV virus CD4 gp41 CCR-5 26 HIV binding and entry into CD4 cell

27. Viral Heterogeneity and why resistant mutations develop  Genome is ~ 10,000 bases of RNA  HIV reverse transcriptase has a high error rate of ~ 1 base mismatch in every 3-5 x 10 4 bases  So for each replicative cycle viruses differing by 2 bases from the input virus are produced and leave the cell.  With its rapid replication cycle, ~ 1-10 billion new virions/day are produced  In an untreated infected patient, a virus with every possible mutation is produced at least once every 24 hours.  Some mutations allow the virus to escape immune response e.g. neutralizing antibodies  Mutations at enzymatic site may impart drug resistance

28. Clavel, F. et al. N Engl J Med 2004;350:1023-1035 Panel A HIV-1 Protease Dimer Binding a Protease Inhibitor Panel B Drug-Sensitive (Wild-Type) Protease Juxtaposed against a Drug-Resistant Protease protease inhibitor 28

29. 6543210 1 2 3 4 5 6 Single Nucleoside Double Nucleoside 2 NRTI+PI or NNRTI Months of Treatment Log RNA copies/mL of blood Baseline Lower limit of measurement ZDV max fall 0.7 log ZDV+3TC max fall 1.8 log ZDV+3TC+PI max fall >3 log 10 Effect of Treatment on HIV Viral Load

31. Summary  Simple retroviruses have just 4 genes – 2 structural and 2 enzymes – and complex Retroviridae also have regulatory gene(s).  Envelope, capsid and RNA are major viral structures.  The virus binds to cell, fuses viral and cell membranes, transcribes RNA to ds DNA (reverse transcription), enters nucleus and integrates into cellular DNA. Sometime later cell produces viral mRNA, and genomic RNA and produces viral proteins, assembles and buds off cell.  There are binding inhibitors, membrane fusion blockers, nucleoside and non-nucleoside reverse transcriptase inhibitors, integration inhibitors, and protease blockers.  HIV RNA develops a mutation nearly every time it goes through RT. These mutations can be at site of action of the antivirals making offspring virus resistant to that drug. To prevent the virus resistant to one drug from overgrowing in the presence of the drug, multiple anti-HIV agents are used in combination. 31

32. Questions?? 32 Dr Michael Para Michael.para@osumc.edu