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MOLECULAR MECHANISMS OF HIV POSTINTEGRATION LATENCY: STRATEGIES FOR ERADICATION

MOLECULAR MECHANISMS OF HIV POSTINTEGRATION LATENCY: STRATEGIES FOR ERADICATION. How is HIV transcribed? What is latency/reservoir? When is it established? How best to eradicate it Mechanistic details. ACTIVE REPLICATION. LATENCY. ELONGATION. Nature, 1987.

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MOLECULAR MECHANISMS OF HIV POSTINTEGRATION LATENCY: STRATEGIES FOR ERADICATION

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  1. MOLECULAR MECHANISMS OF HIV POSTINTEGRATION LATENCY:STRATEGIES FOR ERADICATION • How is HIV transcribed? • What is latency/reservoir? • When is it established? • How best to eradicate it • Mechanistic details

  2. ACTIVE REPLICATION LATENCY

  3. ELONGATION Nature, 1987

  4. SHORT AND LONG TRANSCRIPT FROM HIV LTR

  5. TRANSCRIPTIONAL ELONGATION CONTROL IN PROKARYOTES AND EUKARYOTES LAMBDA N:NutB SITE vs HIV TAT:TAR RNA NELF N-TEF: NELF and DSFI NELF: NELF-A to E (RD) DSIF = Spt4 AND Spt5 P-TEFb = CycT1 and Cdk9

  6. NEW BIOLOGY Important role for control of elongation of transcription in eukaryotic biology: 80% untranscribed genes have paused RNA polymerase II on their promoters Introduced positive and negative elongation factors to transcription (P-TEFb, N-TEF): revealed that they play the key role in HIV replication: epigenetic changes result from the interplay between negative and positive elongation factors on RNA polymerase II on promoters Introduced the control of co-transcriptional processing of RNA, i.e. capping, splicing and polyadenylation: extent of RNA polymerase II phosphorylation dictates alternative splicing, which includes the processing of HIV genomic RNA Provided the first glimpse of HIV proviral latency in the host: stalled RNA polymerase II on inactive proviruses

  7. PCR AMPLIFICATION OF SHORT AND LONG TRANSCRIPTS

  8. Adams et al., PNAS, 1994, 10 individuals, not on HAART, at seroconversion had only short transcripts in the periphery-proviral latency (replication elsewhere)

  9. HIV Latency • HIV can hide in very long lived memory CD4 T cells-insensitive to antiviral drugs which require viral replication • While these cells are rare (105-106/pt), clearing them from the body is predicted to require >60 years of treatment • New approaches are urgently needed to purge or inactivate these latent proviruses, otherwise a “cure” for HIV infection will remain out of reach

  10. HDAC1 P50 P50 P50 P50 S2 S5 S2 S5 S2 RNAPII RNAPII RNAPII S5 Nuc-(+1) Nuc-(+1) CHROMATIN EFFECTS A HDAC1 HIV LTR B Valproic acid HIV LTR

  11. RelA RelA IkB P50 P50 HATs S2 S5 S2 RNAPII RNAPII RNAPII RNAPII S5 LACK OF NF-B A HIV LTR Nuc-(+1) B Prostratin, TNFa S2 S2 S5 S5 HIV LTR Nuc-(+1)

  12. Experimental plan: RT-SHIV and HAART and PRS + VPA Induction RT-SHIV Inoculation (6) Necropsy (2) 6 wks 32 - 35 wks 8 wks 1 wk Rebound (4) HAART (FTC + PMPA + Efavirenz) HAART + Induction HAART 2 wks: 5 cycles 6 wks: 1 cycle per wk Weekly/biweekly analysis: plasma viral RNA, CBC, FACS

  13. Tat P-TEFb S2 S2 S5 S5 S2 RNAPII RNAPII RNAPII RNAPII S5 LACK OF TAT/P-TEFb A HIV LTR B S2 S5 HIV LTR

  14. S2 S2 S2 S5 S5 S5 S2 S2 S5 S5 S2 S2 RNAPII RNAPII RNAPII RNAPII RNAPII RNAPII RNAPII RNAPII RNAPII S5 S5 TRANSCRIPTIONAL INTERFERENCE A polyA signal Host gene promoter HIV 5’ LTR HIV 3’ LTR S2 S5 pA B Activation of HIV 5’LTR (NF-kB) with Prostratin, TNFa polyA signal Host gene promoter HIV 5’ LTR HIV 3’ LTR S2 S5 pA

  15. TRANSCRIPTION FROM HIV LTR DEPENDS ON P-TEFb Cdk9 CycT1 HEXIM1 7SK RNA CycT1 STRESS, UV LIGHT DRB, ACTINOMYCIN D, CELL SIGNALING HMBA, SAHA, ETC. activation Cdk9 Cdk9 CycT1 direct exchange binding Cdk9 Cdk9 CycT1 BRD4 Tat RelA TAR RNA NF-B p50

  16. HOW TO ACTIVATE HIV TRANSCRIPTION FROM LATENCY? 7SK snRNA MePCE LARP7 CycT Cdk9 HEXIM1/2 Active P-TEFb Inactive P-TEFb PARADOX: HMBA INDUCES HEXIM1/2 BUT IS ALSO ONE OF THE MOST POTENT ACTIVATORS OF HIV TRANSCRIPTION, EVEN FROM LATENTLY INFECTED CELL LINES. HMBA IS ALSO ONE OF THE MOST POTENT CELLULAR DIFFERENTIATION AGENTS

  17. HMBA releases P-TEFb from the LC HMBA LC SC Active P-TEFb - 30’ 1h 2h 6h 24h HMBA

  18. Effects of HMBA on P-TEFb and HEXIM1/2 100% levels of HEXIM1/2 inactive P-TEFb 50% 0 3 6 12 24 hrs

  19. HMBA cytoplasm PIP3 PDK1/2 PIP2 PI3K AKT P nucleus HEXIM1 HEXIM1 7SK snRNA 7SK snRNA CycT1 CDK9 Inactive P-TEFb P P CycT1 Active P-TEFb CDK9 P P 2 5 RNAPIIo RNAPIIo HIV LTR transcriptional elongation

  20. SAHA activates the Akt pathway P-TEFb Liu et al. JBC 2006

  21. HMBA/SAHA reactivate HIV in PBMCs Infection in vitro PBMCs from HAART treated patients PBMCs from healthy donors+PHA/IL2 PBMCs + SAHA/HMBA 2/3 days 3 days Wash+Add PBMCs treated PHA/IL2 From healthy donors Infection in vitro HIV-LAI 11 days 15 to 21 days Treatment with SAHA/HMBA P24 ELISA

  22. SAHA

  23. SAHA (+/- NF-B ACTIVATION) • Rhesus macaque trial with SAHA (+/- Prostratin) • (NF-B activation will counteract chromatin effects and transcriptional interference and recruit more RNA polymerase II to the HIV LTR) • SAHA will release and activate P-TEFb transiently from the large, inactive complex • This manipulation will lead to the synthesis of Tat, which can utilize P-TEFb from the inactive complex to sustain HIV replication • Increased levels of HEXIM1/2 and inactive complex will inhibit other viruses (HSV, EBV, CMV, KSVH), attenuate immune activation and help prevent superinfection by HIV.

  24. Matjaz Barboric, Koen Bartholomeeusen, Dalibor Blazek, Xavier Contreras, Jiri Kohoutek, Audrey Low, Tina Lenasi, Fan Zhang COLLABORATION: PPG: Verdin, Greene, Peterlin Steve Deeks, Su Guo, Paul Luciw, Jeffrey Martin, Thomas North

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