HIV transcription, Tat transactivation mrna processing and latency

Transcription

1 HIV transcription, Tat transactivation mrna processing and latency Tat transactivation Roles of CTD kinases in HIV replication Latency and reservoir Strategies to eliminate the reservoir of HIV in the body

2 9 kb genome 46 different spliced transcripts (mrnas) Multiply spliced: Rev-independent Singly spliced and unspliced: Rev-dependent 16 different proteins Gag and Pol polyproteins: cut by viral protease Env polyprotein: cut by furin-like proteases

3 Nature, 1987

4 SHORT AND LONG TRANSCRIPTS FROM HIV LTR

5 PCR AMPLIFICATION OF SHORT AND LONG TRANSCRIPTS

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

7 Tat and TAR: the first important structure in HIV-1 (2/SIV/EIAV) 9

8 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

9 Levels of CycT1 and Cdk9 are vanishingly low in resting T cells, T effector memory and T central memory cells, but they increase following T cell activation and proliferation Small Complex (SC) kinase active CycT1 HMBA, SAHA stress, UV light HEXIM 1/2 CDK 9 CycT1 Large Complex (LC) kinase inactive (7SK snrnp) CDK 9 HEXIM 1/2 MePCE 7SK snrna LaRP7 LARP7 (tumor suppressor), MePCE methyltransferase

10 Small Complex (SC) kinase active CycT1 HMBA, SAHA stress, UV light HEXIM 1/2 CDK 9 CycT1 Large Complex (LC) kinase inactive (7SK snrnp) CDK 9 HEXIM 1/2 MePCE 7SK snrna LaRP7 Tat p65 CycT1 CDK 9 p65 CDK 9 CycT1 Brd4 CTD Tat TAR CycT1 CDK 9 HEXIM 1/2 CTD p50 NF-κB p50 Sp 1 Sp 1 Sp 1 TATA Ii RNAPII HIV LTR Start site

11 NEW BIOLOGY Important role for control of elongation of transcription in eukaryotic biology Introduced positive and negative elongation factors to transcription (P-TEFb, N-TEF): suggested that they play the key role in HIV replication Introduced the control of co-transcriptional processing of RNA, i.e. capping, splicing and polyadenylation Represent the first glimpse of HIV proviral latency in the host

12 cdk9 Cdk 9 and cyclins T1, T2 and K 1 1 cyclin box NLS His-rich PEST cyct1 726 Specialized: binds to CTD of RNAPII, mediates effects of transcriptional enhancers (AIRE, AR, CIITA, NF-κB, cmyc, βcatenin, VP16) 372 Specialized: binds to cyct1 (85%), cyct2 (10%), cyck (5%), phosphorylates serine and threonine residues on Rb, CTD of RNAPII, SP5 and other targets. cyct2 731 Specialized: binds to CTD of RNAPII, other general transcription factors, mediates effects of promoter and enhancer elements (MyoD) cyck 357 Specialized: does not bind to CTD or RNAPII, might function with cellular Tat analogues via RNA?

13 C-TERMINAL DOMAIN (CTD) OF RNA POLYMERASE II 52 HEPTAD REPEATS YSPTSPS Cdk 7: S5, S7 phosphorylation Cdk 9: S2 phosphorylation Extent of CTD phosphorylation regulates co-transcriptional processing: capping, (alternative) splicing, polyadenylation

14 FROM UNPHOSPHORYLATED TO FULLY PHOSPHORYLATED CTD Direct binders contain WW, FF, SH2 and CID domains When inhibiting S2P (P- TEFb) first to go is polyadenylation, next splicing finally elongation, i.e. sterile transcripts are produced Actually, Y1, T4 and S7 are also phosphorylated S7 by Cdk7 (required for snrna transcription) Integrator requires S7P

15 9 kb genome 46 different spliced transcripts (mrnas) Begin transcription Maintain CTD phosphorylation activate ASFs Other kinases for polyadenylation

16 CTD kinases and HIV replication 1. CycH:Cdk7 (TFIIH) is essential for promoter clearance of capping of HIV transcripts. 2. CycT1:Cdk9 (P-TEFb) is essential for elongation of HIV transcription and co-transcriptional processing of viral transcripts. 3. CycK:Cdk12/13 play important roles in elongation and processing of HIV transcripts 4. CycL:Cdk11 plays an important role in HIV polyadenylation and mrna stability.

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

18 MAIN PROBLEM NF-κB CTD kinases

19 HOW TO ACTIVATE HIV TRANSCRIPTION FROM LATENCY? 7SK snrna MePCE LARP7 (LARP7 = La related protein 7 TUMOR SUPPRESSOR, mutated in solid tumors such as breast, gastric CA, etc.) CycT Cdk9 HEXIM1/2 Active P-TEFb Inactive P-TEFb (MePCE, methyl phosphate capping enzyme, previously known as BCDIN3 = New class of methyl transferases, caps 7SK snrna and when activated,protein targets 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.

20 HMBA releases P-TEFb from the LC SC HMBA LC Active P-TEFb LC/SC Cdk9 HEXIM1 HEXIM2 HMBA h 2h 6h 24h

21 Effects of HMBA on P-TEFb and HEXIM1/2 100% 50% levels of HEXIM1/2 inactive P-TEFb hrs

22 Small Complex (SC) kinase active CycT1 HMBA, SAHA stress, UV light HEXIM 1/2 CDK 9 CycT1 Large Complex (LC) kinase inactive (7SK snrnp) CDK 9 HEXIM 1/2 MePCE 7SK snrna LaRP7 Tat p65 CycT1 CDK 9 p65 CDK 9 CycT1 Brd4 CTD Tat TAR CycT1 CDK 9 HEXIM 1/2 CTD p50 NF-κB p50 Sp 1 Sp 1 Sp 1 TATA Ii RNAPII HIV LTR Start site

23 SAHA

24

25 SAHA AND HMBA INHIBIT CYTOPLASMIC CLASS II HDAC6

26 Geldanamycin inhibits SAHA and ST-80 activation of HIV LTR Luciferase ac4vity SAHA ST Geldanamycin (nm)

27 SAHA, Tubacin, ST80 Geldanamycin HDAC6 AC HSP90 AKT PI3K AC AC TUBULIN HSP90 AKT LY AC TUBULIN AC TUBULIN AC HSP90 AKT Kinases AC TUBULIN AC HSP90 AKT AC P tases TUBULIN AI8 P-TEFb HEXIM1/2

28 Synergy between TNFα and HDACis on TI SAHA!"#$% #&'&%(%)"% #&'&%*%)"% TNFα + SAHA

29 ST-80 TNFα + ST80

30 MS-275!"#$% "#&'()%*+)%,"% "#&'()%'+)%,"% TNFα + MS-275

31 HMBA, SAHA, HDAC INHIBITORS Release and activate P-TEFb transiently from the large, inactive complex Modify HEXIM1/2 or CycT1 so they no longer interact Whereas transient activation of PI3K/Akt leads to cell differentiation, sustained activation of PI3K/ Akt to proliferation and cancer Active P-TEFb is a potent inducer of HEXIM1/2, which restores the large, inactive complex These cells become rather refractory to de novo infection by HIV This situation appears analogous to the feedback loop of NF-κB to IκB, where NF-κB potently

32 POSSIBLE COMBINATION THERAPy (HALT) AGAINST HIV RESERVOIR me c me c me c 1 5- azacy4dine HDACi SAHA 2 me c me c Ac Ac Ac HIV LTR Ac Ac Ac Bryosta+n Prostra4n TNF- α p65 CycT1 CDK 9 p65 CDK 9 HMBA, SAHA CycT1 Brd4 CTD Tat TAR CycT1 CDK 9 CTD Tat p50 p50 NF- κb Sp 1 Sp 1 Sp 1 TATA Ii RNAPII HIV LTR Start site

33 Highly Active Anti-Latency Therapy (HALT) No good cell culture model exists, possibly should use GALT, where the major reservoir is. No good small animal model exists, especially with problems on NOD background. Best is SIV-infected rhesus macaques. For transcriptional interference and to increase levels of P- TEFb, some activator of NF-kB will have to be used: BRYOSTATIN, prostratin or TNFalpha. For factor insufficiency, will have to increase levels of active P-TEFb via an HDAC6-specific or pan-hdaci like SAHA. In case of SAHA dose should be diminished and frequency increased.

34 Matjaz Barboric, Dalibor Blazek, Koen Bartholomeeusen, Xavier Contreras, Jiri Kohoutek, Tina Lenasi LABORATORIES: U. of California, San Francisco, Helsinki, Ljubljana, Rio de Janeiro Szechuan