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

Nature, 1987

ELONGATION

SHORT AND LONG TRANSCRIPT FROM HIV LTR

TRANSCRIPTIONAL ELONGATION CONTROL IN PROKARYOTES AND EUKARYOTES

LAMBDA N:NutB SITE vs HIV TAT:TAR RNA

N-TEF: NELF and DSFINELF: NELF-A to E (RD)DSIF = Spt4 AND Spt5P-TEFb = CycT1 and Cdk9

NELF

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

PCR AMPLIFICATION OF SHORT AND LONG TRANSCRIPTS

Adams et al., PNAS, 1994, 10 individuals, not on HAART, at seroconversion had only short

transcripts in the periphery-proviral latency (replication elsewhere)

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

HIV LTR

RNAPII

S5S2

Valproic acid

HIV LTR

RNAPII

S5S2

HDAC1

RNAPII

S5S2

A

B

P50 P50

P50 P50

HDAC1

Nuc-(+1)

Nuc-(+1)

CHROMATIN EFFECTS

HIV LTR

P50 RelAIB

Prostratin, TNF

HIV LTR

RNAPII

S5S2

RNAPII

S5S2

P50 RelA

A

B

Nuc-(+1)

Nuc-(+1)

RNAPII

S5S2

RNAPII

S5S2

HATs

LACK OF NF-B

Experimental plan: RT-SHIV and HAART and PRS + VPA Induction

6 wks 32 - 35 wks 8 wks 1 wk

RT-SHIVInoculation (6)

HAART(FTC + PMPA + Efavirenz)

HAART + Induction

Necropsy (2)

Rebound (4)

HAART

2 wks: 5 cycles 6 wks: 1 cycle per wk

Weekly/biweekly analysis: plasma viral RNA, CBC, FACS

TOM05 Rebounds Normalized to Cessation of Therapy

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18

Weeks post No Treatment

log RT-SHIV copies/ml Plasma

36349(05) Ind36353(05) Ind36160(05) Ind36488(05) Ind36544(05) No IndCutoff

HIV LTR

HIV LTR

RNAPII

S5S2

RNAPII

S5S2

P-TEFb

Tat

A

B

RNAPII

S5S2

RNAPII

S5S2

LACK OF TAT/P-TEFb

HIV 5’ LTR HIV 3’ LTRHost gene promoter

RNAPII

S5S2

RNAPII

S5S2 polyA

signal

pA RNAPII

S5S2

RNAPII

S5S2

RNAPII

S5S2

HIV 5’ LTR HIV 3’ LTRHost gene promoter

RNAPII

S5S2 polyA

signal

pA RNAPII

S5S2

RNAPII

S5S2

Activation of HIV 5’LTR (NF-B) with

Prostratin, TNF

A

B

RNAPII

S5S2

TRANSCRIPTIONAL INTERFERENCE

Tat Cdk9

CycT1

CycT1

Cdk9

Cdk9

RelAp50

CycT1

Cdk9

CycT1

Cdk9

activation

bindingdirect exchange

HEXIM1

7SK RNA

TAR RNA

BRD4

NF-B

TRANSCRIPTION FROM HIV LTR DEPENDS ON P-TEFb

STRESS, UV LIGHTDRB, ACTINOMYCIN D,

CELL SIGNALINGHMBA, SAHA, ETC.

PARADOX: HMBA INDUCES HEXIM1/2 BUT IS ALSOONE OF THE MOST POTENT ACTIVATORS OF HIVTRANSCRIPTION, EVEN FROM LATENTLY INFECTEDCELL LINES. HMBA IS ALSO ONE OF THE MOST POTENT CELLULARDIFFERENTIATION AGENTS

Cdk9

CycT

Active P-TEFb

HEXIM1/2

7SK snRNA

Inactive P-TEFb

LARP7MePCE

HOW TO ACTIVATE HIV TRANSCRIPTION FROM LATENCY?

HMBA releases P-TEFb from the LC

HMBA

LCSC

1h 2h-HMBA 30’ 6h 24h

Act

ive

P-T

EF

b

0 3 6 12 24 hrs

100%

50%inactive P-TEFblevels of HEXIM1/2

Effects of HMBA on P-TEFb and HEXIM1/2

HMBA

PI3K

PIP2 PDK1/2

P

cytoplasm

nucleus

HIV LTR

CycT1CycT1

7SK snRNA

P Inactive P-TEFb

Active P-TEFb

PIP3

AKT

HEXIM1HEXIM1P

CDK9

CDK9

transcriptional elongation

7SK snRNA

P

RNAPIIo

5

RNAPIIo

2 P

SAHA activates the Akt pathway

Liu et al. JBC 2006

P-TEFb

HMBA/SAHA reactivate HIV in PBMCs

Infection in vitro PBMCs from HAART treated patients

PBMCs from healthy donors+PHA/IL2

Infection in vitro HIV-LAI

Treatment with SAHA/HMBA

3 days

11 days

PBMCs + SAHA/HMBA

2/3 daysWash+Add PBMCs treated PHA/IL2

From healthy donors

P24 ELISA

15 to 21 days

Negative Control

SAHA AI8 + SAHA

PHA +IL-2

- + - +

SAHA

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.

Matjaz Barboric, Koen Bartholomeeusen, Dalibor Blazek, Xavier Contreras, Jiri Kohoutek, Audrey Low, Tina Lenasi, Fan Zhang

COLLABORATION: PPG: Verdin, Greene, PeterlinSteve Deeks, Su Guo, Paul Luciw, Jeffrey Martin, Thomas North