The role of PI3K signaling pathways in HIV-1 infection of resting

CD4+T-cells

Suha Saleh, Paul Cameron, Georgina Sallmann, Anthony Jaworowski, and Sharon Lewin

Monash University, Melbourne, Australia

Background:

Persistence of HIV infection in resting CD4+ T-cells remains the major barrier to HIV eradication. -Chun et al., Nat. Med., 1995; Chun et al., Nature, 1997; Finzi et al., Science, 1997; Brenchley et al., J Virol., 2004.

Infection of resting CD4+ T cells is difficult to establish in vitro due to multiple blocks in the viral life cycle.

- Zack et al., J. Virol ., 1992; Zack et al., Cell, 1990; Bukrinsky et al., PNAS., 1992.

Latent infection can be established in resting CD4+ T-cells following incubation with multiple chemokines including the CCR7 ligand, CCL19 .

- Saleh et al., Blood 2007; Cameron et al., PNAS 2010.

chemokinesIn v

itro

Unactivated resting cellsResting CD4+ T-cell

Ex vivo tissue blocks

Eckstein et al, Immunity 2001; 15: 671; Kreisberg et al., J Exp Med 2006; 203:865; Saleh et al., Blood 2007; 110:416; Marini et al., J Immunol 2008; 181: 7713-20; Bosque and Planelle, Blood 2009; 113:58; Cameron et al., Proc Natl Acad Sci 2010 epub Sept 18

Infection of resting CD4+ T-cells

CCL19 ligation activates cofilin and actin polymerisation

CXCR4 + gp120

CCR7 + CCL19

Yoder et al Cell 2008

Cameron et al PNAS 2010

Chemokine signalling pathways: PI3K

PI3K

Chemokine signalling pathways: PLC & JAK/STAT

PLC

JAK/STAT

What roles do these signaling pathways play in HIV integration in

resting T-cells?

Hypothesis and aims

Hypothesis: HIV latent infection can be established in resting CD4+ T-cells through activation of specific chemokine signaling pathways.

Aim:To identify the signaling pathways critical for HIV integration in resting CD4+ T-cells.

What is the role of the PI3K pathway?

WortmanninLY294002

CCL19 (100nM)LY294,002 (50μM)

Wortmannin (100nM)PMA (200nM)

Total Akt

P-Akt

Merge

PI3K Inhibition of CCL19-induced Akt phosphorylation

- + - + - + - - - + + - - - - - - - + + - - - - - - - +

PI3K pathway is critical for integration

Alu-LTR 2-LTR

100

1000

10000

100000

1000000

co

pie

s/m

illio

n c

ell e

qu

ivale

nts

Inhibition of PI3K has little effect on nuclear localisation (2LTR)

SC-514Bay 11-7082

SP600125

SB203580

PD980509

JNK

ER

K

NFB

NFB

P38

P38ERK

JNK

NFB

Inhibition of Erk1/2, Jnk and NF-kB eliminates integration (ALu-LTR)

SC-514Bay 11-7082

SP600125

SB203580

PD980509

C

CL1

9+D

MSO

JNK

ER

K

NFB

NFB

P38

Infection with single round virus gave similar results

pNL4-3 env-Env deficient HIV-1

Env expression vector

pSVIII-HXB2 env

Co-transfected into 293T cells

LTR promoterSingle round Env

pseudotyped viruses

env-

(D. Purcell lab) (M. Churchill lab)

JNK

ER

K

NFB

NFB

P38

Blocking NFAT pathway has no effect on HIV nuclear entry

Cyclosporin

Tacrolimus

PLC

Blocking the NFAT pathway had no effect on integration

Cyclosporin

Tacrolimus

Summary

The Rho A pathway is important for HIV-1 nuclear entry in resting CD4+ T-cells.

Chemokines activate the PI3K pathway and this was critical for integration in resting CD4+ T-cells.

The JNK/ERK and NFB were the most important down stream proteins.

There was no effect of the PLC pathway on integration in resting CD4+ T-cells.

Inhibition of Jnk and NF-kB eliminates integration

NFkB– Critical level required

for integration• Duverger J Virol 2009

– Transcription factors important for integration in active genes

• Felice, Plos One 2009

Jnk– Required for efficient

integrase cleavage via PIN 1

• Managanaro Nat Med 2010

Conclusions

PI3K signaling is critical for HIV integration in chemokine treated resting CD4+ T-cells.

The most downstream critical proteins included both JNK and NF-B.

Strategies that target these pathways may potentially lead to novel interventions to block the establishment of latent infection.

Future directions

To determine the role of the HIV LTR in facilitating integration using mutant viruses that lacks the common NF-kB binding sites in the LTR.

Identifying nuclear factors that are important for integration using a phospho-proteomic screen for kinase substrates activated by PI3K.

Selectively inhibit proteins that have been identified as important for HIV integration using siRNA.

Acknowledgements

Department of Medicine, Monash University–Sharon Lewin–Paul Cameron–Georgina Sallmann

Burnet Institute–Anthony

Jaworowski–Melissa Churchill–Lachlan Gray