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.

Infection of resting CD4+ T-cells Resting CD4+ T-cell Unactivated resting cells Ex vivo tissue blocks chemokines Eckstein et al,

Immunity

2001; 15: 671; Kreisberg et al.,

J Exp Med

110:416; Marini et al.,

J Immunol

2006; 203:865; Saleh et al., 2008; 181: 7713-20; Bosque and Planelle,

Blood Blood

2009; 113:58; 2007; Cameron et al.,

Proc Natl Acad Sci

2010 epub Sept 18

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

JAK/STAT PLC

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?

Wortmannin LY294002

PI3K Inhibition of CCL19-induced Akt phosphorylation Total Akt

P-Akt Merge CCL19 (100nM) LY294,002 (50 μM) Wortmannin (100nM) PMA (200nM ) + + + + + + + +

PI3K pathway is critical for integration

Alu-LTR Un ac tiva te d IL2 /PH A CC L1 9 CC L1 9+L Y-29 CC 400 L1 2 9+Wo rt man an 2-LTR 1000000 100000 10000 1000 100 Un act ivated IL2/ PH A CC L19 CC L19 +L Y-29400 CC L19 2 +Wo rtm ana n

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

ERK P38 JNK NF



B

SC-514 Bay 11-7082 SP600125 PD980509 SB203580

Inhibition of Erk1/2, Jnk and NF-kB eliminates integration (ALu-LTR) SC-514 Bay 11-7082 SP600125 PD980509 SB203580

Infection with single round virus gave similar results

pNL4-3 env-

Env deficient HIV-1

Env expression vector

pSVIII-HXB2

env env-

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

Single round Env pseudotyped viruses Co-transfected into 293T cells

Blocking NFAT pathway has no effect on HIV nuclear entry

PLC

Tacrolimus Cyclosporin

Blocking the NFAT pathway had no effect on integration Tacrolimus Cyclosporin

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 NF  B 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