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.,  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.,  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.

chemokines In vitro Unactivated resting cells Resting 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: ; 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? Wortmannin LY294002

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

Inhibition of PI3K has little effect on nuclear localisation (2LTR) SC-514 Bay SP SB PD JNKERK NF  B P38 ERK JNK NF  B

Inhibition of Erk1/2, Jnk and NF-kB eliminates integration (ALu-LTR) SC-514 Bay SP SB PD CCL19+DMSO JNKERK NF  B 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 promoter Single round Env pseudotyped viruses env- (D. Purcell lab) (M. Churchill lab) JNKERK NF  B 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 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