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The role of PI3K signaling pathways in HIV-1 infection of resting CD4+T-cells

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:.

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The role of PI3K signaling pathways in HIV-1 infection of resting CD4+T-cells

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  1. 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

  2. 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.

  3. Infection of resting CD4+ T-cells Resting CD4+ T-cell Unactivated resting cells Ex vivo tissue blocks In vitro chemokines 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

  4. CCR7 + CCL19 CCL19 ligation activates cofilin and actin polymerisation CXCR4 + gp120 Yoder et al Cell 2008 Cameron et al PNAS 2010

  5. Chemokine signalling pathways: PI3K PI3K

  6. Chemokine signalling pathways: PLC & JAK/STAT JAK/STAT PLC

  7. What roles do these signaling pathways play in HIV integration in resting T-cells?

  8. 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.

  9. What is the role of the PI3K pathway? Wortmannin LY294002

  10. PI3K Inhibition of CCL19-induced Akt phosphorylation Total Akt P-Akt Merge - + - + - + - - - + + - - - - - - - + + - - - - - - - + CCL19 (100nM) LY294,002 (50μM) Wortmannin (100nM) PMA (200nM)

  11. PI3K pathway is critical for integration

  12. Inhibition of PI3K has little effect on nuclear localisation (2LTR) P38 NFB ERK JNK NFB ERK P38 JNK NFB SP600125 SC-514 Bay 11-7082 PD980509 SB203580

  13. Inhibition of Erk1/2, Jnk and NF-kB eliminates integration (ALu-LTR) P38 NFB ERK JNK NFB SP600125 SC-514 Bay 11-7082 CCL19+DMSO PD980509 SB203580

  14. Infection with single round virus gave similar results Env expression vector pSVIII-HXB2 env pNL4-3 env- Env deficient HIV-1 NFB ERK JNK NFB P38 env- (M. Churchill lab) (D. Purcell lab) LTR promoter Single round Env pseudotyped viruses Co-transfected into 293T cells

  15. Blocking NFAT pathway has no effect on HIV nuclear entry PLC Tacrolimus Cyclosporin

  16. Blocking the NFAT pathway had no effect on integration Tacrolimus Cyclosporin

  17. 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.

  18. 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

  19. 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.

  20. 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.

  21. Department of Medicine, Monash University Sharon Lewin Paul Cameron Georgina Sallmann Burnet Institute Anthony Jaworowski Melissa Churchill Lachlan Gray Acknowledgements

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