By: Suha Saleh Supervisor: Prof. Sharon Lewin Monash University,Melbourne, Australia

1. Latent HIV infection can be established in resting CD4+ T-cells in vitro following incubation with multiple diverse chemokines By: Suha Saleh Supervisor: Prof. Sharon Lewin Monash University,Melbourne, Australia

2. INTRODUCTION • HIV latency represents a major barrier to HIV eradication. • HAART successfully suppresses HIV replication but cannot eradicate HIV. • The major stable source of this latent reservoir is resting memory CD4+ T-cells which decays very slowly. - Chun et al., Nat. Med., 1995; Chun et al., Nature, 1997; Finzi et al., Science, 1997. • Infected naïve CD4+ T-cells also represent a long lived reservoir. - Brenchley et al., J Virol., 2004.

3. Pre-integration Resting CD4+ T cell Blood OR Post-integration Lymphoid organs Kreisberg et al., J Exp Med 2006; 203:865; Eckstein et al, Immunity 2001; 15: 671; Audige et al, J Immunol 2004; 172: 2687 Infection of activated and resting T cells Activated CD4+ T cell

4. High frequency of integrated HIV-1 in resting cells incubated with CCR7 ligand (CCL19) Saleh et al., Blood 2007; 110:4161

5. NL4-3 infection [n=6] Unstimulated IL2/PHA CCL19 mock AD8 infection [n=3] Low level productive infection with CCL19 consistent with latent infection 1000 RT CPM/ul 100 1000 RT CPM/ul 100 0 2 3 6 4 5 7 1 Days after infection Saleh et al., Blood 2007; 110:4161

6. Chemokine receptors and T-cells

7. HYPOTHESIS: CCR7 ligation by CCL19 activates a pathway common to multiple chemokine receptors leading to the establishment of latent HIV-1 infection of resting CD4+ T-cells. AIMS: • To determine whether other chemokines that bind receptors expressed on resting CD4+ T-cells can mediate HIV-1 latency. • To determine the effect of CCL19 treatment on pre-integration steps in the virus life cycle.

8. Gene expression of chemokine receptors on resting CD4 T cells with and without CCL19 Tested ligands CCL20 CXCL9, CXCL10 CCL13 CCL1

9. Methods:

10. Multiple CXC and CC chemokines facilitate HIV proviral DNA integration in resting T cells A B

11. No change in the expression of chemokine receptors or activation markersfollowing incubation with different chemokines

12. R U5 gag pol env U3 R PBS Aim 2:Characterise steps pre viral integration in CCL19 treated resting CD4+ T-cells Primer pairs for DNA Viral DNAform R/U5 R/gag 2-LTR Alu-HIV Cytoplasm M667 AA55 + - - - + + - - + + + - + + - + (HIV-1 genomic RNA) Strong-stop M667 M661 Full-length U3 R U5 gag pol env U3 R U5 Nucleus 2-LTR circles Integrated Zack et al., Cell 1990; 61:213, Butler et al., Nat.Med. 2001; 7:631., O’Doherty et al., J Virol. 2002;76:10942

13. Enhanced nuclear localisation in CCL19-treated cells Primer pairs for DNA Viral DNAform R/U5 R/gag 2-LTR Alu-HIV + - - - Strong-stop + + - - Full-length + + + - 2-LTR circles + + - + Integrated

14. Delayed appearance of 2-LTR circles and low level integration in unactivated cells

15. NFkB Chemokine receptor signalling: shared pathways CCR7, CXCR3, CCR6 Survival Chemotaxis Migration NF-kB JNK Cofilin Sanchez-Sanchnez et al ., J. Immunol. 2006; 176:5153 HIV envelope-CXCR4 signalling activates cofilin to overcome cortical action restriction in resting CD4 T cells Yoder et al, Cell 2008; 134: 782

16. Chemokines, cofilin activation and infection of resting T-cells • HIV-induced signaling from CXCR4 activates phosphatase which dephosphorylates and activates cofilin. • This leads to depolymerization of F-actin, releasing HIV-1 reverse transcription complexes and promotes its translocation towards the nucleus. Yoder et al, Cell 2008; 134: 782

17. 10nM PMA 100nM CCL19 pCofilin/ Cofilin Ratio Cofilin pCofilin/ Cofilin Ratio 1 0.5 merge 0 0 2 5 10 15 30 0 2 5 10 15 30 0 10 20 30 0 10 20 Time minutes Time minutes Chemokine induced changes cofilin pCofilin 30 CCL19 CCL19 CCL19 CCL19 100nM CCL19 100nM CCL19 100nM 0 min 0 min 0 min SDF-1 100nM SDF-1 100nM SDF-1 100nM 5 min 5 min 5 min Control Control Control 15 min 15 min 15 min 200 200 200 30 min 30 min 30 min LAT-A LAT-A LAT-A 150 150 150 SDF-1 SDF-1 SDF-1 100 100 100 0 min 0 min 0 min 5 min 5 min 5 min 30 min 30 min 30 min 50 50 50 LAT-A 5 min LAT-A 5 min LAT-A 5 min 0 0 0 20 20 20 30 30 30 1 1 1 10 10 10 2 2 2 3 3 3 4 4 4 0 0 0 Log FITC Phylloidin Log FITC Phylloidin Log FITC Phylloidin

18. MFI phylloidin fluorescence CCL19 100nM SDF-1 100nM LAT-A Cytokine induced changes in F-actin CCL19 0 min 5 min 15 min 200 30 min 150 SDF-1 100 MFI phalloidin fluorescence 0 min 5 min 30 min 50 LAT-A 5 min 0 20 30 1 10 2 3 4 0 Log FITC Phalloidin Time min

19. Summary: • Stimulation of resting memory CD4+ T cells with multiple chemokines leads to a high level of HIV-1 integration and low level of productive HIV-1 infection in the absence of cell activation. • Enhanced integration in chemokine treated resting cells was secondary to increased nuclear localisation and efficient integration. • CCL19 leads to significant activation of cofilin and modest changes in actin polymerisation that may facilitate HIV entry into the nucleus of resting CD4 T-cells. • A novel in vitro model of latent HIV infection.

20. Acknowledgements • Department of Medicine, Monash University • Prof. Sharon Lewin • Dr. Paul Cameron • Georgina Sallman • Ajantha Solomon • Fiona Wightman • Vanessa Evans • Burnet Institute • Johnson Mak • Kate Jones • Jenny Anderson • Anthony Jaworwski • Westmead Millenium Research Institute • Tony Cunningham • Andrew Harman American Foundation for AIDS Research National Health and Medical Research Council of Australia