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Unique integration patterns in an in vitro model of HIV-1 latency.

Unique integration patterns in an in vitro model of HIV-1 latency. . Suha M. Saleh, Dimitrios Vatakis, Andrew Harman, Anthony Cunningham, Paul U. Cameron , and Sharon R Lewin. Background:.

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Unique integration patterns in an in vitro model of HIV-1 latency.

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  1. Unique integration patterns in an in vitro model of HIV-1 latency. Suha M. Saleh, Dimitrios Vatakis,Andrew Harman, Anthony Cunningham, Paul U. Cameron, and Sharon R Lewin

  2. Background: • HIV-1 infection cannot be eradicated with highly active antiretroviral therapy (HAART) because of latent infection of long lived resting memory CD4+ T-cells. - Chun et al., Nat. Med., 1995; Chun et al., Nature, 1997; Finzi et al., Science, 1997; Brenchley et al., J Virol., 2004 • 2009 • Latency is maintained in resting T-cells by factors that largely restrict both transcription and translation. • - Jordan, et al., EMBO J., 2001; Krishnan and Zeichner., J. Virol., 2004; Bennasser et al., Immunity., 2005; Coiras et al., Retrovirology , 2007; Hay et al., Cell Host Microbe., 2008; Kauder et al., Plos Pathogens, 2009 • Integration of HIV-1 in resting CD4+ T cells from patients on HAART occurs within introns of actively transcribed genes. • - Han et al., J Virol. 2004; Shan et al., J Virol 2011

  3. HIV latency and infection of resting T-cells: pre and post activation Resting CD4+ T-cell Activated CD4+ T-cell Post-activation latency Pre-activation latency

  4. Unactivated resting cells Resting CD4+ T-cell Ex vivo tissue blocks Infection of resting CD4+ T-cells In vitro Pre-activation latency 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

  5. Hypothesis: Establishment of latency following direct infection of resting CD4+ T-cells (pre-activation latency) will be associated with a distinct pattern of integration.

  6. Aims • Aim 1: • To compare the sites of integration following infection of CCL19 treated, unactivated and fully activated CD4+ T-cells with latently infected cells from patients on cART. • Aim 2: • To determine the relationship of integration sites to transcription factor binding sites and cellular gene expression.

  7. Methods: (n=4) HIV-1 (+) on cART (Ikeda et al., JID 2007) (n=3) HIV-1 (-) Integration site Gene arrays

  8. Methods Cont’d

  9. Identification of unique integration sites:

  10. Integration in CCL19 treated cells is further from Transcriptional Start Sites (TSS) Patients IL2/PHA CCL19 Unactivated % site of integration Patients CCL19 Unactivated IL2/PHA Distance from TSS

  11. CCL19 treated cells have significantly different integration sites

  12. Integration in CCL19 treated cells is closer to Long Interspersed Nuclear Elements Unactivated CCL19 IL2/PHA Patients

  13. Comparison of HIV integration site distributions ns: not significant

  14. Histone methylation sites H4R3me2 Intermediate transcriptional activity H4K20me3 transcriptional repression Unactivated CCL19 IL2/PHA Patients Unactivated CCL19 IL2/PHA Patients

  15. No differences in expression of genes near integration sites in different in vitro conditions Gene expression (heat map) Unactivate IL2/PHA CCL19 CCL19 CCL19 The majority of the genes near integration sites in all in vitro conditions were involved in cellular housekeeping activities and cell signaling pathways.

  16. Summary: • HIV-1 integration occurred in transcriptionally active genes in all culture conditions. • Sites of integration of HIV-1 in latently infected CCL19 treated cells was different to other in vitro conditions and patients derived cells. • In CCL19 treated cells, integration was • Further from TSS • Closer to • A/T-rich LINE elements • H4K20me3 (heterochromatin marker) • H4K3me2 (involved in priming gene expression).

  17. Sites of integration of HIV-1 might be determined by activation state of the cell at the time of infection. Differing sites of integration may have implications for designing strategies to reverse latency. Conclusions and implications

  18. Future directions • Integration sites in cells with inducible and non-inducible expression of HIV-1 in CCL19 treated latently infected cells using an EGFP reporter virus. • Analysis of latently infected resting CD4 T-cells from blood and tissue in patients on cART.

  19. Acknowledgements • Department of Medicine, Monash University • Sharon Lewin • Paul Cameron • Department of Medicine, UCLA,California • Dimitrios Vatakis • Westmead Millenium Research Institute • Tony Cunningham • Andrew Harman

  20. Activation of virus production from latent infection NL4.3 Salehet al., Retrovirology 2011 n=4

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