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:  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.,  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

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

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 Pre-activation latency

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

 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. Aims

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

Methods Cont’d

SampleUnique Integration site IL2/PHA 432 CCL Unactivated (media alone) 133 Identification of unique integration sites:

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

Chromosomal Feature UnactivatedCCL19IL2/PHAPatients cellsSignificant H2AZ < DNAse hypersensitivity sites < Alu < CCL19 treated cells have significantly different integration sites

Integration in CCL19 treated cells is closer to Long Interspersed Nuclear Elements Unactivated CCL19IL2/PHAPatients

Chromosomal Feature UnactivatedCCL19IL2/PHAPatients cellsSignificant CTCF ++ ns CPG ++ + ns Pol II ++ + ns Comparison of HIV integration site distributions ns: not significant

H4R3me2 Intermediate transcriptional activity Histone methylation sites H4K20me3 transcriptional repression IL2/PHAPatientsCCL19 Unactivated PatientsUnactivatedCCL19IL2/PHA

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

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).

Conclusions and implications 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.

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.

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

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