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., 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

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 cellsResting 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: 7713-20; 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

Sample Unique Integration site

IL2/PHA 432

CCL19 247

Unactivated (media alone)

133

Identification of unique integration sites:

0

50000

100000

150000

200000

250000p<0.001 p<0.001

p<0.001

Base

pai

rsIntegration in CCL19 treated cells is further from Transcriptional Start Sites (TSS)

Unacti

vated

CCL19

IL2/P

HA

Patien

ts 0 50 1000

50

100

CCL19Unactivated

PatientsIL2/PHA

Distance from TSS

% s

ite o

f int

egra

tion

Chromosomal Feature

Unactivated CCL19 IL2/PHA Patients cells Significant

H2AZ ++ +++ ++ + <0.0001

DNAse hypersensitivity sites ++ ++++ ++ + <0.0001

Alu++ +++ ++ ++ <0.0001

CCL19 treated cells have significantly different integration sites

0

500

1000

1500

2000p<0.001 p<0.001

Base

pai

rs

Integration in CCL19 treated cells is closer to Long Interspersed Nuclear Elements

Unactivated CCL19 IL2/PHAPatients

Chromosomal Feature

Unactivated CCL19 IL2/PHA Patients cells Significant

CTCF ++ ++ ++ ++ ns

CPG ++ ++ ++ + ns

Pol II ++ ++ ++ + ns

Comparison of HIV integration site distributions

ns: not significant

H4R3me2Intermediate transcriptional activity

Histone methylation sites

H4K20me3transcriptional repression

IL2/PHA PatientsCCL19 UnactivatedPatientsUnactivated CCL19 IL2/PHA

CCL19CCL19

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.

Una

ctiv

ate

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=4Saleh et al., Retrovirology 2011