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

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.

Infection of activated and resting T cells

Activated CD4+ T cell

Resting CD4+ T cell

Blood

Pre-integration

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

High frequency of integrated HIV-1 in resting cells incubated with CCR7

ligand (CCL19)

Saleh et al., Blood 2007; 110:4161

Low level productive infection with CCL19 consistent with latent infection

100

1000

RT C

PM

/ul

NL4-3 infection [n=6]

100

1000

RT C

PM

/ul

UnstimulatedIL2/PHACCL19mock

AD8 infection [n=3]

0 1 2 3 4 5 6 7Days after infection

Saleh et al., Blood 2007; 110:4161

Chemokine receptors and T-cells

Chemokine receptor

Ligand T-cell expression

CCR5 RANTES Memory

CXCR4 SDF-1 Naive

CCR7 CCL19

CCL21

Memory and naive

CXCR3 CXCL10 (IP-10)

CXCL9 (MIG)

Memory and naive

CCR6 CCL20 (LARC) memory

CCR8 CCL1 memory

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:

1. To determine whether other chemokines that bind receptors expressed on resting CD4+ T-cells can mediate HIV-1 latency.

2. To determine the effect of CCL19 treatment on pre-integration steps in the virus life cycle.

Gene expression of chemokine receptors on resting CD4 T cells with and without CCL19

CCL1

Tested ligands CCL20

CXCL9, CXCL10

CCL13

Methods:

Multiple CXC and CC chemokines facilitate HIV proviral DNA integration in resting T cells

NL43 infection (n=1)

day 0day 4day 7

101

102

103

RT

acti

vit

y C

PM

/ul

NL43 infection (n=2)

A

B

102

103

104

105

106

AD8 infection (n=2)

Co

pie

s/m

illio

n c

ells

No change in the expression of chemokine receptors or activation markers following

incubation with different chemokines

Activation markers

0

1

2

3CD25CD69DR

A

% P

osi

tive

Cel

ls

Receptors expression

Uncultu

red

CXCL9

CXCL10

CCL20

Unactiv

ated

0

20

40

60

80

100CXCR4CCR5CXCR3CCR6

B

Aim 2:Characterise steps pre viral integration in CCL19 treated resting CD4+ T-cells

Nucleus

M667 AA55

Viral DNA

formCytoplasm

Primer pairs for DNAR/U5 R/gag 2-LTR Alu-HIV

(HIV-1 genomic RNA)

U3 R U5 gag pol env U3 R U5

M667 M661

R U5 gag

pol env U3 R

PBS

Strong-stop

Full-length

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

Viral DNA

form

Primer pairs for DNAR/U5 R/gag 2-LTR Alu-HIV

Strong-stop

Full-length

2-LTR circles

Integrated

Enhanced nuclear localisation in CCL19-treated cells

+ + - +

+ + + -

+ + - -

+ - - -

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

unactive

0 20 40102

103

104

105

106

107

copi

es/m

illion

cel

ls

96

CCL19

0 20 40

IL2/PHA

0 20 40 96 96

Alu-LTR

Srtong stop

Full length

2-LTRHours after infection

Chemokine receptor signalling: shared pathways

NFkB

Survival

ChemotaxisMigration

CCR7, CXCR3, CCR6

CofilinNF-B JNKSanchez-Sanchnez et al ., J. Immunol.

2006; 176:5153

Yoder et al, Cell 2008; 134: 782

HIV envelope-CXCR4 signalling activates cofilin to overcome cortical action restriction in resting CD4 T cells

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

Chemokine induced changes cofilin

30

pCofilin

10nM PMA

Time minutes

0

0.5

1

Cofilin

0 10 20

merge

0 10 15 302 5

pCofilin/Cofilin Ratio

0 10 20 30

100nM CCL19

Time minutes0 10 15 302 5

pCofilin/Cofilin Ratio

MF

I phylloidin fluorescence

1 2 3 4Log FITC Phalloidin

CCL19

0 min

Time min0 10 20 30

CCL19 100nM

SDF-1 100nM

LAT-A

0

50

100

150

2005 min

15 min30 min

SDF-1

0 min5 min

30 min

LAT-A 5 min

Cytokine induced changes in F-actin

MF

I phalloidin flu

orescence

• 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.

Summary:

Acknowledgements

American Foundation for AIDS ResearchNational Health and Medical Research Council of Australia

• 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