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
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