mathias lichterfeld, m.d., ph.d. massachusetts general hospital an intrinsic inhibitor of p-tefb and...
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Mathias Lichterfeld, M.D., Ph.D.Massachusetts General Hospital
An intrinsic inhibitor of p-TEFb and HIV-1 transcriptional elongation in CD4 T cells
from elite controllers
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Elite Controllers
• No detectable viremia in the absence of HAART • approx 1/300 HIV-1 infected patients• Model for spontaneous control of HIV-1 • Correlates of immune protection ?
– HIV-specific T cells– Intrinsic resistance of CD4 T cells?
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Reduced susceptibility of CD4 T cells from elite controllers to HIV-1 infection
HIV neg EC VC Progressors103
104
105
106
107
p24
(pg/
ml)
X4-tropic HIV-1
p<0.0001 p=0.0017
p<0.0001
p=0.015
p=0.0065
n=12 n=15 n=17 n=15
R5-tropic HIV-1
p<0.0001 p=0.15
p<0.0001
p=0.0005
p=0.0001
HIV neg EC VC Progressors102
103
104
105
106
107
p24
(pg/
ml)
n=21 n=21 n=17 n=11
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Cyclin dependent kinase inhibitor expressed in T cells and macrophages
Can serve as tumor suppressor
Contributes to resistance of macrophages and hematopoietic stem cells against HIV-1 (Bergamaschi et al, JVI 2009; Zhang et al, JCI 2007).
P21 (cip-1/waf-1)
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ProgressorsHIV neg EC VC0.00001
0.0001
0.001
0.01
0.1
1
p21
mR
NA
exp
ress
ion
p<0.0001
p<0.0001
p<0.0001
p<0.0001
HLA-DR- CD4+ cells
Progressors HIV neg EC VC0.00001
0.0001
0.001
0.01
0.1
1
p21
mR
NA
exp
ress
ion
p<0.0001
p<0.0001
p<0.0001
p<0.0001
HLA-DR+ CD4+ cells
Upregulation of p21 in CD4 cells from elite controllers
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si-RNA mediated silencing of p21 increases HIV-1 replication
R5-tropic HIV-1 X4-tropic HIV-1
ControlsiRNAcells
0 102
103
104
105
0
102
103
104
105
0 102
103
104
105
0
102
103
104
105
0 102
103
104
105
0
102
103
104
105
0 102
103
104
105
0
102
103
104
105C
D4
GFP
0.9%
2.3%
3.5%
10%
p21siRNAcells
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0
5
10
15
20
25
norm
aliz
ed
co
pie
s/ce
ll
Late RT transcripts
Integrated HIV-1 DNA
mRNA transcripts
EC HIV-1 negatives
*
n=9 n=9
0.00
0.05
0.10
0.15
0.20
0.25
norm
aliz
ed
co
pie
s/ce
ll
EC HIV-1 negativesn=6 n=10
**
0.00
0.02
0.04
0.06
0.08
0.10
rela
tive
HIV
-1 m
RN
A le
vel
*
EC HIV-1 negativesn=7 n=9
control p21 inhibitor at day 0
p21 inhibits early viral replication steps
p21 inhibitor at day -2
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• Ex vivo isolation of CD4 T cells from EC, HIV-1 negatives and progressors
• Direct infection with VSV-G pseudotyped HIV-1 without prior in vitro activation
• PCRs-based detection of late RT, 2-LTR and integrated HIV-1 DNA after 48 hours
Methods
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Intrinsic restriction of HIV-1 integration in ex-vivo infected CD4 T cells from elite controllers
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Restriction of HIV-1 integration in CD4 T cells from EC is unrelated to p21
HIV- HIV-- p21
EC EC- p21
HIV- HIV-- p21
EC EC- p21
HIV- HIV-- p21
EC EC- p21
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0.0
0.5
1.0
1.5no
rmal
ized
cop
ies/
cell
Integrated HIV-1 DNA
mRNA transcripts
Late RT transcripts
n=50
10
20
30
40
norm
aliz
ed
co
pie
s/ce
ll
0.0
0.2
0.4
0.6
rela
tive
HIV
-1 m
RN
A le
vel
n=5 n=5
*
p21 inhibitor
control
p21 independently inhibits HIV-1 mRNA transcription from proviral DNA
Infection with VSV-G pseudotyped, single-cycle HIV-1Addition of p21 inhibitor after 48 hours
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• Host protein complex expressed in all CD4 T cells• Phosphorylates RNAPII • Required for elongation of HIV-1 mRNA transcripts• CD4 cells lacking p-TEFb/CDK9 are resistant to HIV-1
Cyclin-dependent kinase 9 CDK9/p-TEFb
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• Isolation of CDK9 from CD4 T cells treated with p21 siRNA or control siRNA
• In vitro kinase to assess ability of CDK9 to phosphorylate RNAPII
• Detection of phosphorylated RNAPII by Western blot
Methods
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GST
Pol II CTD-Phos(Ser-2)
p21
siR
NA
cont
rol s
iRN
A
p21 inhibits enzymatic activity of CDK9
p21 siRNA
control siRNA
0.0
0.2
0.4
0.6
0.8
1.0p=0.0075
rela
tive
sign
al in
tens
ity(C
TD
-P (
Ser
-2)/
GS
T)
Neg
ativ
e co
ntro
l
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p21 inhibits transcriptional elongation of HIV-1
0
2
4
6
fold
incr
ease
of
HIV
-1 m
RN
Ap2
1siR
NA
/con
trol
siR
NA
p=0.007
p=0.06
proximal intermediate distal
HIV-1 mRNA fragments
n=7 n=7 n=7
Transfection of CD4 T cells with control or p21-specific siRNA Quantitative assessment of proximal, intermediate and distal HIV-1 mRNA segments
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• Intrinsic resistance of ex-vivo infected and in-vitro activated CD4 cells from EC against HIV
• Inhibition of HIV-1 integration in ex-vivo infected CD4 T cells from EC is unrelated to p21
• HIV-1 restriction of in-vitro activated CD4 T cells from EC involves p21-dependent inhibition of CDK9/ transcriptional elongation of HIV-1 mRNA
• Evidence for blockages of multiple HIV-1 replication steps in CD4 T cells from EC
Conclusions
Chen et al, JCI, March 2011Buzon et al, JVI, July 2011
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Acknowledgements
MGH ID Division/Ragon Institute
Maria BuzonChun LiKatherine SeissJill BeamonMary F. CarringtonPatrick BurkeXu Yu
Florencia PereyraAbraham BrassBruce Walker
Maha Al-MozainiJennifer RychertEric Rosenberg
FundingDoris Duke Charitable FoundationNIH/NIAIDHarvard CatalystGates FoundationMark and Lisa Swartz Foundation
University of California, DavisRobert Weiss
University of Lausanne, CH Amalio Telenti
Lee Moffitt Cancer Center, Tampa, FLDouglas Cress
VGTI Florida, Port St. Lucie, FLNicholas Chomont