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

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?

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

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)

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

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

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

0

5

10

15

20

25

norm

aliz

ed c

opie

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 c

opie

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

• 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

Intrinsic restriction of HIV-1 integration in ex-vivo infected CD4 T cells from elite controllers

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

0.0

0.5

1.0

1.5no

rmal

ized

copi

es/c

ell

Integrated HIV-1 DNA

mRNA transcripts

Late RT transcripts

n=50

10

20

30

40

norm

aliz

ed c

opie

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

• 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

• 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

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

1.0 p=0.0075

rela

tive

sign

al in

tens

ity(C

TD-P

(Ser

-2)/

GS

T)

Neg

ativ

e co

ntro

l

p21 inhibits transcriptional elongation of HIV-1

0

2

4

6fo

ld in

crea

se o

f HIV

-1 m

RN

Ap2

1siR

NA

/con

trol s

iRN

Ap=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

• 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

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