A new molecular mechanism for HIV-1 latency

HIV latency in primary T cells: natural activation/purging by DCs

Ben BerkhoutAcademic Medical Center University of Amsterdam

Laboratory of Experimental Virology

SOLiD RNA sequences from HIV-1 infected cells

Method: Solid deep sequencing in HIV-infected T cells T-lymphocytes infected with HIV-1 versus uninfected cells Isolate small RNAs SOLiD sequencing: small RNA reads (~35nt), maximum output of

reads (~50 Gb)

Results:16.127.962 raw sequences 5.210.754 small RNAs identified

0,0% 3,8%

9,0%

0,2%

1,1%

80,4%

5,4%

uninfected (451502 reads)

HIV-1

ribosomal RNA, repeats, transposonstRNA

snRNA

scRNA

miRNAs

other

1,0%

7,2%

10,8%0,6%

2,7%

70,9%

6,7%

HIV-1 infected (2522374 reads)

1 1001 2001 3001 4001 5001 6001 7001 8001 9001

A

polgag

env

revtat

vpr

vif

vpu

5' LTR 3' LTR

envnef

po

siti

ve-s

tran

d r

ead

san

tise

nse

rea

ds

HIV-1

Uninfected

HIV-1

Uninfected

PBS

BC

D

E A

dcab

g

f

R R

Origin of vsiRNA of HIV-1

proviral HIV-1 dsDNA

transcription

(+) RNA

(-) RNA

viral LTR

(A)n

(A)n

Dicer

cellular promoter(s)

vsiRNA

pA

pA

- HIV-1 encodes vsiRNAs that suppress virus production- New mechanism for proviral latency: integration next to strong promoter

integration is a chance process!- Resting cell produces set of miRNAs that suppress HIV-1 (Huang et al, 2007)- Permanent latency?

Activation of latent HIV-1 proviruses in primary T cells

by dendritic cells

48 hrs

FACS (intracellular CA-p24)

% HIV-1 producing cells

Activation

24hrs

No Activation

Infection 4 hrs T1249

New latency assay for acutely infected T cells

Fold activation = Latency factor

% CA-p24+ cells activated

% CA-p24+ cells not activated

2 means at least one latently infected cell for each productively infected cell

and 3 means 2 for 1

control 2 5 2 50

1

2

3

4*****

*

Fo

ld a

cti

va

tio

n

control 0.4 4 0.4 40

1

2

3

4 **

Fo

ld a

cti

va

tio

n

control 50 100 50 1000

1

2

3

4 ******

Fo

ld a

cti

va

tio

n

control 1 5 1 50

1

2

3

4 ****

Fo

ld a

cti

va

tio

n

control 0.1 1 0.1 10

1

2

3

4*

Fo

ld a

cti

va

tio

ncontrol 10 50 10 50

0

1

2

3

4 ****

Fo

ld a

cti

va

tio

n

A

primary T lymphocytes

(PHA-activated)

SupT1 T cell line

C

FE

D

B

TNFα [ng/ml] Prostratin [μg/ml]

PMA [ng/ml] PHA [μg/ml]

NaBut [mM] TSA [μM]

From T cell line to primary T cells: all activation modes fail. No latency?

NF-kB, phorbol esters,TCR,HDAC

mock IL-2 CD3/ Iono DC0

1

2

3

4

***

CD28

Fo

ld a

cti

va

tio

n

Diverse HIV activation methods for resting/memory T cells (TCR, NFAT) fail to induce latent HIV-1 in activated/effector T cells

but

co-culture with dendritic cells (iDC) can!

iDC are able to induce HIV-1 in primary T cells

- DC + DCsUninfected

5.2%2.2%0%A

PC

(C

D3

)

PE (CA-p24)

- DC + DC0

1

2

3 **

Fold

act

ivat

ion

control iDC mDC0

2

4

6

8*****

Fo

ld a

cti

va

tio

n

mDC are much less effective2-5 fold activation

Fo

ld a

cti

va

tio

n

control DC0

1

2

3

Fo

ld a

cti

va

tio

ne

xtr

ac

ell

ula

r C

A-p

24

control DC0

1

2

3

fold

MF

I

Raltegravir

- +0

1

2

3 **

Fo

ld a

cti

va

tio

n

2 90

1

2

3 controlDC*** ***

days post infection

Fo

ld a

cti

va

tio

n

Increase in number of CA-p24+ cells, but - also a bit more HIV expression per cell- also more virus produced in supernatant

DC-mediated HIV-1 induction in T cells at the level of gene expression:- T1249 > no transmission/infection effects of DC- Raltegravir >- DC only after 9 days > no early (RT/integration) effects

A B C D E F0

1

2

3

4

5

+ DC- DC

****** ***

***

***

**

CD4+ T cells (donor)

Fol

d ac

tivat

ion

D E0

1

2

3

4

5controlDC donor DDC donor E

******

****

CD4+ T cells (donor)

Fo

ld a

cti

va

tio

n

Donor-to-donor variation

No need for autologous T-DC cells

Cell-cell contact or DC-secreted molecule

A

control mock sup 293T mock sup DC0

1

2

3

4

5

+ 293T + DC

***

*** **

Fo

ld a

cti

va

tio

n

0 0.1 0.3 1 3 100

1

2

3

4

5 *******

(*104 DCs)

Fo

ld a

cti

va

tio

n

CDC:T cell ratio

1:5

B

control sup 293T sup DC0

1

2

3

4

5

***

Fo

ld a

cti

va

tio

n

C 293T DC

supernatant

C 293T 293T DC DC - sup + sup - sup + sup

Soluble Component !

Cell-cell contacts adds to the activation potential !

Which DC-T cell contact: ICAM1-LFA1

control ICAM1 ICAM2 ICAM3 mock ICAM1 ICAM2 ICAM30

2

4

6

8

*****

- iDC + iDC

Fo

ld a

cti

va

tio

n

Same contact important for DC-mediated HIV transmission to T cells

HIV-1 activation from latency – primary T cells – dendritic cells

Signal 1

DC

Signal 2

?

Natural activation mechanism: 1. T-DC synapse2. ?

Early HIV infection (transmission)

DC have well-known role in HIV-1 transmission:Ferry the virus to T cells

New evidence: DC trigger productive T cell infectionat the level of HIV-1 gene expression (CA-p24+)

Anti-latency effect of DC: first T cell infections unlikely to become latent !

Late HIV infection

Latently infected T cells can be activated by circulating DC

Future therapeutics

Identification of the activating molecule to purge reservoirs!

?

van Gogh

Experimental Virology:

Thijs van Montfort

Georgios Pollakis

Rogier Sanders

Rienk Jeeninga

Joost Haasnoot

Nick Schopman

Renée van der Sluis

Biochemistry:Dave Speijer