A new molecular mechanism for HIV-1 latency

1. Laboratory of Experimental Virology A new molecular mechanism for HIV-1 latency HIV latency in primary T cells: natural activation/purging by DCs Ben Berkhout Academic Medical Center University of Amsterdam

2. 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

3. R R B C HIV-1 D A A E positive-strand reads Uninfected PBS tat 3' LTR 5' LTR rev gag nef vif pol env env vpr vpu Uninfected antisense reads f c a d b g HIV-1

4. Origin of vsiRNA of HIV-1 pA proviral HIV-1 dsDNA pA transcription viral LTR cellular promoter(s) (+) RNA (A)n (-) RNA (A)n Dicer vsiRNA • 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?

5. Activation of latent HIV-1 proviruses in primary T cells by dendritic cells

6. No Activation 4 hrs T1249 Infection 24hrs Activation New latency assay for acutely infected T cells 48 hrs FACS (intracellular CA-p24) % HIV-1 producing cells % CA-p24+ cells activated % CA-p24+ cells not activated Fold activation = Latency factor 2 means at least one latently infected cell for each productively infected cell and 3 means 2 for 1

7. B A SupT1 T cell line Prostratin [μg/ml] TNFα [ng/ml] primary T lymphocytes (PHA-activated) D C PHA [μg/ml] PMA [ng/ml] NF-kB, phorbol esters, TCR, HDAC E F NaBut [mM] TSA [μM] From T cell line to primary T cells: all activation modes fail. No latency? Fig 1

8. 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!

9. iDC are able to induce HIV-1 in primary T cells Uninfected - DC + DCs 0% 5.2% 2.2% APC (CD3) PE (CA-p24) Fold activation Fold activation 2-5 fold activation mDC are much less effective

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

11. Donor-to-donor variation No need for autologous T-DC cells

12. B C 293T DC supernatant Cell-cell contact or DC-secreted molecule A Soluble Component ! 1:5 DC:T cell ratio C Cell-cell contacts adds to the activation potential ! C 293T 293T DC DC - sup + sup - sup + sup

13. Which DC-T cell contact: ICAM1-LFA1 Same contact important for DC-mediated HIV transmission to T cells

14. HIV-1 activation from latency primary T cells dendritic cells DC Signal 2 Signal 1 ? • Natural activation mechanism: • 1. T-DC synapse • 2. ?

15. ? 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 infection at 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!

16. Biochemistry: Dave Speijer Experimental Virology: Thijs van Montfort Georgios Pollakis Rogier Sanders Rienk Jeeninga Joost Haasnoot Nick Schopman Renée van der Sluis van Gogh