1. 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 Laboratory of Experimental Virology

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

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

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

6. 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

7. A Fig 1 primary T lymphocytes (PHA-activated) SupT1 T cell line C F E 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

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 - DC+ DCsUninfected 5.2% 2.2% 0% APC (CD3) PE (CA-p24) Fold activation mDC are much less effective2-5 fold activation Fold activation

10. Raltegravir 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

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

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

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 Signal 1 DC Signal 2 ? 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. van Gogh Experimental Virology: Thijs van Montfort Georgios Pollakis Rogier Sanders Rienk Jeeninga Joost Haasnoot Nick Schopman Renée van der Sluis Biochemistry: Dave Speijer