A: Experimental Outline. Jurkat or primary CD4+ T cells were infected with HIV-1 NL4-3 for 2 hours at 37°C and grown at either 37°C or 39.5°C for up to 10 days. B, D. Representative experiments with Jurkat and primary CD4+ T cells, respectively. The levels of intracellular Gag were measured by flow cytometry at the indicated time points. C, E: Mean ± SD of 5 independent experiments in Jurkat and primary CD4+ T cells, respectively. The area under the curve was calculated for all time points in Jurkat cells, and for time points occurring until the appearance of the peak at 39.5°C in primary CD4+ T cells. Statistical significance was assessed by the Wilcoxon test. p<0.05(*).

A: Primary CD4+ T cells were grown at 37°C or 39.5°C for 3 days. Cell growth was assessed every day by direct counting of living cells (Trypan blue exclusion). B: Primary CD4+ T cells were incubated 8 hours at 37°C or 39.5°C. Cell lysates were collected and probed by Western Blot for Hsp90, Hsp70 and Actin. C: Primary CD4+ T cells were grown at 37°C (blue line) or 39.5°C (red line) for 3 days. Cells were stained for the indicated surface markers and fixed in PFA. Isotype-matched monoclonal antibodies were used as negative controls (grey line).

A: Infection of Jurkat cells with single-cycle HIV. One representative experiment is shown. Cells were infected with 5, 20, or 100 ng Gag p24 of Δenv(VSV)/mL/10⁶ cells for 2 hours at 37°C or 39.5°C, washed, and grown at 37°C or 39.5°C. Gag levels were assessed by flow cytometry at 24 hours p.i. B: Infection of P4C5 cells with single-cycle HIV. One representative experiment is shown. 8×10⁴ P4C5 cells were plated in 96-well plates. Cells were infected in triplicates with 1 or 5 ng Gag p24 of Δenv(VSV) and grown at 37°C or 39.5°C. Infection was assessed 36 hours p.i. by measuring β-Galactosidase activity (570 nm OD). C: Mean ± SD of 4 independent experiments. P4C5 cells were infected with 1 ng Gag p24 of the indicated HIV strains. Statistical significance was assessed by the Mann-Withney test. p<0.05(*). D: Infection of P4C5 cells at different temperatures. One of two representative experiments is shown. Data are mean ± SD of triplicates. Using the same protocol as in B, cells were infected with 5 ng Gag p24 of NL4-3 and grown at the indicated temperatures. E: Transduction of P4C5 and Jurkat cells with a GFP expressing lentivector (LV-GFP). One of three representative experiments is shown. P4C5 or Jurkat cells were transduced with 1, 10 or 100 ng Gag p24/mL/10⁶ cells of LV-GFP. GFP levels were measured 24 hours later by flow cytometry.

A: Viral fusion assay. One representative experiment is shown. Jurkat cells were exposed for 2 hours at 37°C or 39.5°C to 5 or 100 ng Gag p24/10⁵ cells/0.1 mL of either WT, or a non fusogenic env mutant (F552Y) NL4-3, both bearing the chimeric protein β-lactamase-Vpr. Viral access to the cytoplasm was assessed by flow cytometry, using the ability of β-lactamase to cleave CCF2-AM, a fluorogenic substrate. B: Mean ± SD of 3 independent experiments. C: In vitro assessment of RT activity. Mean ± SD of 2 independent experiments is shown. HIV-1 NL4-3 virions were lysed and incubated at 37°C or 39.5°C for 1 h or 3 h. RT activity was measured with the Innovagen RetroSys RT Activity Kit (405 nm OD). D: Viral DNA synthesis in Jurkat cells. Mean ± SD of 3 independent experiments is shown. Jurkat cells were infected with 50 ng or 250 ng Gag p24 of Δenv(VSV)//mL/10⁶ cells for 2 hours at 37°C or 39.5°C, washed, and grown at 37°C or 39.5°C. 8 hours p.i., cells were harvested and viral DNA was measured by quantitative PCR. As a control, the RT inhibitor nevirapine (NVP) was added during infection.

A: Levels of Tat in HeLa-LTR-Luc cells. Cells were transfected with 20 ng of Tat-Flag. After 4 hours at 37°C, cells were washed and grown at 37°C or 39.5°C for 48 hours. Tat-Flag and tubulin levels were assessed by Western Blot. B: LTR activity in HeLa cells at 37°C or 39.5°C. HeLa-LTR-Luc and HeLa-LTRΔTAR-Luc cells were transfected with 4 ng or 20 ng of Tat-Flag. After 4 hours at 37°C, cells were washed and grown at 37°C or 39.5°C. 48 hours later, luciferase levels were measured and normalized to protein concentration. Data are mean ± SD of 3 independent experiments. Statistical significance was assessed by the Mann-Withney test. p<0.05(*).

A: Experimental outline. J-Lat 10.6 are Jurkat-derived cells carrying a latent, integrated, provirus, encoding gfp instead of nef. Stimulation of J-Lat 10.6 cells with TNFα triggers HIV-1 reactivation and GFP expression. B: Reactivation of J-Lat 10.6 cells with various stimuli. One representative experiment is shown. J-Lat 10.6 cells were treated with either TNFα, supernatants from IL-2 treated or PHA-activated PBMCs, or co-cultivated with IL-2-treated PBMCs for 48 hours at 37°C or 39.5°C. Viral reactivation was assessed by measuring GFP levels by flow cytometry. C: Mean ± SD of 4 independent experiments. Statistical significance was assessed by a paired t test. p<0.05(*).

A: Visualization of HIV transcription sites in U2OS HIVexo cells. One representative cell (at 37°C), displaying a co-localization of Hsp90 and nascent HIV RNA (followed with a YFP-MS2nls reporter protein) is shown. 2.5×10⁵ U2OS HIVexo cells were plated on coverslips and grown overnight at 37°C. Cells were transfected with 50 ng of Tat and 300 ng of YFP-MS2nls. After 4 hours, cells were washed and grown overnight at 37°C or 39.5°C. B: Mean ± SD of 3 independent experiments. Cells showing a transcription spot were scored for their co-localization with Hsp90. The percentage corresponds to cells in which HIV transcription sites co-localize with Hsp90. Statistical significance was assessed by an unpaired t test p<0.05(*). C: effect of the Hsp90 inhibitor 17-AAG on single-cycle HIV infection in P4C5 cells. Mean ± SD of 4 independent experiments is shown. 2×10⁵ P4C5 cells were infected with 30 ng Gag p24 of Δenv(VSV) for 2 hours at 37°C or 39.5°C, in presence of the indicated doses of 17-AAG. Cells were washed and grown at 37°C or 39.5°C at the indicated doses of 17-AAG. Cells were harvested 24 hours p.i. and Gag levels were assessed by flow cytometry. As a negative control, we used DMSO at a concentration corresponding to 250 nM 17-AAG.