2.2. Dox-Induction and Visualization of USvRNA and GAPDH RNA in HeLa and U2OS Cells

HIV-1 provirus expression was induced in HeLa HIV.Gag-GFP rtTA and HeLa HIV.Gag-GFP rtTA Rev-BFP cells grown on 1.5 mm cover slips by the addition of dox (2 µg/mL) to the cell media for 24 h. Following dox induction, HeLa HIV.Gag-GFP rtTA and HeLa HIV.Gag-GFP rtTA Rev-BFP cells were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, permeabilized in 70% EtOH at 4 °C for a minimum of 1 h, and rehydrated in wash buffer (10% formamide in 2X SSPE) for 20 min. Next, hybridization buffer (10% dextran sulfate, 2X SSPE, 10% formamide) containing a mix of either 48 different Stellaris^® single molecule fluorescence in situ hybridization (smFISH) probes conjugated to Quasar 570 (Biosearch Technologies, Novato, CA, USA) targeting the gag coding region or 80 Stellaris^® smFISH probes targeting human GAPDH transcripts, were added to the rehydrated cells and incubated overnight at 37 °C in a sealed humid chamber. Following the incubation, cells were washed in warm wash buffer for 30 min at 37 °C and then incubated for an additional 30 min at 37 °C in wash buffer containing 5 µg/mL DAPI before being mounted onto slides. The same dox-induction protocol was applied to U2OS and HeLa cells transfected with pHIV.Gag-CFP rtTA or pHIV.Gag-SNAP-tag rtTA.

For cells not subjected to FISH, they were fixed with 3.7% paraformaldehyde and 0.4M KOH in PHEM buffer [3.6% piperazine-N,N = -bis(2-ethanesulfonic acid) (PIPES), 1.3% HEPES, 0.76% EGTA, 0.198% MgSO4, pH to 7.0 with 10 M KOH] [38] for 15 min at room temperature with gentle rocking. Following fixation, cells were then washed in PBS and DAPI stained before mounting onto slides using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific-Invitrogen).

For cells transfected with pHIV.Gag-SNAP-tag rtTA, cells were incubated with 50 nM SNAP-tag ligand Janelia Fluor 549 (JF549) [39] at 37 °C for 1 h. Cells were washed with warm standard buffer and fixed with fixation media prior to DAPI staining and mounting.

During dox-induction, HeLa HIV.Gag-GFP rtTA cells were treated with either 150 µM of 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or 4 µg/mL Actinomycin D (Act D) in cell media to inhibit transcription for 30 or 60 min prior to fixation (i.e., 30 min treatment corresponds to drug being added for last 30 min of the 24 h induction). In separate experiments, HeLa HIV.Gag-GFP rtTA cells were treated with 20 nM of the CRM1 inhibitor LMB for 30, 60, 90, or 120 min prior to fixation.