Rev and CRM1 accumulate at transcription sites of reporters that splice post-transcriptionally. (A) Scheme of the HIV-1 reporter. It contains the two HIV-1 LTRs, the packaging sequence Ψ, the splice donor SD1, the Rev-response element (RRE) within the intron, the splice acceptor SA7, and 24 MS2 repeats for detection in live and fixed cells. (B) Exo1 cells were transfected with Tat. HIV-1 RNAs were visualized by fluorescence in situ hybridization against the MS2 repeats and CRM1 by immunofluorescence. The overlay shows the RNA in red and CRM1 in green. (C) Exo1 cells were cotransfected with Tat and Rev-GFP. The RNA, Rev, and CRM1 were detected as in B. In the overlay, the RNA is depicted in red, CRM1 in green, and Rev-GFP in blue. (D) Same as C but cells were treated with spliceostatin A (SSA) for 3 h prior to fixation and immunolabeling. (E) Bar chart showing the percentage of transcription sites that colocalize with Rev-GFP (light gray) and CRM1 (dark gray) in absence and presence of SSA. Forty to 50 cells were counted in each condition. (Scale bar) 5 µm. Arrows indicate the position of the transcription sites.
