Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Abstract

The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.

Keywords: CRM1; HIV-1 Rev; nuclear export.

FIGURE 1.

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.

FIGURE 2.

Colocalization of Rev, CRM1, and cotranscriptionally spliced HIV-1 reporter RNAs. (A) Scheme of the chimeric HIV-1/MINX reporters. Transcription was driven by the HIV-1 LTR (data not shown), and the RNAs contained the MINX intron followed by the LacZ sequence. MINX_RREin contains the RRE within the intron. The red bar indicates the position of the LacZ probes used for FISH. (B) Cells were transfected with Tat, Rev-GFP, and the MINX reporters as indicated. The RNA was visualized by FISH, Rev by GFP-fluorescence, and CRM1 by immunofluorescence. In the overlay, the RNA is shown in red, Rev in blue, and CRM1 in green. (C) Same as in B except that the cells were treated with SSA for 3 h prior to fixation and immunolabeling. (D) Bar chart showing the percentage of MINX_RREin RNA dots that colocalize with Rev-GFP (light gray) and CRM1 (dark gray) in the absence and presence of SSA. Thirty to 40 cells were counted in each condition. Arrows indicate the position of the transcription sites.

FIGURE 3.

Fast 2D FRAP of Rev and CRM1 in the nucleoplasm and at the HIV-1 transcription site. (A) U2OS cells were transfected with Rev-GFP alone (left), GFP-CRM1 alone (middle), or with both GFP-CRM1 and Rev-Flag (right). The images show GFP-fluorescence in live cells. The arrows indicate the nucleoplasm. (B) 2D FRAP plots of Rev-GFP (open circles) and GFP-CRM1 (cotransfected with Rev-Flag; closed circles) in the nucleoplasm of U2OS cells. (C) Exo1 cells were transfected with either Tat, Rev-GFP, and MS2-mCherry, or with Tat, Rev-Flag, GFP-CRM1, and MS2-mCherry. The micrographs show the GFP and Cherry fluorescence in live cells. Arrows indicate the position of the transcription sites. (D) 2D FRAP plots Rev-GFP (open circles) and GFP-CRM1 (closed circles) at the transcription site of Exo1 cells. The vertical line indicates the transition point between the fast and slow fractions. (Scale bar) 5 µm.

FIGURE 4.

3D FRAP of Rev-GFP and GFP-CRM1 at the transcription site. (A) Exo1 cells were transfected with either Tat and Rev-GFP (upper), or with Tat, GFP-CRM1, and Rev-Flag (lower). FRAP was performed with the 3D FRAP method. Micrographs show selected time points: prebleach and 10 sec, 180 sec, and 598 sec post-bleach. Arrows indicate the position of the transcription sites. (B) Plot of the recovery curves of Rev-GFP and GFP-CRM1 at the transcription site using the 3D FRAP method. The vertical line indicates the transition point between the fast and slow fractions. (Open circles) Rev-GFP; (closed circles) GFP-CRM1; (scale bar) 5 µm.

FIGURE 5.

Analysis of Rev-GFP and GFP-CRM1 3D FRAPs at the transcription site. (A,B) The FRAP data of Rev-GFP and GFP-CRM1 were fitted with a monoexponential, a double, or a three exponential function. Insets show the sizes of the slow, medium, and fast fraction. (C) Comparison of the nucleoplasmic FRAP of Rev and CRM1 with the fast component of Rev-GFP and GFP-CRM1 recoveries at the transcription site. The FRAP data were rescaled for better comparison. (D) Plot of the recoveries of the slow component of Rev-GFP (black), CRM1-GFP (red), and MS2-GFP (green) at the HIV-1 transcription sites. The curves were rescaled from 0 to 1 for better comparison.