FIG. 6.

Analysis of Crm-1 dependence of Gag expression. (A) Schematic representation of a competitor protein for NES-dependent nuclear export (30, 49). The competitor corresponds to a fusion protein containing the NLS of the SV40 large T antigen and the NES of the human T-cell leukemia virus Rex protein lacking the RNA binding domain. This molecule is expected to continuously shuttle between the nucleus and cytoplasm and titrates limiting factors of the NES export pathway. A variant containing an inactivating mutation in the NES domain of the fusion protein (M90) was used as a control. (B) HeLa cells were transiently transfected with plasmid 3-RRE in the absence (−; lane 1) or in the presence (+; lanes 2 to 4) of Rev, with plasmid 3-CTE (lanes 5 to 7), or with plasmid 3-IAPE (lanes 8 to 10). Transfections were performed with or without cotransfected expression plasmids for the NLS-NES competitor (NES) or the M90 variant thereof, as indicated above each lane. Gag expression was determined by immunoblot analysis as described in the legend to Fig. 1. (C) Analysis of Crm-1 dependence of Gag expression using leptomycin B. Cos-7 cells were transiently transfected with plasmid 3-RRE in the absence (lanes 2 and 5) or in the presence (lanes 3 and 6) of Rev or with plasmid 3-IAPE (lanes 1 and 4). Transfection solution was left on the cells for 3 h and then replaced with fresh medium (lanes 1 to 3) or with medium containing 100 nM leptomycin B (lanes 4 to 6). The cells were harvested 16 h after addition of the drug and subsequently analyzed for HIV-1 Gag expression as described in the legend to Fig. 1.