Figure 1.
The nucleocytoplasmic trafficking of Rev.
Summary of the current understanding of molecular events regulating Rev trafficking within the infected cell [3], [4].
Figure 2.
Nullbasic induces Rev redistribution without protein interaction.
(A) HeLa cells expressing Myc-Rev alone (row 1) or with Nullbasic (NB)-mCherry (row 2) were fixed and immunostained with anti-Myc antibody before being analyzed by fluorescence microscopy for Myc-Rev (green) and NB-mCherry (red) subcellular localizations. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc-Rev and NB-mCherry panels. The figure is representative of four fields each from four independent experiments. (B) HeLa cells were transfected to express HIV-eGFP alone (row 1) or with NB-mCherry (row 2). Fixed cells were immunostained with anti-Rev antibody before the subcellular localizations of Rev (cyan) and NB-mCherry (red) were visualized by fluorescence microscopy. Expression of HIV-eGFP was confirmed in the FITC channel (green). Nuclei were stained with DAPI. The overlay panels show the merge of the Nullbasic-mCherry and Rev panels. Figures are representative of five fields each from four independent experiments. (C) HEK293T cells were transfected with either empty vector (pcDNA3.1) or plasmids expressing Nullbasic (NB)-FLAG alone, Tat-FLAG alone, or Myc-Rev with either NB-FLAG or Tat-FLAG, as indicated. The FLAG-tagged proteins and their interacting factors were immunoprecipitated using anti-FLAG beads. Total cell lysates (left) and immunoprecipitated proteins (right) were subjected to western blot analysis using anti-FLAG and anti-Myc antibodies. The endogenous protein CDK9, detected using anti-CDK9 antibody, was used as a positive control for Tat interaction. The figure is representative of four independent experiments. The white bar in last panel is equal to 10 µm.
Figure 3.
Nullbasic-induced redistribution of Rev is CRM1 dependent.
HeLa cells were transfected to express Myc-Rev alone (rows 2 and 3), Nullbasic (NB)-mCherry alone (row 4) or Myc-Rev with NB-mCherry (rows 5 and 6) before being treated with (rows 3 and 6) or without (rows 1, 2, 4 and 6) leptomycin B (LMB) in order to interfere with Myc-Rev and endogenous CRM1 interactions. Cells were fixed and immunostained with anti-Myc (green) and anti-CRM1 (magenta) antibodies and were visualized along with NB-mCherry (red) by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc-Rev panel with CRM1 panel (rows 1, 2, 3, 5 and 6) and the NB-mCherry with the CRM1 panel (row 4). The figure is representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.
Figure 4.
The subcellular localization of the Rev M10 mutant is not altered by Nullbasic.
HeLa cells were transfected to express Myc-RevM10 either alone (row 1) or with Nullbasic (NB)-mCherry (row 2). The subcellular localizations of Myc-RevM10 (green), NB-mCherry (red) and endogenous CRM1 (magenta) were visualized by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc-RevM10 and CRM1 panels. Figures are representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.
Figure 5.
Coexpression of Nullbasic and Rev alters the subcellular localization of B23.
HeLa cells were transfected to express Myc-Rev alone (row 1), Nullbasic (NB)-mCherry alone (row 2) or Myc-Rev with NB-mCherry (row 3). Fixed cells were immunostained with anti-Myc (green) and anti-B23 (magenta) antibodies before the subcellular localizations of Myc-Rev, endogenous B23 and NB-mCherry (red) were analyzed by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc- Rev panel with the B23 panel (rows 1 and 3) and the NB-mCherry panel with the CRM1 panel (row 2). Figures are representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.
Figure 6.
Coexpression of Nullbasic and Rev alters the subcellular localization of C23 but not fibrillarin.
HeLa cells were transfected to express Myc-Rev alone (rows 1 and 4), Nullbasic (NB)-mCherry alone (rows 2 and 5) or cotransfected Myc-Rev with NB-mCherry (rows 3 and 6). Fixed cells were immunostained with anti-Myc (green) and either anti-C23 (magenta, upper panels) or anti-fibrillarin (magenta, lower panels) antibodies before the subcellular localizations of Myc-Rev, endogenous C23 or endogenous fibrillarin, and NB-mCherry (red) were analyzed by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc-Rev panel with either the C23 or fibrillarin panels (rows 1, 3, 4 and 6) and the NB-mCherry panel with either the C23 or fibrillarin panels (rows 2 and 4). All figures are representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.
Figure 7.
Nullbasic does not affect the nuclear import of importin β and transportin 1 cargoes.
HeLa cells were transfected to express the importin β-dependent nuclear import reporter GFP2-cNLS, or the transportin 1-dependent nuclear import reporter GFP2-M9core, either alone (rows 1 and 3, respectively) or with Nullbasic (NB)-mCherry (rows 2 and 4, respectively). The subcellular localizations of the reporter proteins (green) and NB-mCherry (red) were analyzed in fixed cells by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the GFP reporter and DAPI panels. Plasmid expressing GFP2 was also transfected into cells either alone or with NB-mCherry as a vector control (rows 5 and 6, respectively). Figures are representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.