FIG 2.

Evidence for Rev exhibiting position-dependent NES masking. (A) Context-specific NES effects on Rev's subcellular localization. HeLa cells transfected to express E-R-Rev-/Luc and the indicated Rev-mChe variants were fixed, permeabilized, and DAPI stained 24 h posttransfection. Endogenous CRM1 was detected by indirect immunofluorescence using anti-CRM1 antisera. Yellow arrows highlight nucleolar accumulation of Rev and/or CRM1. Scale bars, 10 μm. (B) RevM10-mChe-NES exhibits less accumulation at or near the nucleolus. Rev-mChe subcellular distribution was quantified in individual cells as primarily nuclear (N), cytoplasmic (C), or equivalent in both compartments (N/C). Error bars represent the standard deviations from the mean for three independent transfections. (C) Depictions of RevM10-mChe-LEXY construct and blue light-regulated NES unmasking using the LEXY regulatory module (75). (D) Control experiment demonstrating that the activity of Rev-mChe-LEXY and RevM10-mChe-LEXY variants is equivalent to Rev-mChe constructs lacking LEXY. Viral infectivity was measured as for Fig. 1B. Error bars represent the standard deviations from the mean for three independent experiments. (E) Image panel shows selected frames from a representative time-lapse fluorescence microscopy experiment capturing mCherry fluorescence from RevM10-mChe-LEXY in HeLa cells. Red circles indicate exposure to 572-nm wavelength light (mCherry acquisition wavelength), and green circles indicate exposure to 488-nm wavelength light (LEXY activation wavelength). Black arrows indicate nucleolar Rev accumulation sites, and yellow arrows indicate direction of Rev transitions over time. Scale bars, 10 μm.