Fig 5.
Imaging of the PB2-GFPcomp protein in live 293T cells infected with the WSN-PB2-GFP11 virus. (A) Selected frames from Movie S1A in the supplemental material showing individual PB2-GFPcomp-positive 293T cells at the times postinfection indicated (min). 293T cells transiently expressing GFP1-10 were infected at an MOI of 3 with the WSN-PB2-GFP11 virus, and images (PB2-GFPcomp signal) of live cells throughout the infectious cycle were acquired with a Leica TCS SP2 AOBS microscope, as described in Materials and Methods. Single confocal slices are shown. Brightness and contrast were adjusted for the whole series of images. Arrowheads point to the cell used for quantification shown on Fig. 5C. Scale bar, 10 μm. (B) 293T cells transfected with GFP1-10 and infected with WSN-PB2-GFP11 virus, imaged (PB2-GFPcomp signal) 9 h (left) and 1 day (right) postinfection. Accumulation of PB2-GFPcomp at the plasma membrane is indicated by arrowheads. Scale bars, 10 μm. (C) Quantification of PB2-GFPcomp content over time in representative infected cells treated with 20 nM leptomycin B (red lines) and mock treated (black lines; the corresponding cell is marked with arrowheads in Fig. 5A). PB2-GFPcomp mean fluorescence intensities corrected for background in an ROI in the nucleus (dash-dotted line) and in an ROI in the cytoplasm (dashed line) are represented. Positions and shapes of the ROIs were manually adjusted in each frame to cover significant portions of the appropriate compartments of the cell. au, arbitrary units. (D) Effect of leptomycin B treatment on the intracellular distribution of the PB2-GFPcomp protein in live 293T cells transiently expressing GFP1-10 and infected at an MOI of 3 with the WSN-PB2-GFP11 virus. After viral adsorption, cells were incubated with 15 nM leptomycin B or with the corresponding dilution of dimethyl sulfoxide. Representative images of PB2-GFPcomp-expressing cells acquired at 6 h postinfection are shown. Brightness and contrast were adjusted for presentation purposes. Scale bar, 10 μm. (E) Effect of leptomycin B treatment on the intracellular distribution of the PB2-GFPcomp protein in live infected cells. Values are the ratio of nuclear to cytoplasmic intensities of PB2-GFPcomp fluorescence in the absence (black bars) or presence (gray bars) of leptomycin B. The data are shown as mean values ± SD calculated from >10 cells/condition.