Figure 4.
PIKfyve activity is required for VACV MV entry. A) HeLa cells transfected with EGFP‐FYVEEEA1 for 18 h were infected with WR mCherry‐A4 MVs at an MOI of 2. At the indicated time points, cells were fixed and non‐permeabilized cells subjected to immunostaining with α‐L1R to distinguish bound virions (purple). White arrows represent colocalization events and representative images of the peak time points of colocalization are displayed. Insets display individual colocalization examples from boxed regions in xy, yz and xz planes (imaris). Bars, 5 µm. B) The kinetics of internalized MV colocalization with EGFP‐FYVEEEA1 were quantified and displayed as percent colocalization at each time point. At least 45 cells were analyzed at all time points. Error bars represent the SD of three independent experiments. C) HeLa cells were pretreated with the PIKfyve inhibitor YM201636 for 1 h prior to infection with WR E EGFP at an MOI of 1 in the presence of the inhibitor. At 6 h p.i., cells were prepared for flow cytometry and 10 000 cells scored for infection. Results are means of three independent experiments ± SD. D) HeLa cells were left untreated or were pretreated with YM201636 for 1 h. After which, cells were cooled to 4°C and WR EGFP‐A4 MVs were added at an MOI of 10. After 1 h of binding, cells were harvested for flow cytometry and 10 000 cells scored for associated EGFP fluorescence. Results represent the mean of three independent experiments ± SD. E) HeLa cells were pretreated with IRESSA (40 µM) or YM201636 (20 µM) for 1 h. Cells were cooled to 4°C and WR E/L EGFP MVs (MOI = 1) were bound to cells for 1 h. After binding, cells were washed and treated with 37°C media (pH 7.4 or 5.0) for 5 min. Cells were washed and media containing the inhibitors was added. Cells were then incubated for an additional 4 h at 37°C prior to flow cytometry analysis of 10 000 cells per sample. Results represent means of three independent experiments ± SD.