Mature vaccinia virus WR localizes within endocytic structures that are positive for CD98 in cells. (A to E) Imaging of WR MV kinetics of internalization into CD98-positive vesicles. CD98+/+ MEFs were incubated for 60 min at 4°C with FITC-conjugated anti-CD98 antibody (green) and WR-A4-mCherry (red) at an MOI of 20 PFU/cell. Cells were shifted to 37°C for 0 min (A), 5 min (B), 15 min (C), 30 min (D), or 45 min (E) and individually washed with acidic buffer to remove surface-bound antibody prior to fixation. The confocal images in each panel are enlarged views of the areas outlined by the white squares. Arrowheads point to the CD98-positive vesicles containing WR MV. (F) Percentages of MEF cells showing WR MV localizing in CD98-positive vesicles. For each time point, z sections of 60 cells were analyzed, and the standard deviations are shown. (G) Percentages of WR MV particles per cell that localize within CD98-positive vesicles. The numbers of WR MV particles localizing in CD98-positive structures per cell and total WR MV particles per cell were quantified. For each time point, z sections of 60 cells were analyzed, and the standard deviations are shown.