FIG.3.

Overexpression of dyn^K44A inhibits HRV2 uptake in stably transformed HeLa cells. (A) dyn^K44A or dyn^wt expression was induced in HeLa cells by incubation without tetracycline. After 48 h, cells were incubated with HRV2 (∼30 PFU/cell) for 20 min at 34°C, fixed, and stained for confocal immunofluorescence detection of virus and dynamin mutants. Cells expressing dyn^K44A show strongly reduced virus uptake. In these cells, virus clearly accumulates on the plasma membrane. Cells expressing dyn^wt internalize virus-like untransfected cells. Bar, 10 μm. (B) Cells induced to express HA-tagged dyn^K44A were incubated with HRV2 and rhodamine-transferrin for 20 min at 34°C, washed, fixed, and stained for immunofluorescence by using anti-HRV2 and anti-HA antibodies. HRV2 (green) and transferrin (red) internalization are blocked in cells overexpressing dyn^K44A. Note the colocalization (yellow) of virus and transferrin, presumably in early endosomes of the cell that does not express dyn^K44A. Bar, 10 μm. (C) Cells induced to express HA-dyn^K44A or HA-dyn^wt were infected with HRV2 (∼15 PFU/cell) for 20 min, washed, and incubated overnight at 34°C before immunofluorescence detection of virus-producing cells and of the HA tag. For each experiment, a total of ∼250 cells expressing the respective dynamins was observed and the fraction of cells synthesizing virus was determined (black bars). This was divided by the fraction of virus-producing cells that did not express dynamin (white bars).