FIG. 3.

Both CVB3 isolates require dynamin for infection. (A) Cells transiently transfected with plasmids expressing WT or K44A dynamin 2 (Dyn2 WT or Dyn2 K44A) were infected with CVB3-RD (5 PFU/cell) or CVB3-Nancy (30 PFU/cell). Infection was stopped at 6 h, and new viral protein was detected by staining. Infection was quantified as the percentage of transgene-positive cells that were positive for virus. (B) Cells serially transfected with siRNAs targeting dynamin 2 were infected with CVB3-RD or CVB3-Nancy. In parallel, cells were scored for transferrin (Tfn) uptake as described in Materials and Methods. Neg control, negative control. (C) Immunoblot of cell lysates after serial transfection with negative control (Con) or siRNAs targeting dynamin 2 (Dyn2). The GAPDH blot serves as a loading control. (D) Dynasore-treated cells were infected with CVB3-RD or CVB3-Nancy. Cells were pretreated with dynasore for 45 min, and drug was present throughout binding and for the first 3 h of infection. Complete medium with NuSerum instead of FBS was used. (E) Tfn uptake in dynasore-treated cells. Cells were treated with dynasore for 45 min at 37°C, followed by 1 h at 16°C (to match treatment of virus-infected samples). Alexa Fluor 594-Transferrin was then added to cells in complete medium with NuSerum and allowed to internalize for 40 min at 37°C. Images were captured with a 40× objective. For all infection graphs, data represent the normalized percentages of cells infected ± standard deviations for triplicate samples from each of three independent experiments, except for dynasore experiments, in which duplicate samples were used. P values: *, <0.05; **, <0.01; ***, <0.001.