FIG. 5.

Clathrin and endosomal acidification are not required for infection. (A and B) Cells treated with CPZ (A) or depleted of clathrin heavy chain by serial transfection with siRNAs (B) were infected with CVB3-RD or CVB3-Nancy. In parallel, transferrin (Tfn) uptake was assessed. Cells were pretreated with CPZ for 1 h, and drug was present throughout binding and infection. (C) Immunoblot of cell lysates after serial transfection with negative control (Con) or siRNAs targeting clathrin heavy chain (CHC). GAPDH was used as a loading control. (D and E) Cells were treated with endosomal acidification inhibitor NH₄Cl (D) or bafilomycin A1 (E) and then infected with CVB3-RD, CVB3-Nancy, or VSV (an acidification-dependent virus). Cells were pretreated for 1 h, and drugs were present throughout binding and infection. For all infection graphs, data represent the normalized percentages of cells infected ± standard deviations for duplicate samples from each of three independent experiments, except for clathrin heavy chain siRNA experiments, in which triplicate samples were used. Transferrin uptake data for CPZ-treated cells were from a representative experiment in which four fields (each with 30 to 50 cells) from two wells were counted for each condition. P values: *, <0.05; **, <0.01; ***, <0.001.