Figure 4

PSaV entry and RNA release depend on clathrin-, dynamin-, and cholesterol-mediated endocytosis. A LLC-PK cells grown in 6-well plates were incubated with neutral red (NR)-labeled PSaV Cowden strain for the indicated times and then exposed or not to UV light. The supernatants of each cell lysate were inoculated into fresh LLC-PK monolayers in 6-well plates, overlaid with agar, and incubated for 4 days. Results are shown as percentages to the number of plaques in the unilluminated control. The NR infectious center assay was performed in cells pretreated with DMSO vehicle or 20 μM chlorpromazine (CPZ), or transfected with scrambled siRNA or CHC siRNA (B); pretreated with DMSO vehicle or 100 μM dynasore (Dyna), or transfected with scrambled siRNA or dynamin II (Dyn II) siRNA (C); or pretreated with DMSO vehicle, 20 mM MβCD for 1 h, or MβCD followed by incubation with 200 μM soluble cholesterol for 30 min (D). The cells were then incubated with NR-labeled PSaV for 120 min. The number of plaques was normalized to those obtained with unilluminated controls exposed to the above conditions. Data for panels A–D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. *P < 0.05; **P < 0.001; ***P < 0.0001.