FIG 5.

PEAV entry is dependent on macropinocytosis. (A and B) Vero cells were pretreated with EIPA (A) or wortmannin (B) (15 min, 37°C), followed by inoculation with PEAV (MOI = 1) to allow binding (1 h, 4°C) and entry (1 h, 37°C) in the presence of drugs. (C and D) In the presence of EIPA (50 μM) (C) and wortmannin (400 nM) (D), cells were inoculated with PEAV (MOI = 5) (2.5 h, 37°C) and immunoblotted to detect the early expression of PEAV N protein. Relative amounts of protein were calculated using ImageJ software. (E) Vero cells were inoculated with PEAV (MOI = 5) (1 h, 4°C), pretreated with PMA (positive control) (200 nM, 60 min, 37°C) or maintenance medium (negative control) (1 h, 4°C), and then incubated in maintenance medium containing 0.5 mg/mL Alexa Fluor 488-labeled dextran (15 min, 37°C). Dextran uptake was visualized using immunofluorescence microscopy. (F) Dextran uptake by Vero cells is represented by dextran fluorescence IDO values measured with ImageJ software. The mean ± SD values represent three individual pictures (**, P < 0.01; ***, P < 0.001). (G) Vero cells were inoculated with PEAV (MOI = 5) (1 h, 4°C), washed, incubated (15 min, 37°C), and fixed (4% PFA, 15 min, RT). Vero cells were pretreated with PMA (positive control) (200 nM, 60 min, 37°C) or maintenance medium (negative control) (1 h, 4°C), fixed (4% PFA, 15 min, room temperature), and then incubated with anti-PEAV N (red), and cytoskeletal changes were observed via probing actin with Alexa Fluor 488 phalloidin (green). (H) Vero cells were inoculated with PEAV (MOI = 5) (1 h, 4°C), washed, and incubated in a maintenance medium containing 0.5 mg/mL Alexa Fluor 488-labeled dextran (15 min, 37°C). Cells were fixed (4% PFA, 15 min, room temperature) and immunodetected with an anti-PEAV N protein antibody (red). Confocal microscopy revealed PEAV and dextran colocalization (white arrows).