Fig 3.

Vps28 targets the 3A protein, a vital component of the FMDV replication complex. (A) PK-15 cells were transfected with plasmids encoding HA-EV (2 µg) or HA-Vps28 (0.5, 1, and 2 µg) and Flag-2C (2 µg). Cell lysates were analyzed by western blot after 20 hours post-transfection (hpt). (B) PK-15 cells were transfected with plasmids encoding HA-EV (2 µg) or HA-Vps28 (0.5, 1, and 2 µg) and Flag-3A (2 µg). Cell lysates were analyzed by western blot after 20 hpt. (C) Plasmids encoding HA-Vps28 and Flag-EV or Flag-2C, or Flag-3A were co-transfected to PK-15 cells. At 18 hpt, the cells were fixed and incubated with anti-Flag and anti-HA antibodies and then with secondary antibodies conjugated with FITC (green) and TRITC (red), respectively. Nuclei were counterstained with DAPI (blue), and localization was determined using confocal microscopy. (D) PK-15 cells were transfected with plasmids encoding Vps28 for 24hours and then mock infected or infected with FMDV (MOI = 1) for 6hours. The cell lysates were analyzed by western blot. (E) PK-15 cells were co-transfected with HA-Vps28 or HA-EV, and Flag-3A plasmids, and the transfected cells were maintained in the culture medium in the presence of CHX (100 µg/mL), and the half-life of 3A protein was determined by western blot analysis in the presence or absence of Vps28. (F) The proteasome inhibitor MG132 (10 and 20 µM), the autophagy inhibitor CQ (50 and 100 µM), and the apoptosis inhibitor Z-VAD-FMK (10 and 50 µM) were added to PK-15 cells co-transfected with HA-Vps28 or HA-EV and Flag-3A after 6 hpt, and cell lysates were analyzed by western blot at 20 hpt.