Figure 9. Pak1 is required for FMDV entry into BHK-21cells.

(A,B) FMDV activated Pak1 during early post-infection. (A) Cells were infected (MOI 10), and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot analysis. The level of total Pak1 was measured as the control. Fold induction was determined by densitometry. (B) Cells were infected (MOI 25) and processed for confocal microscopy with anti-phospho-Pak1 (green), anti-FMDV (red), and DAPI (blue). (C,D) IPA-3 inhibited FMDV entry. Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (D) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or IPA-3-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (E–H) FMDV infection was inhibited by IPA-3. (E–G) Pretreated cells (IPA-3) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (E), Western blot (F), and TCID50 assay (G). (H) Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in (C). (I) Effect of IPA-3 on virus entry and post-entry steps. Cells were treated with IPA-3 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P < 0.05; **P < 0.01.