Figure 8. FMDV entry into BHK-21 cells activates Rac1 and depends on Dynamin II.

(A) Activation of Rac1 during FMDV entry. Cells were infected (MOI 10), and Rac1 activation was measured by GST-PAK1-PBD pull-down assay. Fold induction was determined by densitometry. (B,C) Rac1 Inh inhibited FMDV entry. Pretreated cells (200 μM Rac1 Inh) 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). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rac1 Inh-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Rac1 Inh. (D–F) Pretreated cells (Rac1 Inh) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (D), Western blot (E), and TCID50 assay (F). (G) Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in (B). (H) Expression of inactive form of Rac1 inhibited FMDV infection. Transfected cells with Rac1 (WT) and Rac1 (T17N) were infected (MOI 1) for 4 h at 37 °C and analyzed by Western blot. (I) Effect of Rac1 Inh on virus entry and post-entry steps. Cells were treated with Rac1 Inh 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. (J) Dynasore (Dyna) inhibited FMDV entry and multiplication. Cells were treated with indicated concentrations of Dyna as in (I) and then processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P < 0.05; **P < 0.01.