Pak1 is required for ASFV entry in Vero cells.

A) ASFV activates Pak1 at early times post infection. Cells were infected (MOI 5) and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot. Levels of total Pak1 were measured as a control. Fold induction was determined by densitometry (mean ±S.D). B–D) IPA-3 inhibits ASFV entry. B) Cells were pretreated with DMSO or 30 µM IPA-3 and infected (MOI 10) for 60 min to analyze ASFV uptake by FACS. The graph shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 9, performed in duplicate; mean ±S.D.). C) Viral protein synthesis was analyzed in infected cells (MOI 1) at 16 hpi in the presence of IPA-3 at the indicated concentrations. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. D) Supernatants from DMSO or 5 µM IPA-3 treated cells after 48 hpi (MOI 1) were recovered. Lytic viruses were titrated in Vero monolayers and plotted in the table (n = 3). E–F) Pak1 mutant reduces ASFV infection. E) Vero cells were transfected with pEGFP-Pak1-WT, pEGFP-Pak1-AID (Pak D/N form) and pEGFP-Pak1-T423E (Pak C/A form) for 24 h. Then, cells were infected (MOI 1) for 16 h and viral protein synthesis was analyzed by immunoblotting with an anti-ASFV antibody. GFP expression was measured as a control of transfection. β-actin was detected as a load control. F) Fold induction was determined by densitometry and represented in the graphic (mean ±S.D). S.D., standard deviation. * Unspecific cellular protein detected by the antibody.