Rac1 plays a critical role in ASFV entry in Vero cells.

A–B) Activation of Rac1 during ASFV entry. Vero cells were infected (MOI 10) and 0.Rac1 activation was measured by A) Kit Activation Assay (n = 3; mean ±S.D.) and B) Pak1 PBD-Agarose Beads pull down assay. Fold induction was determined by densitometry (mean ±S.D). C) ASFV infection induces clustering of Rac1. Cells were transfected with pEGFP-Rac1, infected (MOI 10) and stained with Topro3 (blue) and anti-p72 (red). Analyzed images by CLSM were represented as a maximum of z-projection. D–E) Rac1 inhibitor blocks viral entry. Pretreated cells (200 µM Rac1 inhibitor) were infected (MOI 10) for 60 min. D) Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal analyzed by FACS (n = 3, performed in duplicate; mean ±S.D.). E) Cells were incubated with Topro3 (blue), TRITC-phalloidin (red) and anti-p72 (green). Images are represented as a maximum z-projection of x-y plane (bottom panels) and x–z plane (upper panels). F) Expression of inactive form of Rac-1 reduces viral infection. Transfected cells with pcDNA or pGFP-Rac1-N17 were infected (MOI 1) for 16 h. Viral protein synthesis was analyzed by immunoblotting with an anti-p32 antibody. GFP and β-actin levels were measured as a control. G–H) Rac-1 inhibitor affects ASFV infection. G) Viral factory formation was analyzed in pretreated and infected cells (MOI 5) for 16 h. Cells were fixed and stained with Topro3, TRITC-phalloidin and anti-p72. Arrowheads: viral factories. Percentage of the infected cells is represented in left graphic (100 cells/condition; n = 3; mean ±S.D.). H) After 48 hpi (MOI 1) supernatants from treated cells were recovered and lytic viruses were titrated (n = 3). I) ASFV-induced ruffles are inhibited by Rac1 inhibitor. Cells were pretreated (200 µM Rac1 inhibitor), infected (MOI 50) for 10 min, fixed and analyzed by FESEM. S.D., standard deviations.