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

<p><b>A–B</b>) Activation of Rac1 during ASFV entry. Vero cells were infected (MOI 10) and 0.Rac1 activation was measured by <b>A</b>) Kit Activation Assay (n = 3; mean ±S.D.) and <b>B</b>) Pak1 PBD-Agarose Beads pull down assay. Fold induction was determined by densitometry (mean ±S.D). <b>C</b>) 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. <b>D–E</b>) Rac1 inhibitor blocks viral entry. Pretreated cells (200 µM Rac1 inhibitor) were infected (MOI 10) for 60 min. <b>D</b>) 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.). <b>E</b>) 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). <b>F</b>) 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. <b>G–H</b>) Rac-1 inhibitor affects ASFV infection. <b>G</b>) 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.). <b>H</b>) After 48 hpi (MOI 1) supernatants from treated cells were recovered and lytic viruses were titrated (n = 3). <b>I</b>) 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.