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.</p

Ruffles induction upon ASFV entry in Vero cells.

<p><b>A</b>) Field Emission SEM of mock-infected and infected cells. Cells were serum starved for 24 h and synchronously infected for 10, 60 and 90 min (MOI 50). A magnification of the cell surface detail (boxes) is shown in the lower right panels. Arrowheads indicate ruffles and arrows indicate bubble-like membrane perturbations. <b>B</b>) TEM of purified viral particle (arrows) localized into ruffles (arrowheads) in the cells after binding for 90 min at 4°C (MOI 3000). <b>C</b>) Ruffle induction upon ASFV binding to Vero cells. After being serum starved for 24 h, virus binding was allowed for 90 min at 4°C (MOI 100), and infected cells were recorded during 30 min after warming at 37°C with a 20× objective. Time stamps indicate min: sec.</p

EIPA treatment inhibits ASFV entry and infection in Vero cells.

<p><b>A–B</b>) EIPA inhibits ASFV uptake. Cells were pretreated (DMSO or EIPA) and infected (MOI 10) for 60 min. <b>A</b>) Infected cells were analyzed by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal. (n = 7, performed in duplicate; mean±S.D.) <b>B</b>) Cells were incubated with Topro3, TRITC-phalloidin and anti-p72 to stain nuclei (blue), actin filaments (red) and viral particles (green), respectively. Images were taken by CLSM and represented as a maximum z- projection of x–y plane (bottom panels) and x–z plane (upper panels). <b>C–E</b>) The infection is inhibited by EIPA. <b>C</b>) Pretreated cells (20 µM EIPA) were infected (MOI 1) for 16 h and analyzed by immunoblotting with an anti-p72 and an anti-ASFV polyclonal antibodies. <b>D</b>) Pretreated cells (60 µM EIPA) were infected (MOI 5) and stained with Topro3, TRITC-phalloidin and anti-p72. Images were taken by CLSM (mid z-section). Arrowheads: viral factories. <b>E</b>) Supernatants from pretreated (20 µM EIPA) and infected cells (MOI 1) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). <b>F</b>) Bypass of EIPA of ASFV infectivity. Acid mediated bypass was performed and samples of pretreated (20 µM EIPA) and infected cells (MOI 1) for 16 h were analyzed by immunoblotting with an anti-ASFV antibody. <b>G–H</b>) ASFV colocalizes with dextran and induces its uptake. <b>G</b>) Cells were pretreated (60 µM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with 647-dextran for 15 min and analyzed by FACS (n = 3; mean ±S.D.). <b>H</b>) After 30 mpi cells were pulsed with Texas-red-dextran for 15 min and incubated with anti-p72 antibody. Images were taken by CLSM (mid z-section) and Nomarsky. Arrowheads: dextran-virus colocalization. β-actin: load control. S.D., standard deviations. ^* Unspecific cellular protein detected by the antibody.</p

Blebs induction upon ASFV entry in IPAM cells.

<p><b>A</b>) Field Emission SEM of mock-infected and infected cells. Cells were synchronously infected for 10 min (MOI 50) with E70 after serum starved for 24 h. Membrane perturbations are indicated by arrowheads. A magnification of the cell surface detail (boxes) is shown in the lower right panels. Bars: 1 µM. <b>B</b>) After synchronic infection at different times (E70, MOI 50), the cells were fixed and blebs formation (arrowheads) was analyzed by Phase Contrast Microscopy using a 63× objective. <b>C</b>) Blebbistatin treatment inhibits ASFV entry. Cells were treated with DMSO or Blebbistatin 60 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection, at indicated concentrations. After 16 hpi (Ba71V, MOI 1) equivalent amounts of protein were analyzed by immunoblotting with an anti-ASFV antibody. β-actin was detected as a load control. Fold induction was determined by densitometry (mean ±S.D) as shown in the graphic below. <b>D</b>) Rock1 colocalizes with ASFV in blebs (arrows). Cells were infected (Ba71V, MOI 50) and fixed at 30 min after infection. Cells were incubated with, anti-Rock1 (red), anti-p72 (green) and Topro3 (blue) to stain blebs, virus and nuclei, respectively. Images were taken by CLSM and represented as a maximum z-projection of x–y plane and Normasky. Magnifications of the bleb containing Rock1 and viruses (boxes) are shown in the corresponding bottom panels. S.D., standard deviations.</p

Actin dynamics is important for first steps during ASFV entry in Vero cells.

<p><b>A–D</b>) Disruption of actin dynamics reduces the entry of ASFV. <b>A</b>) Uptake assays were performed by FACS. Pretreated cells with DMSO or 8 µM Cyto D were infected (MOI 10) for 60 min. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 3, performed in duplicate; mean ±S.D). <b>B</b>) Cells were pretreated (4 µM Cyto D) and infected (MOI 1) for 16 h. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. β-actin was detected as a load control. <b>C</b>) After 48 hpi (MOI 1) supernatants from treated cells (8 µM Cyto D) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). <b>D</b>) Development of viral factories (arrowheads) was analyzed by CLSM after treatment (8 µM Cyto D) and infected (MOI 5) for 16 h. Fixed cells were stained with Topro3 (blue), TRITC-phalloidin (red), and anti-p72 (green) to visualize cell nuclei, actin filaments and viral factories, respectively. Images of a mid z-section are shown. The percentage of infected cells of three independent experiments from CLSM images (100 cells per condition) is represented in graphic format (mean ±S.D.). <b>E–F</b>) ASFV infection induces rearrangements of the actin cytoskeleton. Cells were infected at a MOI of 50 pfu/cell (E) or transfected with pEGFP-actin for 16 h and then infected (MOI 50). For both, E and F, cells were fixed at indicated times post infection and incubated with Alexa Fluor 488-phalloidin (E), anti-p72 and Topro3 (E and F) to stain actin filaments, viral particles and cell nuclei, respectively. Z-slides images were taken by CLSM and represented as a maximum of z-projection. S.D., standard deviation; Cyto D, Cytochalasin D. ^* Unspecific cellular protein detected by the antibody.</p

ASFV entry is independent of clathrin-mediated endocytic pathway.

<p><b>A</b>) ASFV entry is partially inhibited by Dynasore but not by Chlorpromazine. Pretreated Vero cells with DMSO, 100 µM Dynasore (Dyn) and 20 µM Chlorpromazine (CPZ) were infected (MOI 10) for 60 min. Infected cells were analyzed by FACS and the graph shows the percentage of virus entry relative to DMSO control, measured as p72 signal. Bars represent the mean of three independent experiments (mean ±S.D., performed in duplicate). <b>B–C</b>) Dynamin and Clathrin are important for infection progress. Synthesis of viral proteins was measured in infected Vero cells (MOI 1) in the presence of Dyn (B) and CPZ (C) at 6 and/or 16 hpi at the indicated concentrations by Western blot with an anti-ASFV antibody. β-actin was detected as a load control. <b>D</b>) Viral production in the presence of Dynasore and Chlorpromazine. After 48 hpi (MOI 1) supernatants from DMSO, Dyn (100 µM) and CPZ (20 µM) treated cells were recovered. Lytic viruses were titrated in Vero monolayers and plotted in the table (n = 3). S.D., standard deviations; Dyn, Dynasore; CPZ, Chlorpromazine. ^*Unspecific cellular protein detected by the antibody.</p

Pak1 is required for ASFV entry in Vero cells.

<p><b>A</b>) 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>B–D</b>) IPA-3 inhibits ASFV entry. <b>B</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.). <b>C</b>) 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. <b>D</b>) 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). <b>E–F</b>) Pak1 mutant reduces ASFV infection. <b>E</b>) 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. <b>F</b>) 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.</p

Role of PI3K-Akt, EGFR and tyrosin kinases on ASFV entry and infection in Vero cells.

<p><b>A</b>) EGFR and tyrosin kinases are important for ASFV uptake. Virus entry was measured by FACS in cells pretreated with 324674 and Genistein after 60 mpi (MOI 10). Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 3, performed in duplicate; mean ±S.D.). <b>B</b>) PI3K is activated during ASFV entry. Pretreated cells (DMSO or 40 µM LY) were infected (MOI 10) and PI3K activity was measured by ELISA kit at indicated times post infection. <b>C</b>) ASFV infection induces Akt phosphorylation. Cells were infected (MOI 10) and phosphorylation level of Akt was analyzed by immunoblotting with a phospho-Akt Ser473 antibody. Total Akt levels were measured as a control and fold induction was determined by densitometry (mean±S.D). <b>D</b>) PI3K is required for ASFV entry. Cells were pretreated (60 µM LY) and infected (MOI 10) for 60 min to analyze ASFV uptake by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 4, performed in duplicate; mean ±S.D.). <b>E</b>) PI3K plays an important role in viral protein synthesis. Cells were pretreated (20 µM LY) and infected (MOI 1) for 3 and 16 h. Viral protein synthesis was analyzed with anti-p32 and anti-p72 polyclonal antibodies by immunoblotting. Phospho-Akt and β-actin were measured as controls. <b>F</b>) LY inhibits viral factory development. Infected cells (MOI 5) were pretreated (60 µM LY) and viral factories (stained with anti-p72 at 16 hpi) were analyzed by CLSM. 100 cells per condition were counted and data represented by the graphic (n = 3, mean ±S.D). <b>G</b>) Viral production in the presence of LY. Supernatants from treated cells (20 µM LY) were recovered after 48 hpi (MOI 1) and lytic viruses were titrated (n = 3, mean ±S.D). S.D., standard deviations; LY, LY294002.</p