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

A) 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) 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. C) 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). D) 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.). E) 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. F) 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). G) 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.