Fig 3. ASFV does not induce macropinocytosis.

A) Mock and ASFV-infected (MOI 100) macrophages were fixed and processed by transmission EM at 15, 30 and 60 mpi. Representative micrographs of Mock and ASFV-infected macrophages (left panels) and Vero cells (upper right panels) at 15 mpi are shown. ASFV particles are indicated by red arrowheads. Bars, 1 μm. Cells treated as before were quantified for the membrane protrusion density (number of protrusions > 200 nm per μm of plasma membrane from 50 cell profiles). Data display mean values and SD from a representative experiment. B) Macrophages (MAC) or Vero cells were infected (MOI 5; E70 or BA71V strains, respectively) for the indicated times and the phosphorylation levels of Pak1 (Thr423) were determined by immunoblotting. As a control of Pak activation, VACV-infected cells (MOI 5) were analyzed at 30 mpi. Levels of total Pak1 are shown below. C) Mock- and DiD-labeled ASFV-infected Vero cells (MOI 10) were incubated at 37°C for 15 min, fixed, stained for actin (green) and cell nucleus (Hoechst 33258, blue), and analyzed by wide-field fluorescence and DIC microscopy. As control, cells treated with 200 nM PMA, or infected with VACV (MOI 10) are shown. Bar, 5 μm. Incoming virus particles are shown in red. The yellow arrowheads indicate spike-like protrusions in PMA-treated cells while the white arroheads indicate blebbing in VACV-infected cells. Bar, 5 μm. In the bottom panel, the percentage of cells displaying blebbing in control cells (MOCK) or after infection with ASFV or VACV for 15 min is shown. Data indicate mean ± SD from two independent experiments. D) Mock- and ASFV-infected Vero cells (MOI 10) were pulsed for 30 and 60 min at 37°C with 10-kDa dextran-AF-488. As a control, cells treated with 200 nM PMA (30 and 60 min) or infected with VACV (60 min) were analyzed in parallel. Dextran uptake was quantified by flow cytometry and normalized to unstimulated cells (mean percentage ± SD of three independent experiments).