Alphavirus infection induces actin-positive intercellular extensions. (A) Vero cells were mock-transfected (Uninf.) or transfected with WT SINV or Y400K RNA, incubated at 37°C for 8 h, and fixed. Cells were permeabilized and stained with antibodies to detect the viral E2 envelope protein (red) and α-tubulin (blue), and with phalloidin to detect F-actin (green). Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments, which are quantitated in (B). Arrows indicate two examples of intercellular extensions; note that each is positive for all three markers and is in contact with a neighboring cell. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2, actin and tubulin and their contact with a neighboring cell (see methods ). Graph in B shows the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. ** P
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