Fig. 3.

Effects of PAs and GAGs on VACV-induced actin tail formation and EEV entry. (A) PtK₂ cells were infected with VACV strain WR at 5 pfu per cell for 14 h. HP or DS (50 μg/ml) was added to the medium during or after the incubation. Actin and B5 were labeled with TRITC-phalloidin and rat mAb 19C2, respectively, and the samples were processed for immunofluorescent microscopy as described (20). In the presence of HP and DS during incubation, fewer cells (reduced from 55% to 15%, n = 100) were found to make actin tails. Bar chart shows the mean number of actin tails found in cells that made actin tails. Error bar = standard error. (B) Disruption of CEV/EEV membrane by HP. (Upper) Shown are the virions on the surface of VACV-infected RK₁₃ cells −/+ HP (200 μg/ml). Note intact CEV with fully wrapped double envelopes −HP (Upper Left and Inset) and loss of outer membrane +HP (Upper Right and Inset). (Lower) Shown is isolated EEV treated with or without HP then concentrated by centrifugation. (Scale bar, 100 nm.) (C) EM study of VACV EEV entry into L cells and sog9 cells that lack heparan sulfate and CS. Fresh EEV of VACV strain WR was spinoculated onto cells at 4°C and then incubated at 37°C for 10 min. The EEV surface was labeled by rat anti-B5 mAb as in Fig. 1. In two independent experiments, 100 virions were identified, and bound intact EEV, ruptured EEV, and IMV were scored. No rupture of the EEV membrane was seen on sog9 cells.