FIG 9.

Protein pE199L is necessary for viral membrane fusion. (A to D) Vero cells were infected with equivalent amounts (100 PFU/cell) of control vE199Li⁺ and defective vE199Li⁻ purified virus particles. At 3 hpi, the infected cells were fixed, embedded in resin, and examined by EM. Note that defective vE199Li⁻ particles undergo normal endocytosis through clathrin-coated pits (CCP) and clathrin-coated vesicles (CCV) (A) and transit to late multivesicular bodies (MVB) (B) to accumulate eventually in lysosome-like structures (Lys) (C). Results of endosomal virus disassembly, which involves the loss of the outer envelope (oe, depicted in purple) and the outer capsid (ca, green), were similar for control vE199Li⁺ and defective vE199Li⁻ particles. However, the delivery of viral cores (brown) into the cytosol after inner envelope (ie; red) fusion was severely impaired for defective vE199Li⁻ particles, in contrast to that observed for vE199Li⁺ particles (D). Bars, 200 nm. (E) Quantification of virus disassembly. The endocytosed (ENDO) control vE199Li⁺ and defective vE199Li⁻ particles (n > 100 per condition) were classified according to their layer content (ie, inner envelope; ca, capsid; oe, outer envelope). As a reference, the layer content of membrane-bound extracellular particles (EXTRA) was also estimated. Data are expressed as proportions (means ± deviations of results from triplicate experiments) of each virus layer. (F) Quantification of core penetration. The intracellular virus particles (n > 500 for each condition) detected within endocytic vesicles (ENDO) or as cytosolic cores (CYTOSOL) were quantified in triplicate samples for both sets of conditions. Data are expressed as proportions (means ± deviations of results from triplicate experiments) of each virus category relative to the total number of intracellular particles.