FIG. 4.

Quantification of B5R labeling over budding profiles, extracellular particles, and the plasma membrane; the production of the different viral forms in HeLa cells and CEFs; and the quantification of budding and fusion events. (A) HeLa cells were infected with WR, IHD-J, and MVA, and the cells were fixed, labeled with anti-B5R, and embedded as described for Fig. 3. The gold particles over extracellular particles (“free” particles), the cell surface around budding particles, and random pieces of plasma membrane were counted for 30 randomly chosen electron micrographs showing budding profiles and extracellular particles. The values are expressed as number of gold particles per micron of membrane and represent the average and standard deviations. (B) HeLa cells or CEFs were infected at an MOI of 10 and fixed at 16 h postinfection. The total amount of IVs (white bars), IMVs (dense particles in HeLa cells infected with MVA; black bars), IEVs or TGN wrapping profiles (dark gray bars), and CEVs (dense extracellular particles near HeLa cells infected with MVA; light gray bars) were counted in 50 profiles of infected cells and are expressed as a percentage of the total particles counted. (C) HeLa cells were infected and embedded as described for panel A with MVA (black bars), WR (gray bars), and IHD-J (white bars). Fusion and budding profiles, defined asdescribed in the text, at the plasma membrane were counted in 30 randomly chosen electron micrographs and are expressed as events per micron of plasma membrane. Note that the standard deviations are high. This reflects the fact (as described in Results) that in some cells budding is very abundant, whereas in other cells no budding occurs at all. Also note that fusion is barely detectable upon IHD-J infection, which probably reflects the way fusion was defined (see the text).