FIG. 1.

Entry of WR and MVA in HeLa cells (A) and relationship between MOI, intracellular cores, and DNA replication sites (B). (A) HeLa cells grown on coverslips were infected at an MOI of 30 in the presence of 300 μg of cycloheximide per ml with sucrose-purified WR (WR s) or MVA (MVA s), the upper band obtained from MVA purified on Optiprep gradients (MVA OpU) (enriched for EEV), or the lower band of the same gradient (MVA OpL) (containing IMV only). Cells were fixed at the indicated times postinfection and labeled with an anticore antibody followed by anti-rabbit-FITC to visualize intracellular cores. The graph represents the average amounts of cores per cell in 30 cells and the standard errors of the means. (B) HeLa cells grown on coverslips were infected with sucrose-purified WR or MVA at the indicated MOI. After 60 min at 37°C, one set of cells was fixed and labeled with the anticore antibody. A parallel set of cells was washed three times to remove unpenetrated virus and incubated for an additional 3 h before fixation. Intracellular cores and replication sites were visualized by IF (see Materials and Methods) and counted. The values represent the average amounts of cores per cell and standard errors of the means at 60 min postinfection with WR (WR c) or MVA (MVA c) or the average amounts of viral factories (vf) per cell and standard errors of the means at 4 h postinfection (WR vf or MVA vf) in 30 cells. (C) Negative-staining EM and immunolabeling with anti-p42 (B5R; 10-nm-diameter gold) of sucrose-purified WR (panel A) and MVA (panel B) isolated from infected HeLa or BHK cells, respectively. Bars, 300 μm.