FIG 4.

H4 inhibits plaque formation, reduces virus yield, and blocks early VACV infection without impacting particle infectivity. (A) HeLa cells were infected with 150 PFU of WT VACV, and infection was allowed to proceed for 72 h. The plates were stained with crystal violet to visualize plaques. (B) HeLa cells were infected with WT VACV (MOI of 1). Twenty-four hours postinfection, cells were harvested and lysed and virus yield was determined by titration and plaque formation. (C) HeLa cells were infected with WT virus (MOI of 1), and 80 nM H4 was added at the indicated time points. A sample subjected to AraC addition at 6 hpi was included as a positive control for inhibition. Cells were harvested 24 hpi, and virus yield was determined for each sample by serial dilution plaque assay. (D) HeLa cells were infected with WT virus (MOI of 1) in the presence of 80 nM H4. At the indicated time points, cells were washed, and infection was allowed to proceed. A sample subjected to AraC washout at 6 hpi was included as a positive control for inhibition. At 24 hpi, virus yield was determined for each sample by serial dilution plaque assay. (E) WR E EGFP (black bars) or WR L EGFP (white bars) virions were preincubated with 20 or 80 nM H4 for 30 min at room temperature. Virus particles were washed three times and used to infect HeLa cells. Samples were analyzed by flow cytometry for infected EGFP-positive cells at 6 hpi (early) and 8 hpi (late). a.u., absorbance units. (F) WR E/L EGFP virions were preincubated with 2 μM H4 for 2, 3, or 4 h. Virions were washed and used to infect HeLa cells prior to fixation and analysis by Plaque 2.0 for total nuclei and EGFP-positive infected cells. (A to F) All experiments were performed in triplicate; representative images are shown (A), or results are displayed as means ± SD (B to F).