FIG. 5.
Single-cycle growth curves and viral DNA replication of origin mutant viruses in vitro. Replicate monolayers of Vero cells or PRN were infected with a calculated multiplicity of 2.5 PFU/cell of WT, DoriL-ILR, DoriL-ILR-R, DoriS-I, and DoriS-I-R. Infected cells were incubated at 37°C and harvested at the indicated times. (A) Infectious virus was quantified by a standard plaque assay on Vero cell monolayers. The titers shown are the means ± SD (error bars) of three independent experiments. (B) Total cellular DNA was extracted, slot blotted on a nylon membrane, and hybridized to a cocktail of 32P-labeled HSV-1 DNA fragments as probes. Hybridized counts were quantitated by PhosphorImager analysis, and the genome copy number per cell was determined by comparing the experimental values to a standard curve of known quantities of purified viral DNA and dividing by the number of cells infected. The results of one of two independent assays are shown.