ICP0 is required for reactivation of VP16 gene expression and viral DNA replication. Confluent monolayers of HEL cells were infected with 6 PFU/cell of KM110-R to establish quiescence. Four days later the cells were mock infected or superinfected with the indicated HSV strains (MOI of 10). Samples harvested 18 hours later were then analyzed for VP16 expression by Western Blot (panel A) or viral DNA replication by Southern blot (panel B). (A) Samples were scored for VP16 and cellular β-actin by Western blot. (B) Total cellular DNA was cleaved with Bam HI and Nhe I, then analyzed by Southern blot hybridization using an HSV-1 VP16 probe. U2OS cells: samples extracted from permissive U2OS 24 hours after infection infected with KM110-R (MOI of 10). wt: wild-type VP16 protein or gene; mt: mutant VP16 protein or gene.