Figure 4

ICP0-deficient HSV fails to reactivate VP16 expression or viral DNA replication following high MOI infection. Confluent monolayers of HEL cells were mock infected or infected with 6 PFU/cell of KM110-R to establish quiescence. Four days later the cells were mock infected or superinfected with 30 PFU/cell of n212 or 10 PFU/cell of KOS. 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. Lane U2OS cells: samples extracted from U2OS cells 24 hours after infection with 10 PFU/cell KM110-R; Lanes KOS 1 hr and n212 1 hr: samples harvested from HEL cells one hour postinfection with KOS or n212 (MOIs of 10 and 30 respectively), documenting that the input virus does not interfere with detection of newly synthesized VP16 or viral DNA. wt: wild-type; mt: mutant