FIG. 5.

Consecutive stages of replication-independent and -dependent histone H3 deposition on the CMV genome. (A) MRC-5 cells were infected with CMV for 0.5 to 72 h, relative amounts of viral and cellular DNAs were determined at the UL32-T and GAPDH loci by quantitative PCR, and the results were normalized to GAPDH at 0.5 h (set to 1). The data present the mean amounts of DNA from three independent experiments with standard deviations. (B) MRC-5 cells were pretreated with PAA (200 μg/ml) or aphidicolin (2 μM) for 1 h or left untreated prior to infection with CMV. Infection continued for 2 h in the presence of the inhibitors. ChIP was performed using an antibody against the C-terminal domain of histone H3, and the amounts of input and coprecipitated DNA were determined by quantitative PCR with eight specific primer pairs, as indicated. PCR results from two independent experiments, each quantified in duplicate, are presented as mean output-to-input DNA fractions normalized to the output-to-input ratio of GAPDH without drug treatment (set to 1). The error bars indicate standard deviations. (C) MRC-5 cells were infected with CMV and either left untreated or treated with PAA (200 μg/ml) immediately following infection. After 24 h, the culture medium was changed, and fresh drug was added where applicable. Samples were collected at 2 and 48 h postinfection. Relative amounts of viral (means of all seven viral indicator loci) and cellular (GAPDH) DNAs were determined by quantitative PCR and normalized to GAPDH at 2 h postinfection (set to 1). The bars represent mean values with standard deviations from three experiments. (D) The samples from panel C were subjected to ChIP as described for panel B. PCR results from two independent experiments, each quantified in duplicate, are presented as mean output-to-input DNA fractions normalized to the output-to-input ratio of GAPDH at 2 h (set to 1). The error bars indicate standard deviations.