E2F activation is detrimental for MCMV replication in human cells.

(A) Multistep growth kinetic in human RPE-1 cells. Cells were infected with MCMV-M117-FL and the recombinant viruses at an MOI of 0.2 TCID50/cell. Supernatant of infected cells were harvested at the indicated time point and titrated. The experiments were done in triplicate. Means ±SEM are shown. (B) Multistep growth kinetic in human embryonic lung fibroblasts (MRC-5). Cells were infected with MCMV-M117-FL and the recombinant viruses at an MOI of 1 TCID50/cell. Supernatants of infected cells were harvested at the indicated time points and titrated with centrifugal enhancement. The experiments were done in triplicate. Means ±SEM are shown. (C) RPE-1 cells were infected with WT MCMV or MCMV-M117-HA. Cell lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Co-precipitating proteins were detected by Western blot analysis. (D) RPE-1 cells were synchronized by serum starvation for 48 hours, infected at an MOI of 3 TCID50/cell (without serum) and harvested at 24 hpi for RNA extraction and qRT-PCR with primers specific for the indicated genes. Means ±SEM of 3 independent experiments are shown. Transcripts were quantified using the ΔΔCt method and normalized to a housekeeping gene (Gapdh). (E) RPE-1 cells were infected at an MOI of 0.5 TCID50/cell with MCMV-GFP. Three hpi, the medium was removed and the E2F inhibitor HLM006474 was added. At 3 hpi (Input) or after 3 days, the cell lysates were harvested for DNA extraction and viral DNA quantification by qPCR. MCMV genome copies were normalized to β-actin copies. Means ±SEM are shown. (F) RPE-1 cells were infected at an MOI of 2 TCID50/cell for 24h. Cell lysates were harvested 24 hpi and subjected to SDS-PAGE. (G) Multistep growth kinetic in human RPE-1 cells. Cells were infected with WT MCMV, MCMV-ΔM117 and MCMV-ΔM117-E4orf6/7 at an MOI of 0.2 TCID50/cell. Supernatants of infected cells were harvested at the indicated time points and titrated. The experiments were done in triplicate. Means ±SEM are shown. DL, detection limit; ns, not significant; *, p<0.05; **, p< 0.01; ***, p<0.001.