Fig. 5. M97 causes shutoff of host DNA synthesis by Cyclin A sequestration.

Serum-starved 3T3 cells were infected with the indicated recombinant viruses and subjected to subcellular fractionation (a), cell cycle analysis (b, c), or confocal microscopy (d). a The levels of Cyclin A, E, and Cdk2 proteins was determined at 24 h post infection in nuclear and cytosolic fractions by immunoblotting. The soluble viral nuclear protein IE1 and the cytosolic marker GAPDH served as controls. The immunoblots are representative of three independent experiments with similar results. b, c The DNA content of infected cells was analyzed by propidium iodide staining followed by flow cytometry and plotted as DNA histograms. b The accumulation of viral and cellular DNA was monitored over the time course of infection. c To discriminate viral from cellular DNA replication, infected cells were treated with ganciclovir (GCV) or left untreated. d, e At 30 hpi, infected cells were pulse-labeled with EdU. EdU staining via click-chemistry (green fluorescence) served to determine sites of cellular and viral DNA synthesis. EdU was combined with immunofluorescence detection of the viral replication factor M57 (red fluorescence) that marks sites of viral DNA synthesis. DAPI was used for nuclear counterstaining (blue fluorescence). Scale bars: 10 μm. e The indicated number of cells were categorized based on the (co-)localization of EdU and M57 fluorescence within the nucleus. f Mouse embryonic fibroblasts were infected with the indicated viruses at an MOI of 0.02. At the indicated days post infection, the infectious supernatant was harvested and subjected to virus titration. Means (center of the error bars) and standard errors of the mean of n = 3 (3, 5, 7 days post infection) biological replicates are depicted. For 0 days post infection: n = 2. Two-sided t-tests without multiple hypothesis correction were performed comparing the indicated viruses at 5 days post infection.