Fig 2.

Nuclear entry of viral genomes is unaffected by cyclin A2. Cyclin A2-overexpressing cells and the indicated control U373 cells were grown to confluence and infected with BrdU-labeled HCMV. (A) At 3 h postinfection, cells were fixed and analyzed for IE1/IE2 protein expression and subcellular localization of viral genomes by confocal immunofluorescence microscopy. DAPI was used as a nuclear counterstain. (B and C) In a parallel approach, cells were harvested at 3 h postinfection and fractionated into nuclei (nu) and cytoplasm (cy) by hypotonic lysis. (B) The purity of fractions was confirmed and compared to that of whole-cell lysates (wh) by immunoblot analysis of β-tubulin (cytoplasmic marker) and lamin A/C (nuclear marker). In addition, the nucleocytoplasmic distribution of exogenous cyclin A2 was controlled using HA and cyclin A2 antibodies. (C) Total DNA isolated from whole cells and from purified nuclei was analyzed for the relative amount of viral DNA by quantitative real-time PCR using UL112/113-specific primers. The ratios of nuclear and whole-cell viral DNA contents are given as mean values of two independent experiments.