For most infections, subconfluent MRC-5 cells in serumcontaining medium were inoculated with recombinant HCMV virus at an input genome number equivalent to that of 3-5 infectious units of wild type virus/cell for 1 hour, unless otherwise indicated. Inoculum was removed and fresh medium was replenished. For infection of G0-synchronized cells, MRC-5 cells were incubated in serum-free medium for 72 hours, infected as described above, and maintained in serum-free media throughout the infection. For shRNA knockdown experiments, subconfluent MRC-5 cells were transduced with lentivirus for 24 hours, incubated in fresh medium for additional 48 hours, and infected as described above. When necessary, PAA (100 mg/ml) was added immediately following infection, and MG132 (10 mM) was added 12-14 hours prior to harvest. For viral growth analysis, virus production in the media of infected cultures was determined by TCID 50 , plaque assay, or qPCR. For qPCR analysis, virion DNA was prepared as previously described [29] . Briefly, cell-free supernatants were treated with DNase I to remove contaminating DNA, and virions were lysed with proteinase K and SDS. DNA was extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The DNA was subjected to qPCR using primers and a taqman probe specific for UL54.