To analyze the effects of SUMO-1 overexpression on virus production, virus yield assays were performed as described previously [47] , with some modifications. Briefly, U373-Neo and U373-SUMO-1 cells transduced with either shUbc9 or nonsilencing lentiviral particles (see below) or non-transduced, 5 610 4 cells per well were seeded in 24-well plates, incubated overnight, and infected with HCMV AD169 at an MOI of 1. At 120 h p.i., cells were subjected to one cycle of freezing and thawing, and titers were determined by transferring 100-ml aliquots from each of the wells to a fresh 96-well monolayer culture of HFF cells followed by 1:5 serial dilution across the plate. After incubation at 37uC for 7 days, cell monolayers were stained with crystal violet and plaques were counted.