We also examined the effects of SUMO-1 overexpression on virus yield. The titers of viral particles produced from nontransduced U373-SUMO-1 and from transduced U373-SUMO-1 shUbc9 and U373-SUMO-1 NS cells after infection with HCMV at an MOI of 1 were determined and compared to those produced from infected U373-Neo and U373-Neo shUbc9 control cells. A 2-3-fold increase in viral progeny titers was observed in U373-SUMO-1 and U373-SUMO-1 NS with respect to U373-Neo, while the U373-SUMO-1 shUbc9 cells exhibited yields of Ubc9, and anti-vinculin antibodies (left panel). Cell lysates were incubated with anti-FLAG-M2-Agarose beads and the immunoprecipitated samples were analyzed by western blotting with anti-UL44 and anti-Ubc9 antibodies (right panel). (E) The sumoylation in vitro of wild-type 6His-UL44 and mutant 6His-UL44Dloop and 6His-UL44L86A/L87A proteins was carried out as in (A) and analyzed by western blotting with an anti-UL44 antibody. (F) The sumoylation in vitro of a UL44 mutant bearing the K167R substitution in the flexible loop of UL44 involved in DNA binding was carried out in the presence of DNA and compared to that of wild-type UL44. For all panels, the arrowhead indicates the unmodified form of UL44 or free SUMO-1 and the asterisks indicate the sumoylated forms. doi:10.1371/journal.pone.0049630.g004 infectious virus similar to those of the U373-Neo and U373-Neo shUbc9 cells (Fig. 6D) .