Here we report that the association of Ubc9 and UL44 leads to conjugation of SUMO molecules on multiple lysine residues. Both SUMO-1 and SUMO-2/3 were found to be conjugated to UL44. Sumoylation of UL44 was detected not only in vitro and in transiently transfected cells but, more importantly, also in HCMVinfected human cells during virus replication. Interestingly, we observed that binding of UL44 to DNA greatly stimulates SUMO conjugation to the protein both in vitro and in cells. In addition, we show that overexpression of SUMO-1 alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both viral DNA replication and virus production in an Ubc9-dependent manner. These data represent the first report of sumoylation of a viral processivity factor and show that there is a complex interplay between the HCMV UL44 protein and the cellular sumoylation system.