The observation that UL44 interacts with the SUMO-conjugating enzyme Ubc9 prompted us to investigate the possibility that UL44 may be sumoylated. This hypothesis was first tested in a cellfree system by incubating a purified 6His-UL44 fusion protein with purified Aos1/Uba2 (E1), Ubc9 (E2), and SUMO-1. As a control, sumoylation of human p53 was also examined by the same assay. As expected [52, 53] , p53 was readily modified to give mainly a single mono-sumoylated product that reacted with both anti-p53 ( Fig. 2A , left bottom panel) and anti-SUMO-1 ( Fig. 2A , right bottom panel) antibodies (the bands appearing at high molecular weight, which are particularly visible in the anti-SUMO-1 panel, correspond to SUMO-E1 and -E2 enzymes conjugates). In a western blot analysis with an anti-UL44 antibody ( Fig. 2A , left top panel), three main slower migrating forms of UL44 were observed (lane 3). The appearance of the ,65, 80, and 95-kDa forms of UL44 was strictly dependent on the presence of SUMO-1, as the substitution of wild-type SUMO-1 with a mutant form which bears the Gly97-to-Ala change (SUMO-1 mut) and hence cannot be attached to target proteins, eliminated formation of these products ( Fig. 2A, left top panel, lane 4 ). In addition, SUMO-1 modification was abolished if either SUMO-1, Aos1/ Uba2, or Ubc9 was omitted from the reaction (not shown). A western blot analysis with an anti-SUMO-1 antibody confirmed that the slower migrating UL44 bands contained SUMO-1 ( Fig. 2A, right top panel) . Taken together, the above results established that UL44 is a substrate for in vitro SUMO-1 conjugation.