We next sought to identify the SUMO-1 acceptor sites of UL44. A prediction analysis with the SUMOplot program (Abgent) identified seven residues in UL44 with a certain probability to be sumoylated: K73, K172, K224, K339, K371, K410, and K431. To test whether one or more of these lysines could be a SUMO-1 acceptor site, each of the candidate residues was conservatively mutated to arginine, both individually and in combination, and the mutant proteins were tested for in vitro sumoylation. None of the single point mutants exhibited a consistently altered SUMO-1 modification pattern as compared to wild-type UL44 (Fig. S4A ). Mutants carrying two or three K/R substitutions also showed a SUMO-1 modification pattern identical to that of wild-type UL44 ( Fig. S4A and data not shown) . Similar results were obtained when in vitro sumoylation reactions with SUMO-2/23 were performed (data not shown). We decided to also test the ability of these K/R mutants to be modified by SUMO-1 in mammalian cells. Consistent with the in vitro data, none of the tested mutations significantly affected the ability of UL44 to undergo SUMO-1 conjugation ( Fig. S4B and data not shown) .