These results suggested that UL44 might possess multiple lysine residues that could alternatively serve as SUMO-1 acceptors, as reported for other proteins [57, 58] , and/or that UL44 might be sumoylated on lysine residues other than those predicted by SUMOplot. UL44 contains 31 lysines, most of which are solventexposed in the crystal structure [9] and therefore potentially accessible to SUMO molecules, which makes it difficult to identify the target lysines by mutational approaches. Therefore, we attempted to map the sites of UL44 where SUMO-1 is conjugated by mass spectrometry analysis. To this end, we expressed UL44 in an E. coli expression/modification system that produces SUMOconjugated proteins [30] . The 6His-tagged UL44 construct was co-expressed in E. coli with the pTE1E2S1 plasmid, which contains a linear fusion of genes for E1 and E2 enzymes and SUMO-1 under the control of an IPTG-inducible promoter [30] . To confirm sumoylation of UL44 with this system, UL44 was purified from the bacterial cultures expressing UL44 alone or in combination with the SUMO conjugation system and analyzed by western blotting with both the anti-UL44 (Fig. S5, left panel) and the anti-SUMO-1 (Fig. S5, right panel) antibody. Shifted bands with an apparent molecular mass of ,65, 80, and 95 kDa, similar to those detected in in vitro reactions ( Fig. 2A) and in cells (Fig. 3) , were observed in UL44 purified from bacteria cotransformed with pTE1E2S1, but not in UL44 purified from bacteria expressing only UL44 (Fig. S5) . Then, to identify lysine acceptor sites by mass spectrometry, sumoylated UL44 was separated by SDS-PAGE, in-gel-digested with trypsin and peptides were analyzed by LC-MSMS. Upon application of the software ''ChopNSpice'' for database search, we identified 16 sumoylation sites in UL44, including the predicted sites K172, K339, K371, K410, and K431. Table 2 summarizes the UL44 peptides that have been found to be sumoylated. The corresponding MS spectra are shown in Fig. S6 .