Intriguingly, higher molecular mass forms of the Epstein-Barr virus DNA polymerase processivity factor BMRF1 compatible with sumoylated products have been observed [73] , suggesting that such post-translational modification could be a general feature of the DNA polymerase accessory subunit in herpesviruses. In addition, sumoylation of Vaccinia Virus G8R protein has been recently predicted on the basis of structural similarities to PCNA [74] , but not yet experimentally demonstrated. Thus, our findings could stimulate further studies on sumoylation of DNA polymerase subunits in other herpesviruses or, more in general, in other viral systems. Figure S5 Sumoylation of UL44 in E. coli. The pRSET44 plasmid, encoding 6His-tagged UL44, was introduced into E. coli together with the pTE1E2S1 plasmid, which expresses E1 and E2 sumoylation enzymes and SUMO-1. As a control, bacteria were also transformed only with pRSET44. The 6His-tagged UL44 was purified from bacterial cultures expressing UL44 alone or in combination with the SUMO conjugation system and analyzed by western blotting with anti-UL44 and anti-SUMO-1 antibodies. (TIF) Figure S6 Mass spectrometry analysis of sumoylation sites of UL44. MSMS analysis of tryptic peptides conjugated to a tryptic peptide of SUMO-1 (ELGMEEEDVIEVYQEQTGG or IADNHTPKELGMEEEDVIEVYQEQTGG, 1 tryptic miscleavage) derived from E. coli-expressed, sumoylated UL44. Sequence of the conjugate is listed in each table above the spectra. Y-type and b-type fragment ions are assigned in the spectra. The table below each spectrum summarizes the database search. Highlighted are the m/z values that match the fragment ions obtained from in silico fragmentation of the conjugate. Conjugated lysine residues to SUMO-1 are highlighted in red and the position in UL44 is listed. Modifications, measured (observed) m/z values, the actual mass (in Da), the charge state, and the mass error (ppm, parts per million) are listed as well in the table above each spectrum. The different colors represent the various measured and calculated fragment ions of each conjugate in the spectrum and table underneath, respectively. The question marks in some of the spectra indicate fragment ions that do not match to any calculated y-and b-type ions of the conjugate. (PDF) Figure S7 Sumoylation in vitro of a GST-UL44 fusion is stimulated by DNA. E. coli-expressed, purified GST-tagged UL44 was incubated with purified sumoylation enzymes in the absence or presence of SUMO-1 and/or DNA. The samples were analyzed by western blotting with an anti-UL44 antibody. (TIF) Figure S8 UL44 is sumoylated in HCMV-infected U373 cells. (A) U373-Neo and U373-SUMO-1 cells were either mockinfected or infected with HCMV at MOI of 5 PFU/cell for 72 h. Cell lysates were then analyzed by western blotting with an anti-UL44 antibody. The arrowhead indicates the unmodified form of UL44 and the asterisks indicate the sumoylated UL44 forms. (B) Control U373-Neo cells, and U373-Neo cells transduced with lentiviral particles expressing either a Ubc9-silencing shRNA (U373-Neo shUbc9) or a non-silencing shRNA sequence (U373-Neo NS) were mock-infected or infected with HCMV at an MOI of 5 or 1 PFU/cell. At 72 h p.i., cells were fixed and stained with a primary antibody against UL44 and successively with a secondary fluorescein-conjugated antibody (green) which contained Evans Blue to counterstain cells (red). Cell samples were then analyzed by CLSM.