FIG. 2.

Arginines 138, 148, and 150 within the ICP27 RGG box are methylated in vivo. (A) ICP27 was immunopurified from nuclear (Nuc) and cytoplasmic (Cyt) fractions of wild-type KOS-infected HeLa cell extracts. The immunoprecipitates were resolved by SDS-PAGE and visualized by Coomassie blue staining. Plus signs indicate ICP27 protein, and asterisks denote heavy-chain IgG. M, molecular mass. (B) ICP27 was cut from the Coomassie-stained gels and in-gel digested. The resulting peptides were analyzed for protein methylation by liquid chromatography-MALDI-TOF-TOF MS. WT, wild type; monoisotopic mass, the observed monoisotopic mass of the peptide; Mass Diff, the difference between the theoretical mass and the monoisotopic mass; Mod Diff, the theoretical mass of a particular posttranslational modification; MA (ppm), mass accuracy, which is Mass Diff divided by the theoretical mass in parts per million; modification, the number of methyl groups on each arginine residue at a particular position in the peptide; #MC, the number of cleavage sites that were not cleaved by the enzyme. (C) Schematic diagram of the ICP27 coding region, illustrating structural motifs, including the leucine-rich amino terminus (LRR), nuclear localization signal (NLS), RGG box RNA-binding motif (RGG), three predicted KH domains, and a zinc finger-like motif (CCHC). Specific amino acid positions of methylated arginine residues are shown within the RGG box motif.