Phosphorylation site analysis of Semliki Forest virus nonstructural protein 3

Nonstructural protein 3 (Nsp3) is an essential subunit of the alphavirus RN A replication complex, although its specific function(s) has yet to be well defined. Previously, it has been shown that Semliki Forest virus Nsp3 (482 amino acids) is a phosphoprotein, and, in the present study, we have mappe d its major phosphorylation sites. Mass spectrometric methods utilized incl uded precursor ion scanning, matrix-assisted laser desorption/ionization ma ss spectrometry used in conjunction with on-target alkaline phosphatase dig estions, and tandem mass spectrometry, Two-dimensional peptide mapping was applied to separate tryptic P-32-labeled phosphopeptides of Nsp3, Radiolabe led peptides were then subjected to Edman sequencing, and phosphoamino acid analysis. In addition, radiolabeled Nsp3 was cleaved successively with cya nogen bromide and trypsin, and microscale iron-chelate affinity chromatogra phy was used to enrich phosphopeptides. By combining these methods, we show ed that Nsp3 is phosphorylated on serine residues 320, 327, 332, 335, 356, 359, 362, and 367, and is heavily phosphorylated on peptide Gly(338)-Lys(41 5), which carries 7-12 phosphates distributed over its 13 potential phospho rylation sites. These analytical findings were corroborated by constructing a Nsp3 derivative devoid of phosphorylation, The results represent the fir st determination of phosphorylation sites of an alphavirus nonstructural pr otein, but the approach can be utilized in phosphoprotein analysis in gener al.