Phosphorylation Site Analysis of Semliki Forest Virus Nonstructural Protein 3

• Published in: The Journal of biological chemistry Vol. 275; no. 36; pp. 27775 - 27783
• Main Authors: Vihinen, H, Saarinen, J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 08.09.2000
• Subjects: Alkaline Phosphatase; Amino Acid Sequence; Cyanogen Bromide; Mass Spectrometry; Molecular Sequence Data; NS3 protein; Peptide Fragments - chemistry; Peptide Mapping; phosphatase; Phosphorylation; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Semliki Forest virus; Semliki forest virus - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002195200

Nonstructural protein 3 (Nsp3) is an essential subunit of the alphavirus RNA 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 mapped its major phosphorylation sites. Mass spectrometric methods utilized included precursor ion scanning, matrix-assisted laser desorption/ionization mass spectrometry used in conjunction with on-target alkaline phosphatase digestions, and tandem mass spectrometry. Two-dimensional peptide mapping was applied to separate tryptic 32 P-labeled phosphopeptides of Nsp3. Radiolabeled peptides were then subjected to Edman sequencing, and phosphoamino acid analysis. In addition, radiolabeled Nsp3 was cleaved successively with cyanogen bromide and trypsin, and microscale iron-chelate affinity chromatography was used to enrich phosphopeptides. By combining these methods, we showed 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 415 , which carries 7–12 phosphates distributed over its 13 potential phosphorylation sites. These analytical findings were corroborated by constructing a Nsp3 derivative devoid of phosphorylation. The results represent the first determination of phosphorylation sites of an alphavirus nonstructural protein, but the approach can be utilized in phosphoprotein analysis in general.