Ser(2194) is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A

Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of p hosphorylation of NS5A have not been determined, and the functional signifi cance of phosphorylation remains unknown. Here, we showed by two-dimensiona l phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype Ib NS5A from insect cells on identical serine residues. We identified a maj or phosphopeptide (corresponding to amino acids 2193-2212 of the HCV Ib pol yprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatography-mass spectrometry, The elution time of t he phosphopeptide determined by negative-ion electrospray ionization-mass s pectrometry corresponded with the elution time of the majority of P-32-labe l that was incorporated into the phosphopeptide by an in vitro kinase react ion. Subsequent analysis of the peak fraction by automated positive-ion ele ctrospray ionization-tandem mass spectrometry revealed that Ser(2194) was t he major phosphorylated residue on the phosphopeptide Gp-SPPSLASSSASQLSAPSL K. Substitution for Ser(2194) with Ala resulted in the concomitant disappea rance of major in vivo phosphorylated peptides. Ser(2194) and surrounding a mino acids are highly conserved in all HCV genotypes, suggesting NS5A phosp horylation at Ser(2194) may be an important mechanism for modulating NS5A b iological functions. (C) 2000 Academic Press.