Ser2194 is a highly conserved major phosphorylation site of the hepatitis c virus nonstructural protein NS5A

Abstract

Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193-2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatographymass spectrometry. The elution time of the phosphopeptide determined by negative-ion electrospray ionization-mass spectrometry corresponded with the elution time of the majority of 32P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionizationtandem mass spectrometry revealed that Ser2194 was the major phosphorylated residue on the phosphopeptide Gp-SPPSLASSSASQLSAPSLK. Substitution for Ser2194 with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser2194 and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser2194 may be an important mechanism for modulating NS5A biological functions. © 2000 Academic Press.