Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321.

The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser(2321), a result verified by two additional, independent methods: (i) substitution of Ala for Ser(2321) and the concomitant disappearance of the major in vivo phosphorylated peptides and corresponding in vitro phosphorylated peptides; and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser(2321) with the major in vivo phosphorylated NS5A peptides. Site-directed mutagenesis of Ser(2321) suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser(2321) (PLPPPRS(2321) PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.