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.
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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D016384:Consensus Sequence; D006224:Cricetinae; D016174:Hepacivirus / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*
D010446:Peptide Fragments; D010455:Peptides; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase