Reed K E, Rice C M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
J Biol Chem. 1999 Sep 24;274(39):28011-8. doi: 10.1074/jbc.274.39.28011.
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.
丙型肝炎病毒（HCV）NS5A蛋白由一种细胞丝氨酸/苏氨酸激酶磷酸化。为了确定NS5A磷酸化的主要位点，对放射性标记的HCV-H NS5A磷酸肽进行了纯化，并进行了磷酸氨基酸分析和埃德曼降解。这些数据确定HCV-H NS5A蛋白中的主要细胞内磷酸化位点为Ser(2321)，这一结果通过另外两种独立方法得到验证：（i）将Ser(2321)替换为Ala后，主要的体内磷酸化肽和相应的体外磷酸化肽随之消失；（ii）Ser(2321)磷酸化的合成肽的消化产物与主要的体内磷酸化NS5A肽共迁移。Ser(2321)的定点诱变表明，NS5A的磷酸化对于先前描述的与NS4A和PKR（一种细胞抗病毒激酶，似乎不催化NS5A磷酸化）的相互作用并非必需。Ser(2321)侧翼氨基酸序列（PLPPPRS(2321)PPVPPPR）富含脯氨酸的性质表明，脯氨酸定向激酶负责大部分HCV NS5A的磷酸化，这与先前的激酶抑制剂研究一致。
