Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: Properties of the NS5A-associated kinase
Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: Properties of the NS5A-associated kinase
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DOI：
10.1128/jvi.71.10.7187-7197.1997
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发表时间：
1997-10-01
影响因子：
5.4
通讯作者：
Rice, CM
中科院分区：
文献类型：
--
作者：
Reed, KE;Xu, J;Rice, CM
源自丙型肝炎病毒 (HCV) 基因型 Ib 分离株的 NS5A 先前已被证明在丝氨酸残基上发生磷酸化 (T, Kaneko, Y, Tanji, S. Satoh, M. Hijikata, S, Asabe, K, Kimura, and K, Shimotohno, Biochem, Biophys, Res, Commun, 205:320-326, 1994)。证明了 NS5A 源自 la 和远缘相关 2a 基因型的 HCV 分离株。对来自 la 分离株的 NS5A 进行磷酸化氨基酸分析表明，磷酸化主要发生在丝氨酸上，小部分苏氨酸残基也被磷酸化，在多种细胞类型中观察到 NS5A 磷酸化，包括 COS-1、BHK-21、HeLa 和肝癌细胞系 HuH-7。体外激酶测定，当反应的 pH 为中性或微碱性时，这种活性似乎最高，并且显示出对 Mn2+ 的偏好超过 Mg2+，最佳浓度约为 10 mM Mn2+。有些令人惊讶的是，在含有 Mn2+ 和/或 Mg2+ 的反应缓冲液中添加大于或等于 0.25 mM Ca2+ 可以抑制 NS5A 的体外磷酸化，NS5A 磷酸肽图谱的比较体外和培养细胞中的磷酸化表明大多数磷酸肽发生共迁移，表明体内和体外参与 NS5A 磷酸化的一种或多种激酶是相同的。各种激酶抑制剂对 NS5A 磷酸化的影响与属于丝氨酸-苏氨酸激酶 CMGC 组的激酶活性一致。NS5A 磷酸化的体外激酶测定的发展应有助于鉴定负责其磷酸化的激酶和可能影响 NS5A 在 HCV 传播中的功能的磷酸化位点。
NS5A derived from a hepatitis C virus (HCV) genotype Ib isolate has previously been shown to undergo phosphorylation on serine residues (T, Kaneko, Y, Tanji, S. Satoh, M. Hijikata, S, Asabe, K, Kimura, and K, Shimotohno, Biochem, Biophys, Res, Commun, 205:320-326, 1994), In this report, phosphorylation of NS5A derived from HCV isolates of the la and distantly related 2a genotypes is demonstrated. Phosphoamino acid analysis of NS5A from the la isolate indicated that phosphorylation occurs predominantly on serine, with a minor fraction of threonine residues also being phosphorylated, NS5A phosphorylation was observed in diverse cell types, including COS-1, BHK-21, HeLa, and the hepatoma cell line HuH-7, Phosphorylation of a glutathione S-transferase (GST)/HCV-H NS5A fusion protein was also demonstrated in an in vitro kinase assay, This activity seemed to be highest when the pH of the reaction was neutral or slightly alkaline and displayed a preference for Mn2+ over Mg2+, with an optimum concentration of approximately 10 mM Mn2+, Somewhat surprisingly, in vitro phosphorylation of NS5A was inhibited by the addition of greater than or equal to 0.25 mM Ca2+ to reaction buffer containing Mn2+ and/or Mg2+, Comparison of phosphopeptide maps of NS5A phosphorylated in vitro and in cultured cells showed that most of the phosphopeptides comigrated, suggesting that one or more kinases involved in NS5A phosphorylation in vivo and in vitro are the same, The effects of various kinase inhibitors on NS5A phosphorylation were consistent with a kinase activity belonging to the CMGC group of serine-threonine kinases, The development of an in vitro kinase assay for NS5A phosphorylation should facilitate identification of kinase(s) responsible for its phosphorylation and of phosphorylation sites which may influence the function of NS5A in HCV propagation.