丙型肝炎病毒非结构蛋白 5A 的连续 S232/S235/S238 磷酸化。
Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A.
机构信息
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
出版信息
J Virol. 2018 Sep 26;92(20). doi: 10.1128/JVI.01295-18. Print 2018 Oct 15.
The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A. The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.
丙型肝炎病毒 (HCV) 蛋白 NS5A 是一种磷酸化蛋白，在病毒复制和组装中起着关键作用。人们认为 NS5A 在一系列保守丝氨酸残基上依次发生磷酸化；然而，磷酸化级联仍然不清楚。使用三种磷酸化特异性抗体，我们发现 HCV 感染的 Huh7.5.1 细胞中 S232、S235 和 S238 的磷酸化是平行发生的，提示由酪蛋白激酶 Iα (CKIα) 从 S232 到 S235 再到 S238 对 NS5A 进行分子内顺序磷酸化。与此一致的是，S225、S229 或 S232 的丙氨酸突变降低了 NS5A 在 S232、S235 和 S238 的磷酸化，而相同位点的天冬氨酸突变则挽救了 NS5A 的磷酸化。相比之下，S235 或 S238 的丙氨酸或天冬氨酸突变对 S232 或 S235 的磷酸化几乎没有影响。与分子内顺序磷酸化级联一致，单个 NS5A 分子上共存 S232、S235 和 S238 的磷酸化。一个 NS5A 分子中 NH 末端丝氨酸残基的磷酸化不能挽救另一个 NS5A 分子中 COOH 末端丝氨酸残基的磷酸化。CKIα 抑制降低了 NS5A 在 S232、S235 和 S238 的磷酸化。总之，我们的结果表明，丙型肝炎病毒 NS5A 蛋白的 CKIα 介导的分子内顺序磷酸化级联从 S232 到 S235 再到 S238。S225 和 S229 也对 NS5A 的上述顺序磷酸化级联有很大贡献。丙型肝炎病毒的非结构蛋白 5A (NS5A) 被认为在一系列丝氨酸残基上依次发生分子内磷酸化；然而，直接证据缺失。我们提供了第一个关于 CKIα 介导的 NS5A 从丝氨酸 232 到 235 再到 238 的分子内顺序磷酸化级联的直接证据。这种顺序磷酸化级联发生在无序的低复杂度 I 区，该区域与 I 区结构域一起形成 NS5A 二聚体中的 RNA 结合槽。无序区的顺序磷酸化增加了 RNA 结合槽中的电荷排斥，并可能因此调节 NS5A 的 RNA 结合能力和在病毒 RNA 复制和组装中的功能。
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