Hsu Shih-Chin, Lo Chieh-Wen, Pan Ting-Chun, Lee Kuan-Ying, Yu Ming-Jiun
Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan.
Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
J Virol. 2017 Jun 26;91(14). doi: 10.1128/JVI.00194-17. Print 2017 Jul 15.
The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase Iα, which requires a priming phosphorylation at S235. It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.
丙型肝炎病毒（HCV）的非结构蛋白5A（NS5A）是一种具有两种磷酸化状态的磷蛋白：低磷酸化和高磷酸化。基因突变研究表明，存在一组负责NS5A高磷酸化以及在病毒复制和组装中发挥作用的丝氨酸残基；然而，这些丝氨酸残基之间的磷酸化水平和潜在相互作用尚不清楚。我们使用三种特异性抗体来检测在我们之前的蛋白质组学研究中鉴定出的S222、S235和S238位点的NS5A磷酸化情况。在HCV（J6/JFH-1）感染的Huh7.5.1细胞中，几乎检测不到S222磷酸化，而S235磷酸化和S238磷酸化在时间和细胞内空间上总是同时被检测到。S235A突变消除了S238磷酸化，而S238A突变不影响S235磷酸化，这表明S235磷酸化独立于S238磷酸化发生，而S238磷酸化依赖于S235磷酸化。与此一致的是，免疫沉淀结合免疫印迹显示，S235磷酸化单独存在而没有S238磷酸化，而S238磷酸化仅在同一NS5A分子上的S235被磷酸化时才存在。S235磷酸化的NS5A构成了主要的高磷酸化NS5A物种。S235A突变使病毒复制减弱，而S238A突变不影响复制。我们得出结论，S235是HCV复制所需的主要NS5A高磷酸化位点。S238可能由酪蛋白激酶Iα磷酸化，而这需要在S235处进行起始磷酸化。多年来已知丙型肝炎病毒非结构蛋白5A（NS5A）在两种磷酸化状态之间转变：低磷酸化和高磷酸化。还已知一组丝氨酸残基负责NS5A高磷酸化及其功能；然而，负责NS5A高磷酸化的主要丝氨酸残基尚不清楚。在此，我们首次表明丝氨酸235磷酸化的NS5A构成了病毒复制所需的主要高磷酸化NS5A物种。我们还表明丝氨酸残基之间的NS5A磷酸化是相互依赖的，并且以定向方式发生，即丝氨酸235处的磷酸化导致丝氨酸238处的磷酸化。我们的数据提供了首个原理证明证据，即NS5A经历了一个顺序磷酸化级联反应。
