Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A
Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A
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DOI：
10.1128/jvi.00068-10
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发表时间：
2010-08-15
影响因子：
5.4
通讯作者：
Lai, Michael M. C.
中科院分区：
文献类型：
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作者：
Chen, Yung-Chia;Su, Wen-Chi;Lai, Michael M. C.
丙型肝炎病毒(丙型肝炎病毒)复制涉及许多病毒和宿主因素。在这里，我们采用了基于慢病毒的RNA干扰(RNAi)筛选方法来寻找可能的细胞因素。通过使用一个激酶-磷酸酶RNAi文库和一个丙型肝炎病毒复制子报告系统，我们鉴定了一个丝氨酸-苏氨酸激酶，Polo-like kinase1(Plk1)，它是一个潜在的调节丙型肝炎病毒复制的宿主因子。Plk1基因敲除降低了丙型肝炎病毒复制子细胞和丙型肝炎病毒感染细胞中的丙型肝炎病毒RNA复制和非结构蛋白(NS)的产生，但对宿主细胞的生长和细胞周期没有明显影响。基因敲除细胞中Plk1的过表达挽救了丙型肝炎病毒的复制。有趣的是，在Plk1基因敲除细胞和Plk1激酶抑制剂处理的细胞中，NS5A的过度磷酸化形式(P58)和基础磷酸化形式(P56)的比率低于对照组。进一步的研究表明，Plk1可以与NS5A一起免疫沉淀。这两种蛋白部分共存于核周区域。此外，在体外检测系统中，Plk1可以将NS5A磷酸化为p58和p56两种形式；其磷酸化效率与已报道的酪蛋白激酶相当。综上所述，本研究表明Plk1是一种NS5A磷酸激酶，从而间接调节丙型肝炎病毒RNA的复制。由于Plk1对丙型肝炎病毒复制和宿主细胞生长的不同影响，Plk1有可能成为抗丙型肝炎病毒治疗的靶点。
Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.