Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein
Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein
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DOI：
10.1074/jbc.m508175200
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发表时间：
2005-10-28
影响因子：
4.8
通讯作者：
Cameron, CE
中科院分区：
文献类型：
--
作者：
Huang, LY;Hwang, J;Cameron, CE
丙型肝炎病毒 (HCV) 非结构蛋白 5A (NS5A) 已被证明可以拮抗许多细胞途径，包括抗病毒干扰素-α 反应。然而，这种蛋白质与病毒聚合酶相互作用的能力表明 NS5A 在基因组复制中具有更直接的作用。在这项研究中，我们利用两种细菌表达的 NS5A 可溶性衍生物来探索该蛋白质的新功能。我们发现 NS5A 能够结合 HCV 正链和负链 RNA 的 3' 端。正链 RNA 3' 端 NS5A 的高亲和力结合位点映射到聚嘧啶束，这是已知对基因组复制和感染性至关重要的元件。 NS5A 偏爱含有尿苷或鸟苷片段的单链 RNA。高亲和力结合位点的平衡解离常数值在 10 nM 范围内。二维凝胶电泳和蛋白质印迹显示稳定表达亚基因组复制子的 Huh-7 细胞中存在未磷酸化的 NS5A。此外，RNA免疫沉淀和NS5A Pull-down实验显示复制子衍生的NS5A分别与合成RNA和HCV基因组结合的能力。 NS5A 中所有酪蛋白激酶 II 磷酸化位点的删除支持 Huh-7 中亚基因组复制子的稳定复制。然而，该衍生物不能用无机磷酸盐标记，这表明 NS5A 的复制功能不需要 NS5A 的广泛磷酸化。 NS5A 是一种 RNA 结合蛋白，这一发现为开发治疗 HCV 感染的药物和一类新的 RNA 结合蛋白结构定义了一个新的功能靶点。
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral interferon-alpha response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the 3'-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the 3'-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nM range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.