National Research Laboratory of Hepatitis C Virus, Ilsong Institute of Life Science, Hallym University, Anyang, Korea.
J Biol Chem. 2011 Apr 1;286(13):11290-8. doi: 10.1074/jbc.M110.194472. Epub 2011 Feb 5.
Hepatitis C Virus (HCV) nonstructural 5A (NS5A) is a pleiotropic protein involved in viral RNA replication and modulation of the cellular physiology in HCV-infected cells. To elucidate the mechanisms of the HCV life cycle, we identified cellular factors interacting with the NS5A protein in HCV-infected cells. Huh7.5 cells were electroporated with HCV Jc1 RNA. Cellular factors associated with HCV NS5A were identified by immunoprecipitation with Dynabead-conjugated NS5A antibody and LC-MS/MS. Phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) was identified as a binding partner for the NS5A protein. NS5A derived from both genotypes 1b and 2a interacted with PI4KIIIα. NS5A interacted with PI4KIIIα through amino acids 401-600 of PI4KIIIα and domain I of NS5A. Interference of the protein interaction between NS5A and PI4KIIIα decreased HCV propagation. Knockdown of PI4KIIIα significantly reduced HCV replication in Huh7 cells harboring the subgenomic replicon and in Huh7.5 cells infected with cell culture grown virus (HCVcc). Silencing of PI4KIIIα further inhibited HCV release into the tissue culture medium. NS5A may recruit PI4KIIIα to the HCV RNA replication complex. These data suggest that PI4KIIIα is an essential host factor that supports HCV proliferation and therefore PI4KIIIα may be a legitimate target for anti-HCV therapy.
丙型肝炎病毒（HCV）非结构 5A（NS5A）是一种多功能蛋白，参与 HCV 感染细胞中的病毒 RNA 复制和细胞生理学调节。为了阐明 HCV 生命周期的机制，我们鉴定了与 HCV 感染细胞中 NS5A 蛋白相互作用的细胞因子。使用 HCV Jc1 RNA 对 Huh7.5 细胞进行电穿孔。通过用 Dynabead 缀合的 NS5A 抗体进行免疫沉淀和 LC-MS/MS，鉴定与 HCV NS5A 相关的细胞因子。鉴定出磷脂酰肌醇 4-激酶 IIIα（PI4KIIIα）是 NS5A 蛋白的结合伴侣。来自基因型 1b 和 2a 的 NS5A 均与 PI4KIIIα相互作用。NS5A 通过 PI4KIIIα 的氨基酸 401-600 和 NS5A 的结构域 I 与 PI4KIIIα 相互作用。干扰 NS5A 和 PI4KIIIα 之间的蛋白相互作用会降低 HCV 的复制。PI4KIIIα 的敲低显著降低了携带亚基因组复制子的 Huh7 细胞和用细胞培养生长的病毒（HCVcc）感染的 Huh7.5 细胞中的 HCV 复制。PI4KIIIα 的沉默进一步抑制了 HCV 释放到组织培养基中。NS5A 可能将 PI4KIIIα 募集到 HCV RNA 复制复合物中。这些数据表明 PI4KIIIα 是支持 HCV 增殖的必需宿主因子，因此 PI4KIIIα 可能是抗 HCV 治疗的合理靶点。
