Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA
Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA
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DOI：
10.1099/vir.0.19305-0
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发表时间：
2003-10-01
影响因子：
3.8
通讯作者：
Luo, GX
中科院分区：
文献类型：
--
作者：
El-Hage, N;Luo, GX
生化研究表明，丙型肝炎病毒（HCV）的非结构蛋白彼此相互作用，并与细胞内膜相关。本研究的目的是确定非结构病毒蛋白是否共定位于 HCV RNA 复制的特定细胞内位点，并鉴定 HCV 复制复合物 (RC) 的病毒成分。进行免疫荧光和亚细胞分级分离研究，以确定人肝癌细胞系 Huh7 中非结构 HCV 蛋白和复制 RNA 的细胞内共定位，其中亚基因组 HCV RNA 持续复制。复制的 HCV RNA 用 5-溴尿苷 5'-三磷酸 (BrUTP) 标记。结果表明，每种非结构 HCV 蛋白主要与新合成的用 BrUTP 标记的 HCV RNA 和内质网 (ER) 蛋白钙连接蛋白共定位。与这些发现一致的是，亚细胞分级分离和蛋白质印迹分析表明，非结构性 HCV 蛋白主要与 HCV RNA 共定位于膜组分中。相反，用去污剂处理后，病毒非结构蛋白和 RNA 仍保留在可溶部分中，证实了 HCV RC 的膜关联。膜结合 RC 中的 HCV RNA 对 RNase 处理具有抗性，而一旦膜被去垢剂处理破坏，它就会对 RNase 敏感，这表明 HCV RC 在膜结构内组装。总的来说，这些发现表明 HCV RNA 复制发生在核周 ER 膜结合的 HCV RC 中，含有非结构病毒蛋白和 RNA。
Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5'-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.