Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts
Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts
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DOI：
10.1016/j.virol.2004.03.034
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发表时间：
2004-07-01
期刊：
影响因子：
3.7
通讯作者：
Lai, MMC
中科院分区：
文献类型：
--
作者：
Aizaki, H;Lee, KJ;Lai, MMC
丙型肝炎病毒 (HCV) RNA 复制的机制和机制仍然知之甚少。我们之前的研究表明，HCV RNA 合成发生在脂筏膜结构上 [J.病毒。 77（2003）77 4160]。在这项研究中，我们进一步鉴定了 Huh-7 细胞中这些复制复合物 (RC) 的特征，这些复制复合物支持亚基因组 HCV 复制子的主动 RNA 复制。生化分析表明，这些膜结构在 4 摄氏度下对 Nonidet P-40 或 Triton X-100 (TX-100) 具有抗性，同时在 4 摄氏度下被 β-辛基葡萄糖苷或在 37 摄氏度下被 Triton TX-100 溶解，这是脂筏的特征。胆固醇封存测定进一步证明了 HCV 非结构 (NS) 蛋白与富含胆固醇的脂筏之间的关联。 RC 含有负链和正链 HCV RNA，正链 RNA 的丰度大约是负链的 10 倍。此外，HCV RNA 和 NS 蛋白分别对 RNase 和蛋白酶消化具有抗性，但在用筏破坏剂处理后变得敏感。这些结果表明 HCV RC 在脂筏内受到保护。含有 NS 蛋白和病毒 RNA 的耐去垢剂膜 (DRM) 级分能够使用内源性 HCV RNA 模板合成 HCV RNA。 NS 蛋白分布于 ER 和高尔基体中，但大多数活性 RC 在高尔基体衍生膜中检测到。细胞胆固醇的消耗选择性地减少了 HCV RNA 的复制。这些发现为HCV体内复制机制提供了进一步的见解。 (C) 2004 Elsevier Inc. 保留所有权利。
The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly characterized. Our previous study has shown that HCV RNA synthesis occurs on a lipid raft membrane structure [J. Virol. 77 (2003) 77 4160]. In this study, we further characterized these replication complexes (RCs) in Huh-7 cells that support active RNA replication of a subgenomic HCV replicon. Biochemical analysis showed that these membrane structures were resistant to Nonidet P-40 or Triton X-100 (TX-100) at 4 degreesC while solubilized by beta-octylglucoside at 4 degreesC or Triton TX-100 at 37 degreesC, characteristic of lipid rafts. Cholesterol sequestration assay further demonstrated the association between HCV nonstructural (NS) proteins and cholesterol-rich lipid rafts. The RCs contained both minus- and plus-strand HCV RNA, with the plus-stranded RNA being approximately 10-fold more abundant than the minus-strand. Furthermore, the HCV RNA and NS proteins were resistant to RNase and protease digestion, respectively, but became sensitive after treatment with the raft-disrupting agents. These results suggested that the HCV RCs are protected within lipid rafts. Detergent-resistant membrane (DRM) fractions containing NS proteins and viral RNA were capable of HCV RNA synthesis using the endogenous HCV RNA template. NS proteins were distributed in both the ER and the Golgi, but the majority of the active RCs were detected in the Golgi-derived membrane. Depletion of cellular cholesterol selectively reduced HCV RNA replication. These findings provide further insights into the mechanism of HCV replication in vivo. (C) 2004 Elsevier Inc. All rights reserved.