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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >An in vitro model of hepatitis C virion production
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An in vitro model of hepatitis C virion production

机译：丙型肝炎病毒粒子生产的体外模型

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The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5′ and 3′ ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5′ and 3′ ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of ≈50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.
机译：丙型肝炎病毒（HCV）是全球范围内肝脏疾病的主要原因。缺乏令人满意的细胞培养系统阻碍了对病毒生命周期的理解。 HCV复制子系统的开发已取得重大进展，但该系统不产生病毒体。在这项研究中，我们在两个核酶之间构建了一个可感染的HCV基因型1b cDNA，该酶被设计为产生HCV的确切5'和3'末端。产生在病毒RNA依赖性RNA聚合酶（RdRp）的活性位点具有突变的第二种构建体作为对照。将HCV-核酶表达构建体转染到Huh7细胞中。 HCV结构蛋白和非结构蛋白均通过免疫荧光和蛋白质印迹检测。 RNase保护性检测显示出正链和负链HCV RNA。 5'和3'末端的序列分析提供了病毒复制的进一步证据。培养基的蔗糖密度梯度离心显示，HCV RNA和结构蛋白的共定位浓度为1.16 g / ml，即HCV病毒体的假定密度。电子显微镜显示直径约50 nm的病毒颗粒。培养基中的HCV RNA水平高达每毫升1000万个拷贝。在RdRp中具有失活突变的HCV-核酶构建体未显示出病毒复制，组装和释放的证据。该系统支持高水平HCV病毒体的生产和分泌，并扩展了可用于HCV生物学研究的工具库。

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