Targett-Adams Paul, Boulant Steeve, McLauchlan John
MRC Virology Unit, Church Street, Glasgow G11 5JR, United Kingdom.
J Virol. 2008 Mar;82(5):2182-95. doi: 10.1128/JVI.01565-07. Epub 2007 Dec 19.
The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.
丙型肝炎病毒（HCV）RNA复制所涉及的机制尚不清楚，该病毒生命周期的这一方面尚未被了解。据认为，病毒编码的非结构蛋白与RNA基因组在内质网（ER）重排膜上相互作用，形成复制复合物，据信该复合物是RNA合成的场所。我们报告称，通过使用针对双链RNA（dsRNA）的特异性抗体，在含有复制型HCV JFH-1基因组的Huh-7细胞中很容易检测到dsRNA，而在对照细胞中则不存在。因此，与其他RNA病毒基因组一样，HCV基因组的复制可能涉及双链RNA复制中间体的产生。在支持HCV RNA复制的Huh-7细胞中，dsRNA表现为离散的病灶，与病毒编码的NS5A和核心蛋白相关，其形态和分布与通过基于荧光的杂交方法可视化的含有HCV RNA的结构相同。对病毒感染细胞的去卷积z-stack图像进行三维重建，详细揭示了dsRNA病灶、NS5A、内质网和脂滴（LDs）之间的关系。该分析表明，dsRNA病灶位于内质网表面，常被内质网结合的NS5A蛋白网络部分或完全包围。此外，病毒诱导的dsRNA病灶与附着在内质网上的脂滴并列。因此，我们报告了HCV诱导的dsRNA病灶的可视化，这可能是病毒RNA复制的位点，并提出HCV基因组合成发生在病毒感染细胞中与内质网相连的脂滴相关位点。
