Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

已知所有正链RNA病毒（positive strand RNA virus）的基因组复制过程均与细胞内膜结构紧密结合。以黄病毒科（Flaviviridae）成员丙型肝炎病毒（hepatitis C virus, HCV）为例，感染该病毒的细胞内会积累大量囊泡，形成所谓的膜网（membranous web, MW），该结构被认为是病毒RNA复制的核心场所。然而，学界目前对膜网的生物发生过程及其三维结构仍知之甚少。本研究联合使用基于免疫荧光（immunofluorescence）与电子显微镜（electron microscopy, EM）的技术方法，对HCV感染细胞中病毒诱导的膜结构进行了系统分析。研究发现，膜网主要源自内质网（endoplasmic reticulum, ER），其组分包含糙面内质网（rough ER）标记物，以及早期内体（early endosomes）、晚期内体（late endosomes）、COP囊泡（COP vesicles）、线粒体（mitochondria）与脂滴（lipid droplets, LDs）的标记蛋白。膜网的主要构成成分为单膜囊泡与双膜囊泡（double membrane vesicles, DMVs），其中双膜囊泡占主导，其出现的动力学特征与病毒RNA复制的动力学过程高度相关。双膜囊泡主要由NS5A诱导产生，而单膜囊泡则由NS4B诱导，这提示膜网的形成需要HCV多种复制酶蛋白（replicase proteins）的协同作用。三维重构（three-dimensional reconstructions）结果显示，双膜囊泡是从ER膜向胞质溶胶伸出的突起结构，且常通过类似颈部的结构与ER膜相连。此外，在感染后期可观察到多膜囊泡（multi-membrane vesicles）的形成，推测这是病毒感染引发细胞应激反应的结果。综上，HCV感染细胞中诱导的膜重排形态与无亲缘关系的小RNA病毒（picornaviruses）、冠状病毒（coronaviruses）及动脉炎病毒（arteriviruses）相似，但与亲缘关系较近的黄病毒（flaviviruses）则存在显著差异。本研究结果揭示了HCV与远缘正链RNA病毒之间此前未被发现的相似性，这大概率反映了这些病毒在建立膜状复制工厂（replication factories）时所劫持的细胞通路（cellular pathways）具有共性。