Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
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DOI：
10.1371/journal.ppat.1003056
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Bartenschlager R
中科院分区：
文献类型：
--
作者：
Romero-Brey I;Merz A;Chiramel A;Lee JY;Chlanda P;Haselman U;Santarella-Mellwig R;Habermann A;Hoppe S;Kallis S;Walther P;Antony C;Krijnse-Locker J;Bartenschlager R
已知所有正链RNA病毒复制其基因组与细胞内膜密切相关。在丙型肝炎病毒（HCV）（黄病毒科的一个成员）的情况下，感染的细胞含有形成膜网（MW）的囊泡的积累，该膜网被认为是病毒RNA复制的位点。然而，很少有人知道的生物成因和三维结构的MW。在这项研究中，我们使用了免疫荧光和电子显微镜（EM）为基础的方法相结合，以分析感染细胞中的HCV诱导的膜结构。我们发现，MW主要来自内质网（ER），并包含粗糙ER的标记以及早期和晚期内涵体，COP囊泡，线粒体和脂滴（LD）的标记。MW的主要成分是单膜囊泡和双膜囊泡（DMV）。后者占主导地位，其出现的动力学与病毒RNA复制的动力学相关。DMV主要由NS 5A诱导，而NS 4 B诱导单膜囊泡，认为MW的形成需要几种HCV复制酶蛋白的协同作用。三维重建鉴定DMV作为从ER膜到胞质溶胶中的突起，经常通过颈样结构连接到ER膜。此外，在感染后期，多膜囊泡变得明显，可能是由于应激诱导的反应。因此，在HCV感染的细胞中诱导的膜重排的形态类似于不相关的小核糖核酸病毒、冠状病毒和动脉炎病毒，但与密切相关的黄病毒明显不同。这些结果揭示了HCV和远亲正链RNA病毒之间意想不到的相似性，推测反映了这些病毒建立膜复制工厂所利用的细胞途径的相似性。所有正链RNA病毒在细胞质中在不同的膜隔室中作为“复制因子”复制。构建这些工厂的膜是从不同来源招募的，并作为组装多亚基蛋白复合物（“复制酶”）的平台，催化病毒RNA基因组的扩增。在这项研究中，我们发现，丙型肝炎病毒（HCV），慢性肝病的主要致病因素，诱导主要内质网衍生膜的深刻重塑。令人惊讶的是，这些膜重排的3D结构与不相关的小核糖核酸病毒和冠状病毒诱导的结构相似，但与密切相关的黄病毒形成鲜明对比。在感染早期，HCV诱导双膜囊泡（DMV），其作为ER的突起出现;随后，HCV诱导另外的多膜囊泡，其可能是细胞应激反应的结果并且使人联想到自噬反应。这些深刻的膜重排是由HCV编码的非结构蛋白的协同作用诱导的，其中NS 5A是唯一能够诱导DMV的蛋白。这些结果为HCV诱导的膜改变的3D结构提供了重要的见解，并揭示了HCV与非常遥远的小核糖核酸病毒和冠状病毒之间意想不到的相似性。
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. All positive-strand RNA viruses replicate in the cytoplasm in distinct membranous compartments acting as ‘replication factories’. Membranes building up these factories are recruited from different sources and serve as platforms for the assembly of multi-subunit protein complexes (the ‘replicase’) that catalyze the amplification of the viral RNA genome. In this study we found that hepatitis C virus (HCV), a major causative agent of chronic liver disease, induces profound remodeling of primarily endoplasmic reticulum-derived membranes. Surprisingly, the 3D architecture of these membrane rearrangements is similar to those induced by the unrelated picorna- and coronaviruses, but in striking contrast to the closely related flaviviruses. Early in infection HCV induces double membrane vesicles (DMVs) that emerge as protrusions of the ER; later on, HCV induces in addition multi-membrane vesicles that are probably the result of a cellular stress reaction and that are reminiscent to an autophagic response. These profound membrane rearrangements are induced by the concerted action of HCV-encoded nonstructural proteins of which NS5A is the only one capable to induce DMVs. These results provide important insights into the 3D architecture of the membrane alterations induced by HCV and reveal unexpected similarities between HCV and the very distantly related picorna- and coronaviruses.
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影响因子：
5.4
作者：
Gosert, R;Kanjanahaluethai, A;Baker, SC
通讯作者：
Baker, SC
影响因子：
5.4
作者：
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影响因子：
5.4
作者：
BIENZ, K;EGGER, D;PASAMONTES, L
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PASAMONTES, L
影响因子：
5.4
作者：
BIENZ, K;EGGER, D;TROXLER, M
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TROXLER, M
影响因子：
3.7
作者：
Guévin C;Manna D;Bélanger C;Konan KV;Mak P;Labonté P
通讯作者：
Labonté P