The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types.
The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types.
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DOI：
10.1371/journal.ppat.1006774
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发表时间：
2017-12
期刊：
影响因子：
6.7
通讯作者：
Cosset FL
中科院分区：
文献类型：
--
作者：
Denolly S;Mialon C;Bourlet T;Amirache F;Penin F;Lindenbach B;Boson B;Cosset FL
病毒孔蛋白是一种小的跨膜蛋白，具有调节细胞膜内离子通道活性的特性，具有多种前病毒功能。丙型肝炎病毒（HCV）编码一种毒孔蛋白p7，在病毒颗粒（VP）的组装、包膜和分泌过程中起作用。HCV p7通过信号肽酶在E2-p7和p7- ns2连接处的裂解从病毒多蛋白中释放出来，但也作为E2p7前体存在，性质不明确。在这里，我们发现hcvcc感染细胞中的异位p7表达减少了颗粒相关E2糖蛋白的分泌。通过生化分析，我们发现p7剂量依赖性地减缓了er到高尔基体的传输，导致细胞内E2的保留，这表明及时的E2p7切割和p7释放是控制E2水平的关键事件。通过研究E2p7加工加速的HCV突变体，我们发现E2p7切割控制E2在无核衣壳亚病毒颗粒和感染性病毒粒子的细胞内表达和分泌水平。此外，我们的成像数据显示，在p7释放后，p7的氨基端暴露于细胞质，并协调装载E1E2糖蛋白的NS5A和基于ns2的组装位点之间的相遇，从而导致核衣壳包膜。我们在p7膜界面发现了准时突变体，通过取消NS2/NS5A相互作用，由于核心和RNA的分泌减少以及非/部分包膜颗粒的分泌增加，导致传染性传播缺陷。总之，我们的研究结果表明，延迟的E2p7前体切割是通过p7介导的细胞分泌途径调节细胞内和分泌E2水平以及揭示位于p7氨基末端的关键新组装功能所必需的。病毒孔蛋白是一种小的跨膜病毒蛋白，具有调节细胞膜内离子通道活性的特性，影响一些基本的生物过程，如运输、离子通量以及细胞器之间的连接和交换。丙型肝炎病毒（HCV）编码一种毒孔蛋白p7，在病毒颗粒的组装、包膜和分泌过程中起作用。HCV p7是由HCV多蛋白裂解产生的，但也作为E2p7前体存在，性质不明确。在这项研究中，我们探索了E2糖蛋白和p7病毒孔蛋白之间的延迟裂解如何调节它们与病毒粒子组装和/或细胞膜过程扰动相关的功能。具体来说，我们证明p7能够调节细胞分泌途径，从而诱导HCV糖蛋白在细胞内的保留，并有利于HCV颗粒的组装。我们的研究还发现了位于p7氨基末端的一种新的组装功能，该功能通过e2p7调节的加工被揭示，并控制不同类型释放的病毒颗粒的感染性。总之，我们的研究结果强调了HCV颗粒组装和分泌的关键翻译后控制，控制其特异性感染性。
Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus. Viroporins are small transmembrane viral proteins with ion channel activities modulating properties of intracellular membranes, which impacts several fundamental biological processes such as trafficking, ion fluxes as well as connections and exchanges between organelles. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles. HCV p7 is produced by cleavage from the HCV polyprotein but also exists as an E2p7 precursor, of poorly defined properties. In this study, we have explored how the retarded cleavage between E2 glycoprotein and p7 viroporin could regulate their functions associated to virion assembly and/or perturbation of cellular membrane processes. Specifically, we demonstrate that p7 is able to regulate the cell secretory pathway, which induces the intracellular retention of HCV glycoproteins and favors assembly of HCV particles. Our study also identifies a novel assembly function located at p7 amino-terminus that is unmasked through E2p7-regulated processing and that controls the infectivity of different types of released viral particles. Altogether, our results underscore a critical post-translational control of assembly and secretion of HCV particles that governs their specific infectivity.
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