RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
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DOI：
10.1371/journal.ppat.1000702
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发表时间：
2009-12
期刊：
影响因子：
6.7
通讯作者：
Randall G
中科院分区：
文献类型：
--
作者：
Coller KE;Berger KL;Heaton NS;Cooper JD;Yoon R;Randall G
丙型肝炎病毒（HCV）通过一系列复杂的受体相互作用进入肝细胞，最终通过网格蛋白介导的内吞作用内化。然而，除了受体外，对感染性HCV进入的细胞分子要求知之甚少。因此，我们分析了靶向140个细胞膜运输基因的siRNA文库，以鉴定感染性HCV产生和HCV假颗粒进入所需的宿主基因。这种方法确定了16个宿主辅助因子的HCV进入，主要在网格蛋白介导的内吞作用，包括组件的网格蛋白内吞机制，肌动蛋白聚合，受体内化和分选，和内体酸化。我们接下来开发了高感染性荧光HCV颗粒的单颗粒追踪分析，以检查HCV病毒体与内吞作用的细胞辅因子的共运输。我们观察到HCV病毒粒子与肌动蛋白细胞骨架的多个连续相互作用，包括沿着丝状伪足的回缩、内化过程中的肌动蛋白成核以及内化颗粒沿沿着肌动蛋白应力纤维的迁移。HCV在内化之前与网格蛋白和泛素连接酶c-Cbl共定位。进入HCV颗粒与受体分子CD 81和紧密连接蛋白claudin-1相关;然而，HCV-claudin-1相互作用并不限于Huh-7.5细胞-细胞连接。令人惊讶的是，HCV内化通常发生在Huh-7.5细胞-细胞连接之外，这可能反映了当前HCV细胞培养模型的极化性质差。内化后，HCV颗粒与GFP-Rab 5a阳性内体一起转运，这与运输到早期内体一致。这项研究提出了成像HCV入境的技术进展，除了确定新的HCV感染的宿主辅因子，其中一些可能是抗病毒的目标。丙型肝炎病毒（HCV）慢性感染1.3亿人，是肝硬化和肝癌的主要原因。目前的抗病毒治疗聚乙二醇干扰素-2 α +利巴韦林是成功的，只有一半的治疗患者。这导致了设计改进的治疗策略的密集努力。HCV感染的细胞辅因子的鉴定极大地扩展了药物设计的潜在靶点库。在本文中，我们结合联合收割机RNA干扰分析的HCV内吞作用的发展，活细胞成像的高度感染性的HCV颗粒。我们确定了16个宿主辅助因子的HCV条目，其中大部分功能在网格蛋白介导的内吞作用的连续阶段。我们观察荧光HCV颗粒与这些细胞辅因子及其相关途径，包括肌动蛋白细胞骨架，已知的受体CD 81和紧密连接蛋白claudin-1，网格蛋白，E3泛素连接酶和早期内体的贩运。令人惊讶的是，考虑到紧密连接蛋白作为HCV进入因子的作用，病毒体进入通常发生在细胞-细胞连接之外。本文确定了新的治疗开发的主机目标，描述了技术，图像HCV的进入，并提供了深入了解HCV-细胞相互作用的进入过程。
Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets. Hepatitis C virus (HCV) chronically infects 130 million people and is a major cause of cirrhosis and liver cancer. The current antiviral therapy of pegylated interferon-2 alfa + ribavirin is successful in only half of treated patients. This has led to an intensive effort to design improved therapeutic strategies. The identification of cellular cofactors of HCV infection greatly expands the pool of potential targets for drug design. In this paper, we combine RNA interference analysis of HCV endocytosis with the development of live cell imaging of highly infectious HCV particles. We identify 16 host cofactors of HCV entry, most of which function in sequential stages of clathrin-mediated endocytosis. We observe the trafficking of fluorescent HCV particles with these cellular cofactors and their related pathways, including the actin cytoskeleton, known receptors CD81 and the tight junction protein claudin-1, clathrin, an E3 ubiquitin ligase, and early endosomes. Surprisingly, given the role of tight junction proteins as HCV entry factors, virion entry generally occurred outside of cell-cell junctions. This paper identifies novel host targets for therapeutic development, describes techniques to image HCV entry, and provides insights into HCV-cell interactions in the entry process.
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影响因子：
4.8
作者：
Bartosch, B;Vitelli, A;Cosset, FL
通讯作者：
Cosset, FL
影响因子：
9.8
作者：
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影响因子：
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作者：
Koutsoudakis, George;Kaul, Artur;Bartenschlager, Ralf
通讯作者：
Bartenschlager, Ralf
影响因子：
5.4
作者：
Grove, Joe;Huby, Thierry;McKeating, Jane A.
通讯作者：
McKeating, Jane A.
影响因子：
64.8
作者：
Krishnan, Manoj N.;Ng, Aylwin;Sukumaran, Bindu;Gilfoy, Felicia D.;Uchil, Pradeep D.;Sultana, Hameeda;Brass, Abraham L.;Adametz, Rachel;Tsui, Melody;Qian, Feng;Montgomery, Ruth R.;Lev, Sima;Mason, Peter W.;Koski, Raymond A.;Elledge, Stephen J.;Xavier, Ramnik J.;Agaisse, Herve;Fikrig, Erol
通讯作者：
Fikrig, Erol