Hepatitis C Virus Trafficking in Hepatocytes

摘要 丙型肝炎病毒的进出异常复杂，涉及许多宿主辅助因子和 独特的贩运过程。进入因子包括基侧膜蛋白CD81 和SRB1，紧密连接蛋白CLDN和OCLN，以及EGFR信号转导所必需的。 丙型肝炎病毒受体独特的亚细胞定位导致了丙型肝炎病毒要么(I) 在进入期间前往紧路口的车辆，或(Ii)扰乱紧路口以进入CLDN 和OCLN。我们已经开发出丙型肝炎病毒进入极化三极化体的单粒子成像 维Huh-7.5有机化合物来回答这个问题。有机化合物执行基本的肝脏 并在体内形成适当的极化、肝细胞结构。使用这个系统， 我们已经定义了进入丙型肝炎病毒的步骤。丙型肝炎病毒粒子首先定位于“早期受体”(CD81， SR-B1和EGFR)，然后交通到紧密交界处 与肌动蛋白细丝结合。令人惊讶的是(与目前进入丙型肝炎病毒的模型形成对比)， 贩运到紧密连接不需要EGFR信号。在EGFR存在的情况下 抑制物，丙型肝炎病毒粒子仍然定位在与“晚期受体”相关的紧密连接处 CLDN和OCLN，并且不能将笼状蛋白招募到丙型肝炎病毒/受体复合体中。有趣的是，EGFR是 在紧密连接处有选择地激活。我们提出了一个模型，其中丙型肝炎病毒与 早期受体激活依赖于CD81或SRB1的迁移到紧密连接。EGFR， 与丙型肝炎病毒受体复合体相关的蛋白在紧密连接时通过 与CLDN和/或OCLN相互作用，然后招募网状蛋白内吞机械用于 病毒粒子内化。我们将在目标1和目标2中测试该模型。 我们先前对丙型肝炎病毒出口的研究结合了RNA干扰(RNAi)筛查和LIVE 对丙型肝炎病毒衣壳运输的细胞成像发现细胞外丙型肝炎病毒从 肝细胞通过分泌途径。越来越多的证据表明，一条次要途径 丙型肝炎病毒的释放，细胞间的传播，也很重要。对丙型肝炎病毒的这一途径知之甚少 释放，但其受体要求除外。我们已经开发了肝脏器官系统 如上所述，除了使用经工程设计的HepG2细胞表达 丙型肝炎病毒协同因子CD81和miR-122加上一个荧光报告程序来检测感染。在目标3中，我们 将使用这些极化的细胞系统来定义丙型肝炎病毒细胞间传播的途径。

ABSTRACT Hepatitis C virus entry and egress are unusually complex, involving many host cofactors and distinctive trafficking processes. Entry factors include the basolateral membrane proteins CD81 and SRB1, the tight junction proteins CLDN and OCLN, and a requirement for EGFR signaling. The distinct subcellular localization of HCV receptors has led to proposals that HCV either (i) traffics to the tight junction during entry, or (ii) disrupts tight junctions to gain access to CLDN and OCLN. We have developed single particle imaging of HCV entry into polarized three- dimensional Huh-7.5 organoids to answer this question. The organoids perform basic liver functions and form the appropriate in vivo polarized, hepatocyte architecture. Using this system, we have defined the steps of HCV entry. HCV virions first localize with “early receptors” (CD81, SR-B1, and EGFR) at the basolateral membrane and then traffic to the tight junction in association with actin filaments. Surprisingly (and in contrast to current models of HCV entry), EGFR signaling is not required for trafficking to the tight junction. In the presence of EGFR inhibitors, HCV virions remain localized at the tight junction in association with “late receptors” CLDN and OCLN and fail to recruit clathrin to the HCV/receptor complex. Interestingly, EGFR is selectively activated at the tight junction. We propose a model wherein HCV association with early receptors activates a CD81- or SRB1-dependent migration to the tight junction. EGFR, which is associated with the HCV receptor complex becomes activated at the tight junction via an interaction with CLDN and/or OCLN, which then recruits the clathrin endocytic machinery for virion internalization. We will test this model in Aims 1 and 2. Our previous study of HCV egress combined an RNA interference (RNAi) screen with live cell imaging of HCV capsid trafficking to discover that extra-cellular HCV is released from the hepatocyte via the secretory pathway. Increasing evidence indicates that a secondary pathway of HCV release, cell-cell spread, is also important. Little is known about this pathway of HCV release, except for its receptor requirements. We have developed the hepatic organoid system described above, in addition to a polarized system using HepG2 cells engineered to express the HCV cofactors CD81 and miR-122 plus a fluorescent reporter to detect infection. In Aim 3, we will use these polarized cell systems to define the pathways of HCV cell-cell spread.