Hepatitits C Virus Trafficking in Infected 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素和独特的 贩运过程。进入因子包括基侧膜蛋白CD81和SRB1，致密的 连接蛋白CLDN和OCLN，以及对EGFR信号的要求。明显的亚细胞定位 丙型肝炎病毒受体的变化导致了这样的假设：(I)在进入时，丙型肝炎病毒会进入紧要关口，或者(Ii) 中断紧密连接以访问CLDN和OCLN。我们已经开发出丙型肝炎病毒的单颗粒成像 进入极化三维HUH-7.5有机体来回答这个问题。有机化合物执行基本的 并在体内形成适当的极化、肝细胞构筑。使用这个系统，我们有 明确了进入丙型肝炎病毒的步骤。丙型肝炎病毒粒子首先与“早期受体”(CD81、SR-B1和EGFR)一起定位在 基侧膜，然后运输到与肌动蛋白细丝相关的紧密连接。令人惊讶的是 (与目前进入丙型肝炎病毒的模型不同)，EGFR信号不是贩运到TECH所必需的 交叉口。在EGFR抑制剂存在的情况下，丙型肝炎病毒病毒粒子仍然定位在紧密连接处。 与“晚期受体”CLDN和OCLN结合，并且不能将胞质蛋白招募到丙型肝炎病毒/受体复合体中。有趣的是， EGFR在紧密连接处被选择性地激活。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的向紧密连接的迁移。EGFR，关联到 与丙型肝炎病毒受体复合体通过与CLDN和/或相互作用在紧密连接处被激活 OCLN，然后招募网状蛋白内吞机器进行病毒粒子内化。我们将在以下方面测试此模型 目标1和2。 我们先前对丙型肝炎病毒出口的研究结合了RNA干扰(RNAi)筛查和活细胞成像 丙型肝炎病毒衣壳运输发现细胞外丙型肝炎病毒通过分泌从肝细胞中释放出来 路径。越来越多的证据表明，丙型肝炎病毒释放的第二条途径，细胞间传播，也是 很重要。除了它的受体要求外，人们对丙型肝炎病毒释放的这一途径知之甚少。我们有 开发了上述肝脏器官系统，以及使用HepG2细胞的极化系统 设计表达丙型肝炎病毒辅助因子CD81和miR-122，外加一个荧光报告程序来检测感染。在……里面 目的3，我们将使用这些极化细胞系统来确定丙型肝炎病毒细胞-细胞传播的途径。

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.