Hepatitits C Virus Trafficking in Infected Hepatocytes

摘要 丙型肝炎病毒的进入和排出异常复杂，涉及许多宿主辅因子和独特的 贩运过程。进入因子包括基底外侧膜蛋白CD 81和SRB 1，紧密连接蛋白CD 81和SRB 1。 连接蛋白CLDN和OCLN，以及EGFR信号传导的需要。不同的亚细胞定位 丙型肝炎病毒受体的研究已经导致了这样的建议，即丙型肝炎病毒要么（i）在进入时运输到紧密连接处，要么（ii） 破坏紧密连接以进入CLDN和OCLN。我们已经开发了HCV的单粒子成像 进入极化三维Huh-7.5类器官来回答这个问题。类器官执行基本的 肝功能和形成适当的体内极化的肝细胞结构。使用这个系统，我们有 定义了HCV进入的步骤。HCV病毒粒子首先定位于“早期受体”（CD 81、SR-B1和EGFR）， 基底外侧膜，然后运输到与肌动蛋白丝相关的紧密连接。人惊讶 (and与目前的HCV进入模型相反），EGFR信号传导不需要运输到紧密的细胞中。 交界处。在EGFR抑制剂的存在下，HCV病毒体仍然定位在紧密连接处， 具有“晚期受体”CLDN和OCLN，并且无法将网格蛋白招募到HCV/受体复合物中。有趣的是， EGFR在紧密连接处被选择性激活。我们提出了一个模型，其中HCV与早期 受体激活CD 81或SRB 1依赖性迁移到紧密连接。EGFR，与 HCV受体复合物通过与CLDN的相互作用在紧密连接处被激活，和/或 OCLN，然后招募网格蛋白内吞机制用于病毒体内化。我们将测试这个模型， 目标1和2。 我们先前的研究结合了RNA干扰（RNAi）筛选和肝细胞成像， HCV衣壳运输，发现细胞外的HCV通过分泌性 通路越来越多的证据表明，HCV释放的第二途径，细胞-细胞传播，也是HCV感染的一个重要途径。 重要.除了对受体的需求外，对HCV释放的这一途径知之甚少。我们有 除了使用HepG 2细胞的极化系统外，还开发了上述肝类器官系统 工程化以表达HCV辅因子CD 81和miR-122加上荧光报告基因以检测感染。在 目的3，我们将利用这些极化细胞系统来确定HCV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.