Hepatitis C Virus Trafficking in Hepatocytes

丙型肝炎病毒在肝细胞中的贩运

基本信息

批准号：
10542648
负责人：
Glenn C Randall
金额：
$ 8.43万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-05 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10542648
关键词：
3-Dimensional Architecture CD81 gene Capsid Cell Culture System Cell Culture Techniques Cell Line Cells Chronic Clathrin Complement Complex Data Defect Early Endosome Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Golgi Apparatus HepG2 Hepatic Hepatitis C Hepatitis C virus Hepatocyte Image Infection Knock-out Membrane Proteins Microfilaments Microscopic Modeling Morbidity - disease rate Organoids Pathway interactions Persons Phosphorylation Process Proteins RNA interference screen Reporter Role Signal Transduction Specific qualifier value System Tail Technology Testing Tight Junctions Tyrosine Phosphorylation Very low density lipoprotein Virion base basolateral membrane cellular engineering cofactor extracellular in vivo live cell imaging liver function migration mortality mutant novel particle polarized cell receptor recruit trafficking

项目摘要

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.

抽象的丙型肝炎病毒的进入和排出异常复杂，涉及许多宿主辅助因子和独特的运输过程包括基底外侧膜蛋白 CD81。 SRB1、紧密连接蛋白 CLDN 和 OCLN，以及 EGFR 信号转导的要求。 HCV 受体独特的亚细胞定位导致有人提出 HCV 要么 (i) 在进入期间到达紧密连接点的流量，或 (ii) 中断紧密连接点以获得对 CLDN 的访问权限我们开发了 HCV 进入偏振三层的单粒子成像。维 Huh-7.5 类器官来回答这个问题类器官执行基本肝脏功能。使用该系统发挥功能并形成适当的体内极化肝细胞结构，我们已经定义了 HCV 病毒粒子首先定位于“早期受体”（CD81、 SR-B1 和 EGFR）位于基底外侧膜，然后运输至紧密连接令人惊讶的是（与目前的 HCV 进入模型相反），当 EGFR 存在时，运输至紧密连接不需要 EGFR 信号转导。抑制剂，HCV 病毒体仍定位于与“晚期受体”相关的紧密连接处 CLDN 和 OCLN 无法将网格蛋白募集至 HCV/受体复合体。我们提出了一个 HCV 与紧密连接相关的模型。早期受体激活 CD81 或 SRB1 依赖性迁移至 EGFR，与 HCV 受体复合物相关的物质通过紧密连接处被激活与 CLDN 和/或 OCLN 相互作用，然后招募网格蛋白内吞机制我们将在目标 1 和 2 中测试这个模型。我们之前的 HCV 流出研究将 RNA 干扰 (RNAi) 筛查与活体病毒筛查结合起来。 HCV 衣壳运输的细胞成像，发现细胞外 HCV 从越来越多的证据表明，肝细胞通过分泌途径。 HCV 释放、细胞间传播的过程对于 HCV 的这一途径知之甚少。释放，除了其受体要求外，我们开发了肝类器官系统。如上所述，除了使用经改造的 HepG2 细胞表达 HCV 辅因子 CD81 和 miR-122 加上荧光报告基因来检测感染。将使用这些极化细胞系统来定义 HCV 细胞间传播的途径。