Hepatitis C Virus Trafficking in Hepatocytes

丙型肝炎病毒在肝细胞中的贩运

• 批准号: 10574536
• 负责人: Glenn C Randall
• 金额: $ 39.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-05 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574536
• 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; basolateral membrane; cellular engineering; cofactor; extracellular; in vivo; live cell imaging; liver function; migration; mortality; mutant; novel; particle; polarized cell; receptor; recruit; trafficking

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.

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， 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CLDN和OCLN，以及EGFR信号传导的需要。 HCV受体的不同亚细胞定位导致了以下建议： 在进入期间，交通进入紧密连接处，或（ii）破坏紧密连接处以进入CLDN 和OCLN。我们已经开发了HCV进入极化三- 维度Huh-7.5类器官来回答这个问题。类器官执行基本的肝脏 功能并形成适当的体内极化肝细胞结构。使用这个系统， 我们已经定义了HCV进入的步骤。HCV病毒体首先定位于“早期受体”（CD 81， SR-B1和EGFR），然后运输到基底外侧膜的紧密连接， 与肌动蛋白丝结合。令人惊讶的是（与目前的HCV进入模型相反）， 运输到紧密连接处不需要EGFR信号传导。在EGFR存在下 在抑制剂的作用下，HCV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，并且不能将网格蛋白募集到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依赖性迁移至紧密连接。EGFR， 与HCV受体复合物相关的蛋白质在紧密连接处被激活， 与CLDN和/或OCLN的相互作用，然后招募网格蛋白内吞机制， 病毒粒子内化我们将在目标1和2中检验这一模式。 我们以前的研究HCV出口结合RNA干扰（RNAi）屏幕与活 HCV衣壳运输的细胞成像，以发现细胞外的HCV是从 肝细胞通过分泌途径。越来越多的证据表明， HCV的释放，细胞间的传播，也很重要。对HCV的这一途径知之甚少 释放，除了其受体的要求。我们已经开发出了肝脏类器官系统 除了使用经工程化以表达所述蛋白的HepG 2细胞的极化系统之外， HCV辅因子CD 81和miR-122加荧光报告基因检测感染。在目标3中，我们 将使用这些极化细胞系统来定义HCV细胞-细胞传播的途径。