Hepatitis C Virus Trafficking in Hepatocytes

丙型肝炎病毒在肝细胞中的贩运

• 批准号: 10382070
• 负责人: Glenn C Randall
• 金额: $ 4.54万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382070
• 关键词: 3-Dimensional; Architecture; CD81 gene; Capsid; Cell Culture System; Cell Culture Techniques; Cells; Chronic; Clathrin; Complex; Epidermal Growth Factor Receptor; HepG2; Hepatic; Hepatitis C; Hepatitis C virus; Hepatocyte; Image; Infection; Membrane Proteins; Microscopic; Modeling; Morbidity - disease rate; Organoids; Pathway interactions; Process; Proteins; RNA interference screen; Reporter; Role; Signal Transduction; System; Technology; Testing; Tight Junctions; Virion; basolateral membrane; cellular engineering; cofactor; extracellular; in vivo; live cell imaging; liver function; migration; mortality; 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 SR-BI, the tight junction proteins: CLDN and OCLN, and a requirement for EGFR signaling. The distinct subcellular localizations of HCV receptors have 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. We propose a model wherein HCV association with early receptors activates a CD81- and/or SR-BI-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. Since the tight junction proteins CLDN and OCLN are required for cell-cell spread, this strongly suggests the need to study cell-cell spread in a polarized cell culture system that actually forms tight junctions. 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. The specific aims are: Aim 1. Define the role of early receptors in HCV entry: how does HCV get to the tight junction? Aim 2. Define the role of EGFR signaling and late receptors in HCV internalization at the tight junction. Aim 3. Characterize the pathways of HCV egress and cell-cell spread in polarized 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， 独特的贩运过程。进入因子包括基底外侧膜蛋白：CD 81和 SR-BI，紧密连接蛋白：CLDN和OCLN，以及EGFR信号传导的要求。的 HCV受体的不同亚细胞定位导致了以下建议：HCV（i）运输至 在进入过程中的紧密连接，或（ii）破坏紧密连接以进入CLDN和OCLN。我们 已经开发了HCV进入偏振三维Huh-7.5的单粒子成像 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。使用该系统，我们定义了以下步骤： HCV进入。我们提出了一种模型，其中HCV与早期受体的结合激活了CD 81- 和/或SR-BI依赖性迁移至紧密连接。EGFR与HCV相关 受体复合物，通过与CLDN和/或OCLN的相互作用在紧密连接处被激活， 其然后募集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。 我们以前的研究结合了RNA干扰（RNAi）屏幕与活细胞 成像HCV衣壳运输，以发现细胞外HCV从肝细胞释放 通过分泌途径。越来越多的证据表明，HCV释放的第二途径， 细胞间的扩散也很重要关于HCV释放的这一途径知之甚少，除了其 受体需求。由于紧密连接蛋白CLDN和OCLN是细胞间连接所必需的， 扩散，这强烈表明需要研究极化细胞培养系统中的细胞-细胞扩散， 形成紧密连接我们已经开发了上述肝类器官系统， 除了使用经工程改造以表达HCV辅因子CD 81和CD 82的HepG 2细胞的极化系统之外， 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。具体目标是： 目标1。定义早期受体在HCV进入中的作用：HCV如何到达紧密连接？ 目标二。明确EGFR信号传导和晚期受体在HCV内在化中的作用， 交界处。 目标3.描述极化肝细胞中HCV流出和细胞间扩散的途径。