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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：
10356096
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
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/10356096
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.

摘要