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.

Public Health Relevance

130 million people are chronically infected with hepatitis C virus with significant morbidity and mortality. We have developed advanced microscopic approaches to image HCV infection on 3-dimensional organoid cell cultures. We will apply this technology to uncover the pathways of HCV entry and spread.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56AI080703-06A1
Application #
9408767
Study Section
Virology - B Study Section (VIRB)
Program Officer
Koshy, Rajen
Project Start
2017-02-09
Project End
2018-01-31
Budget Start
2017-02-09
Budget End
2018-01-31
Support Year
6
Fiscal Year
2017
Total Cost
$397,545
Indirect Cost
$147,545
Name
University of Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
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Shulla, Ana; Randall, Glenn (2016) (+) RNA virus replication compartments: a safe home for (most) viral replication. Curr Opin Microbiol 32:82-88
Shulla, Ana; Randall, Glenn (2015) Spatiotemporal analysis of hepatitis C virus infection. PLoS Pathog 11:e1004758
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Coller, Kelly E; Berger, Kristi L; Heaton, Nicholas S et al. (2009) RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathog 5:e1000702