# Hepatitis C Virus Trafficking in Hepatocytes

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2022 · $399,048

## Abstract

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.

## Key facts

- **NIH application ID:** 10356096
- **Project number:** 5R01AI137514-04
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Glenn C Randall
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $399,048
- **Award type:** 5
- **Project period:** 2019-03-05 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10356096

## Citation

> US National Institutes of Health, RePORTER application 10356096, Hepatitis C Virus Trafficking in Hepatocytes (5R01AI137514-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10356096. Licensed CC0.

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