Hepatitis C Virus Trafficking in Hepatocytes

RESEARCH ABSTRACT: HEPATITIS C VIRUS TRAFFICKING IN HEPATOCYTES Many viruses have evolved distinct pathways of transmitting their infection between cells. This viral spread involves canonical pathways that release unprotected virions from the infected cell (thus potentially exposing the virion to neutralizing antibodies) and/or pathways that shield the virion from the humoral response (cell-cell spread, syncytia formation, and the release of virions in extra-cellular vesicles). Viral egress pathways that evade the humoral response are especially important to understand given the potential importance of manipulating viral susceptibility to this critical arm of adaptive immunity. Most viruses of the Flaviviridae appear to use both mechanisms of viral egress. We and others previously characterized the pathway of canonical hepatitis C virus (HCV) release, while canonical dengue virus (DENV) secretion has also been well characterized. In contrast, noncanonical pathways of virion spread in the Flaviridae are poorly understood. Our lab has a longstanding interest in Flaviviridae entry and spread. In our prior funding period, we developed a multi-pronged analysis (host genetics, pharmacological inhibition and single particle imaging) of polarized hepatoma organoids to characterize HCV entry and egress. The work established the most complete, detailed pathway of HCV entry, in addition to extensive preliminary data on HCV cell-cell spread. We expanded these studies into the non-canonical spread of DENV, focusing on a process we discovered years ago: DENV-induced lipophagy, which is the selective autophagic mobilization of lipid droplet stores to stimulate lipid metabolism. This process has been associated with the non-canonical release of DENV into extra-cellular vesicles. We have identified a central cellular player in this process: the selective autophagy adaptor NBR1. In this R01 renewal application, we will focus on extending and expanding our studies on HCV and DENV non-canonical virion spread. In the case of HCV, we developed quantitative cell-cell spread assays and identified putative cellular secretion pathways and cargo adaptors that modulate cell-cell spread. We propose to characterize the significance of these pathways using imaging of our 3-dimensional polarized hepatoma spheroid system. Additionally, we will collaborate for an ultra-structural analysis of HCV-infected hepatocyte cell-cell junctions cryoelectron microscopy tomography (cryo-ET). For DENV non-canonical spread, we will validate a rove for NBR1 and lipophagy in the cell-cell spread of DENV. We will then characterize the pathway of non-canonical release of DENV virions. Finally, we will perform ultrastructural analysis of the NBR1-dependent DENV containing extra-cellular vesicles using cryo-ET. The specific aims are: Aim 1. Characterize the pathway(s) of HCV cell-cell spread in infected hepatocytes. Aim 2. Characterize the NBR1-dependent pathway of non-canonical DENV egress.