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.