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Abstract

The goal of this thesis was to define the mechanism of HCV entry using three-dimensional hepatoma cells. Although in vivo, HCV infection takes place in the context of highly polarized hepatocytes, HCV research has, up until now, been performed in unpolarized cell systems. We therefore sought to examine HCV entry in a more relevant system, both to broadly define the entry pathway of HCV and to define the individual roles of its many entry factors. We first developed a system for single particle tracking of fluorescently labeled HCV in three dimensional organoids. These organoids display markers of polarity, hepatocyte function, and correct localization of HCV entry factors. We optimized this system for both live and fixed cell microscopy in order to capture interactions with cellular factors. We then used this system to follow labeled HCV during infection. We found HCV colocalizes with early factors SR-BI, CD81, and EGFR at the basolateral, then traffics to the tight junction in an actin-dependent manner. The receptor complex then colocalizes with CLDN1 and OCLN as well. These observations support the tight junction targeting model proposed by Evans et al. Although other groups proposed EGFR signaling mediated tight junctional relocalization, we find that in polarized cells, EGFR and its associated signaling are required for internalization. Finally, we explored the internalization pathway of the viral particle. In the polarized cell system, viruses are internalized via clathrin-mediated endocytosis, traffic through early endosomes, and undergo fusion in a pH-dependent fashion. EGFR is activated at the tight junction at time points associated with internalization. Residues on its cytoplasmic tail, potentially sites for components of the clathrin endocytic machinery, are also required for HCV infection. Finally, we demonstrate EGFR signaling is also required for recruitment of such adaptors to HCV.

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