Sequential Entry of Hepatitis C Virus into Polarized Hepatoma Organoids

Hepatitis C virus (HCV) is a hepatotropic RNA virus. An estimated 58 million people are chronically infected with HCV, while HCV-associated hepatocellular carcinoma and cirrhosis accounted for 290, 000 deaths in 2019. HCV entry into hepatocytes is highly complex with distinct stages and multiple host cofactors. However, the precise functions of most of the host factors in HCV entry are not fully appreciated. Moreover, studies of entry using polarized cell culture that physiologically resembles hepatocytes in vivo are limited. Using single particle imaging of HCV infection of polarized hepatoma organoids, we observed that epidermal growth factor receptor (EGFR) performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. EGFR is required for the recruitment of clathrin endocytic proteins to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into early endosomes, which form the site of virion uncoating. The tight junction proteins, claudin-1 (CLDN1) and occludin (OCLN), function at two distinct stages of HCV entry. CLDN1 is required for efficient HCV virion accumulation at the tight junction. OCLN is required for the internalization of virions. Our findings provide insights into the mechanism of HCV entry and highlight potential interventional strategies.