CELL-CELL CONTACT-MEDIATED HEPATITIS C VIRUS INFECTION AND ITS REQUIREMENT FOR HCV RECEPTORS

Purpose: Hepatitis C virus (HCV) leads to chronic infection in 80% of infected people. In these patients, HCV evades the immune system with the presence of neutralizing antibodies (nAbs), yet the underlying mechanisms have not been completely understood. Recently discovered cell-cell contact-mediated (CCCM) HCV transmission, in contrast to cell-free infection, was found to be nAbs-resistant, suggesting its important role in immune evasion. The objective of the current study was to characterize CCCM HCV infection and to determine the roles of the four known HCV receptors (CD81, scavenger receptor B1, claudin-1 and occludin) in this process. Methods: Infected donor hepatocyte Huh7.5.1 were either co-cultured with uninfected and fluorescently labeled target Huh7.5.1, or seeded into the upper chamber of a trans-well, where uninfected Huh7.5.1 were seeded in the lower chamber. After different time of culturing, the mixed donor and target cells in the co-culture assay or the target cells in the trans-well assay was immunostained for HCV core and analyzed by flow cytometry for percentage of newly infected target cells. To determine the roles of HCV receptors and cytoskeleton in CCCM HCV transfer, co-culture assay was also performed with target cells where one or all HCV receptors were knocked-down by siRNA, or with the presence of cytoskeleton inhibitors. Results: In this study, we have demonstrated that HCV spreading among hepatocytes leads to the formation of infection foci dependent upon cell density and that HCV is directly transferred from infected hepatocytes to uninfected hepatocytes. Compared to cell-free infection in the trans-well assay (undetectable within 20 hr), CCCM HCV transfer in the co-culture assay occurs very rapidly (3 hr). Knock-down of HCV receptors in target cells and cytochalasin D treatment of the co-culture greatly inhibited CCCM HCV transfer, suggesting the important roles of all four HCV receptors and intact actin cytoskeleton in this process. With a fluorescently labeled replication-competent HCV, the CCCM transfer process is further dissected by 3D live cell imaging into four steps: donor cell-target cell contact, formation of viral puncta-target cell conjugation, transfer of viral puncta, and post transfer. Conclusions: The study shows for the first time that CCCM HCV transfer constitutes a rapid and vital route for HCV infection and dissemination. These findings will aid in the development of new and effective strategies for preventing and treating HCV infection.