4). Given that CD81 engagement by HCV E2 protein induced SYK phosphorylation (Fig. 3B), we tested the functional impact of these signaling events in HCV infection. Using the HCV J6/JFH-1 and Huh7.5 experimental system, we found that transient knockdown of SYK by small interfering RNA (siRNA), or use of a potent and reversible SYK inhibitor, BAY 61-3606, significantly reduced HCV core and NS3 protein expression in Huh7.5 cells, suggesting the involvement of SYK in HCV infection (Fig.
3E,F). Because SYK activation and ezrin phosphorylation result in F-actin reorganization,[25] use of a specific F-actin reorganization inhibitor, cytochalasin B, resulted in a dose-dependent inhibition of HCV infectivity at the HCV RNA (Fig. 4A) and NS3 protein levels (Fig. 4B). The chemical agents used showed no cellular toxicity (Supporting Fig. 5A,B). The HCV life cycle involves multiple events including cell VX-809 entry, postentry trafficking, intracellular replication of viral RNA and proteins, assembly, and release.[37] To determine the role of EMR proteins in HCV infectivity and replication we took advantage of the HCV J6/JFH-1, HCV E1/E2 pseudo-particles (HCVpp), and HCV Con1 replication systems. Because chronic HCV infection resulted in decreased moesin and radixin expression, we asked if decreases in moesin or radixin prior to infection could modulate target cell susceptibility to infection.
Indeed, siRNA knockdown of moesin (Fig. 5A) and radixin (Fig. 5B) prior to infection with HCV J6/JFH-1 virus led Ibrutinib to significantly higher HCV NS3 protein (Fig. 5A,B) and HCV RNA expression (Supporting Fig. 6). In contrast, overexpression of moesin or radixin prior to
HCV J6/JFH-1 infection significantly reduced Huh7.5 cell susceptibility to infection demonstrated Tau-protein kinase by reduced HCV NS3 protein levels (Fig. 5C,D). Given that SYK inhibition decreased HCV infection via ezrin, we tested the role of ezrin in regulating HCV infection. Transient knockdown of ezrin prior to HCV J6/JFH-1 infection resulted in significantly lower HCV NS3 (Fig. 5E) protein and RNA (Supporting Fig. 6) in Huh7.5 hepatoma cells compared to controls. These observations suggest that ezrin, which is the only EMR protein that has been shown to associate and redistribute with F-actin,[25] can be exploited by HCV to mediate postentry trafficking within the cell, similar to observations with other viruses for effective infection.[38, 39] However, overexpression of ezrin prior to HCV J6/JFH-1 infection of Huh7.5 hepatoma cells had no significant effect on HCV NS3 protein expression (Fig. 5F), suggesting that in the presence of excess ezrin, the virus multiplicity of infection (MOI) determines the level of virus infection. Next, we assessed at which level in the HCV life cycle EMR proteins exerted their effect using HCVpp. We found that transient knockdown of moesin and radixin resulted in increased HCVpp infection of Huh7.5 cells (Fig. 6A).
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