Multiple functions of cellular FLIP are essential for replication of hepatitis B virus

Hepatitis B virus (HBV) infection is a leading cause of liver diseases; however, the host factors which facilitate the replication and persistence of HBV are largely unidentified. Cellular FLICE inhibitory protein (c-FLIP) is a typical antiapoptotic protein. In many cases of liver diseases, the expression level of c-FLIP is altered, which affects the fate of hepatocytes. We previously found that c-FLIP and its cleaved form interact with HBV X protein (HBx), which is essential for HBV replication, and regulate diverse cellular signals. In this study, we investigated the role of endogenous c-FLIP in HBV replication and its underlying mechanisms. The knockdown of endogenous c-FLIP revealed that this protein regulates HBV replication through two different mechanisms. (i) c-FLIP interacts with HBx and protects it from ubiquitin-dependent degradation. The N-terminal DED1 domain of c-FLIP is required for HBx stabilization. (ii) c-FLIP regulates the expression or stability of hepatocyte nuclear factors (HNFs), which have critical roles in HBV transcription and maintenance of hepatocytes. c-FLIP regulates the stability of HNFs through physical interactions. We verified our findings in three HBV infection systems: HepG2-NTCP cells, differentiated HepaRG cells, and primary human hepatocytes. In conclusion, our results identify c-FLIP as an essential factor in HBV replication. c-FLIP regulates viral replication through its multiple effects on viral and host proteins that have critical roles in HBV replication.