The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) and responsible for replicating the whole HCV genome with help of viral and cellular proteins. However, how cellular factors influence NS5B and, in turn, regulating HCV replication are still poorly defined. The well known tumor suppressor Fbw7, a component of E3 ubiquitin ligase SCFFbw7, targets oncoproteins or cellular regulatory proteins for ubiquitin-mediated degradation through a highly conserved binding site called a Cdc4 phosphodegron (CPD). But little is known about whether Fbw7 plays a role in regulation of viral proteins. In this study, we revealed that the conserved CPD is shared by NS5B of almost all genotype of HCV and our data demonstrated that NS5B is a bona fide substrate of Fbw7. Forced expression of Fbw7 promoted the ubiquination of NS5B and negatively regulated its turnover in the proteasome dependent manner. We further revealed the interaction between NS5B and Fbw7, which resulted in the relocation of Fbw7 from nucleus to cytoplasm. During HCV replication, ectopic expression of Fbw7 could strongly down-regulate NS5B level and consequently inhibited the virus replication. When endogenous Fbw7 was knocked down, both NS5B protein abundance and HCV replication were remarkably up-regulated. The results provide more insights into the interplay of HCV and cellular factors and shed light on molecular mechanisms of HCV replication and pathogenesis. (C) 2016 Elsevier Inc. All rights reserved.