Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNAdependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-␣)-induced NF-B and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-␣-induced NF-B and JNK activation. In this study, we have demonstrated that TNF-␣-induced NF-B activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2-and IKK-induced NF-B activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKK␣. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKK␣, and then TNF-␣-mediated IKK␣ kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-␣-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-␣ signaling pathways and may contribute to HCV pathogenesis.Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma (1, 14). More than 170 million individuals worldwide are infected with HCV. HCV belongs to a member of the Flaviviridae family and contains a single-stranded, positivesense RNA genome of ϳ9,600 nucleotides in length. The HCV genome encodes a single polyprotein precursor of approximately 3,010 amino acids that is cleaved by both cellular signal peptidase and viral protease to generate structural and nonstructural proteins (19,21,33,35). The N-terminally localized core and envelope proteins (E1 and E2) are viral structural proteins, and the remainders of the genome are nonstructural proteins. The nonstructural protein 5B (NS5B) is an RNAdependent RNA polymerase. The NS5B is the key enzyme that catalyzes the replication of HCV. We have previously demonstrated that NS5B is a phosphoprotein that is predominantly localized in the perinuclear region in the cytoplasm (24). Although RNA-dependent RNA polymerase activities have been demonstrated using both bacterially and baculovirus-expressed NS5B proteins (8,16,34,39,40,60), the detailed mechanism of HCV replication is poorly understood by the lack of an efficient cell culture system. The NS5B has been extensively characterized at the biochemical (8, 34) and structural levels (10, 30) and has been a prime target for inhibitors of HCV replication.The nuclear transcription factor NF-B plays a critical role in regulating the expression of many cytokines and immunoregulatory proteins (4-6). The NF-B complex is composed of homodimers or heterodimers of Rel and NF-B proteins, including NF-B1, NF-B2, p65, Rel B, and c-Rel (4). The activity of NF-B can be elevated by various stimuli, including tumor necrosis factor (TNF), interleukin 1 (IL-1), and phorbol esters (55). In most cells,...