Our previous study indicated that the core protein of hepatitis C virus (HCV) can associate with tumor necrosis factor receptor (TNFR)-related lymphotoxin-β receptor (LT-βR) and that this protein-protein interaction plays a modulatory effect on the cytolytic activity of recombinant form LT-βR ligand (LT-α1β2) but not tumor necrosis factor alpha (TNF-α) in certain cell types. Since both TNF-α/TNFR and LT-α1β2/LT-βR are also engaged in transcriptional activator NF-κB activation or c-Jun N-terminal kinase (JNK) activation, the biological effects of the HCV core protein on these regards were elucidated in this study. As demonstrated by the electrophoretic mobility shift assay, the expression of HCV core protein prolonged or enhanced the TNF-α or LT-α1β2-induced NF-κB DNA-binding activity in HuH-7 and HeLa cells. The presence of HCV core protein in HeLa or HuH-7 cells with or without cytokine treatment also enhanced the NF-κB-dependent reporter plasmid activity, and this effect was more strongly seen with HuH-7 cells than with HeLa cells. Western blot analysis suggested that this modulation of the NF-κB activity by the HCV core protein was in part due to elevated or prolonged nuclear retention of p50 or p65 species of NF-κB in core protein-producing cells with or without cytokine treatment. Furthermore, the HCV core protein enhanced or prolonged the IκB-β degradation triggering by TNF-α or LT-α1β2 both in HeLa and HuH-7 cells. In contrast to that of IκB-β, the increased degradation of IκB-α occurred only in LT-α1β2-treated core-producing HeLa cells and not in TNF-α-treated cells. Therefore, the HCV core protein plays a modulatory effect on NF-κB activation triggering by both cytokines, though the mechanism of NF-κB activation, in particular the regulation of IκB degradation, is rather cell line and cytokine specific. Studies also suggested that the HCV core protein had no effect on TNF-α-stimulated JNK activity in both HeLa and HuH-7 cells. These findings, together with our previous study, strongly suggest that among three signaling pathways triggered by the TNF-α-related cytokines, the HCV core protein potentiates NF-κB activation in most cell types, which in turn may contribute to the chronically activated, persistent state of HCV-infected cells.