Interleukin-1b (IL-1b) is a potent pro-inflammatory cytokine involved in the pathogenesis of HCV, but the sensors and underlying mechanisms that facilitate HCV-induced IL-1b proteolytic activation and secretion remains unclear. In this study, we have identified a signalling pathway leading to IL-1b activation and secretion in response to HCV infection. Previous studies have shown the induction and secretion of IL-1b through the inflammasome complex in macrophages/monocytes. Here, we report for the first time the induction and assembly of the NALP3-inflammasome complex in human hepatoma cells infected with HCV (JFH-1). We demonstrate that activation of IL-1b in HCVinfected cells involves the proteolytic processing of pro-caspase-1 into mature caspase-1 in a multiprotein inflammasome complex. Next, we demonstrate that HCV is sensed by NALP3 protein, which recruits the adaptor protein ASC for the assembly of the inflammasome complex. Using a small interfering RNA approach, we further show that components of the inflammasome complex are involved in the activation of IL-1b in HCV-infected cells. Our study also demonstrates the role of reactive oxygen species in HCV-induced IL-1b secretion. Collectively, these observations provide an insight into the mechanism of IL-1b processing and secretion, which is likely to provide novel strategies for targeting the viral or cellular determinants to arrest the progression of liver disease associated with chronic HCV infection.

In this study, we demonstrated the molecular mechanisms of TGF-β1 induction as well as proteolytic activation in HCV (JFH-1)-infected cells. Our studies showed the synthesis and secretion of TGF-β1 in HCV-infected cells which was reduced in the presence of Ca2+ chelators, an inhibitor of mitochondrial Ca2+ uptake, and antioxidants. We also showed that the expression of HCV NS proteins NS3/4A, and NS5A can induce TGF-β1 by cell-based luciferase assay. Furthermore, mutational analysis revealed that the functionally active protease domain of NS3 and N-terminus domain of NS5A are required for TGF-β1 activity. Using siRNA approach we demonstrated that HCV-induced furin and thrombospondin-1 (TSP-1) are involved in the proteolytic activation of TGF-β1. Our results also suggest that TGF-β1 positively regulates HCV RNA replication. Collectively, these observations provide insight into the mechanism of TGF-β1 activation, which likely manifest in liver fibrosis associated with hepatitis C infection.