OBJECTIVE: To explore the influence of the retinoic acid indicible gene-I (RIG-I) on hepatitis C virus (HCV) replication and the molecular mechanism of action of RIG-I. METHODS: We constructed an RIG-I expression vector and co-transfected it into Huh-7 cells along with HCV-replicon RNA. We assayed HCV replication and NS5A protein synthesis via real-time polymerase chain reaction (RT-PCR) and western blotting. Also, we performed an enzyme-linked immunosorbent assay (ELISA) to measure the level of interferon (IFN)-alpha/-beta secretion. Additionally, we examined, via western blotting, the phosphorylation state of p38, Erk1/2, and nuclear factor (NF)-kappaB p65. RESULTS: Overexpression of RIG-1 in Huh-7 cells co-transfected with an HCV-replicon RNA significantly inhibited HCV replication and NS5A protein synthesis. Co-transfected cells had increased production of IFN-alpha/-beta production and had higher levels of phosphorylated p38, Erk1/2, and NF-kappaB p65. CONCLUSIONS: RIG-I significantly inhibits HCV replication and NS5A protein synthesis by inducing type I IFN production. The underlying molecular mechanism for this effect appears to be mediated by increased phosphorylation of NF-kappaB p65, p38-mitogen-activated protein kinases (MAPK), and Erk1/2.
OBJECTIVE: To explore the influence of the retinoic acid indicible gene-I (RIG-I) on hepatitis C virus (HCV) replication and the molecular mechanism of action of RIG-I. METHODS: We constructed an RIG-I expression vector and co-transfected it into Huh-7 cells along with HCV-replicon RNA. We assayed HCV replication and NS5A protein synthesis via real-time polymerase chain reaction (RT-PCR) and western blotting. Also, we performed an enzyme-linked immunosorbent assay (ELISA) to measure the level of interferon (IFN)-alpha/-beta secretion. Additionally, we examined, via western blotting, the phosphorylation state of p38, Erk1/2, and nuclear factor (NF)-kappaB p65. RESULTS: Overexpression of RIG-1 in Huh-7 cells co-transfected with an HCV-replicon RNA significantly inhibited HCV replication and NS5A protein synthesis. Co-transfected cells had increased production of IFN-alpha/-beta production and had higher levels of phosphorylated p38, Erk1/2, and NF-kappaB p65. CONCLUSIONS: RIG-I significantly inhibits HCV replication and NS5A protein synthesis by inducing type I IFN production. The underlying molecular mechanism for this effect appears to be mediated by increased phosphorylation of NF-kappaB p65, p38-mitogen-activated protein kinases (MAPK), and Erk1/2.