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Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

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Viral infections activate cellular expression of type I interferons (IFNs). These responses are partly triggered by RIG-I and mediated by Cardif, TBK1, IKKe and IRF-3. This study analysed the mechanisms of dsRNA-induced IFN responses in various cell lines that supported subgenomic hepatitis C virus (HCV) replication. Transfection of dsRNA into Huh7, HeLa and HEK293 cells induced an IFN expression response as shown by IRF-3 dimerization, whilst these responses were abolished in corresponding cell lines that expressed HCV replicons. Similarly, RIG-I-dependent activation of the IFN-stimulated response element (ISRE) was significantly suppressed by cells expressing the HCV replicon and restored in replicon-eliminated cells. Overexpression analyses of individual HCV non-structural proteins revealed that NS4B, as well as NS34A, significantly inhibited RIG-I-triggered ISRE activation. Taken together, HCV replication and protein expression substantially blocked the dsRNA-triggered, RIG-I-mediated IFN expression response and this blockade was partly mediated by HCV NS4B, as well as NS34A. These mechanisms may contribute to the clinical persistence of HCV infection and could constitute a novel antiviral therapeutic target. INTRODUCTIONType I interferon (IFN) plays a central role in eliminating virus, not only following clinical therapeutic application but also as a cellular immune response (Samuel, 2001;Taniguchi & Takaoka, 2002). Hepatitis C virus (HCV) infection is characterized by persistence and replication of the virus in the liver, despite an intact host immune system (Alter, 1997). Indeed, even after administration of the currently most potent IFN reagents, as many as half of the patients are refractory to the treatment and fail to eradicate the virus (Fried et al., 2002). These features have led to speculation that HCV escapes from or attenuates the host antiviral response . Cellular antiviral responses are primarily mediated by IFN and IFN-stimulated genes (ISGs), including 2,5-oligoadenylate synthetase, dsRNA-dependent protein kinase R (PKR) and MxA proteins, as well as by as yet uncharacterized genes Stark et al., 1998). A study of experimental chimpanzee HCV infection has shown that various cytokines and chemokines are induced in the liver during the course of acute HCV infection and its clearance, and that a considerable proportion of the genes is induced by type I IFN (Bigger et al., 2001).Control of expression of ISGs is mediated by binding of type I IFNs to their receptors. Following receptor binding, STAT1 and STAT2 are phosphorylated to form ISGF-3, which translocates to the nucleus and binds the IFN-stimulated response element (ISRE), located in the promoter/enhancer region of ISGs, and activates transcription of ISGs (Samuel, 3These authors contributed equally to this work. Plasmid pcDNA3.1 (Invitrogen) was used as an empty vector for mock transfection. pRL-CMV (Promega), which expressed the Renilla luciferase protein, was used for correction of transfection efficiency.Cell culture. HCV strain JFH1-...