The combination of peginterferon and ribavirin is the standard treatment for chronic hepatitis C. Data from our recent clinical study suggests that ribavirin augments the induction of interferon stimulated genes (ISGs) in patients treated for HCV infection. In order to further characterize the mechanisms of action of ribavirin in combination therapy, we examined the effect of ribavirin treatment on ISG induction in mammalian cells grown in tissue culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was also studied. In order to further characterize the mechanisms of action of ribavirin, we examined the effect of ribavirin treatment on ISG induction in cell culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was also studied. Similar to interferon-alfa, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative PCR assays demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs. These ISGs, including IRF7 and IRF9 are known to play an important role in anti-HCV responses. When ribavirin is used in conjunction with interferon, induction of specific ISGs is synergistic when compared to either drug applied separately. Direct up-regulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with interferon signaling and intracellular double stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and NF-KappaB pathways in the action of ribavirin. In conclusion, our study suggests that ribavirin, acting via a novel innate mechanism, potentiates the anti-HCV effect of interferon. Understanding the mechanism of action of ribavirin would be valuable in identifying novel antivirals. As mentioned above, ribavirin appears to augment induction of IFN-stimulated genes (ISGs). The effect of ribavirin on liver gene expression in humans has not been examined. To elucidate the mechanism of action of ribavirin, we studied its effects on serum enzymes, HCV RNA levels, serum cytokines and hepatic gene expression. 42 treatment-nave patients with chronic hepatitis C were randomized to receive weight-based ribavirin (1200 or 1000 mg/d) or no treatment for 4 weeks, after which standard combination therapy with Peg-IFN-alfa-2a and ribavirin was started. RNA was extracted from liver biopsies of 6 patients on no treatment, 6 patients after 4 weeks of ribavirin alone prior to the first Peg-IFN injection and 6 patients 6 hours after the first Peg-IFN injection (and no ribavirin). Gene expression was assessed (genotype 1 only) using the Affymetrix U133 Plus 2.0 array. Serum HCV titers, ALT and serum levels of selected cytokines including interferon-gamma-inducible protein-10 (IP10), monokine induced by interferon-gamma (MIG), and monocyte chemoattractant protein 1 (MCP1) were measured before and at the end of 4 weeks of ribavirin. The groups were similar in baseline factors and genotype distribution. After 4 weeks of ribavirin treatment, HCV RNA levels decreased by a mean of 0.59 log10 (p<0.001) and serum ALT by 30 U/L (p<0.001). Patients with genotype 2/3 had a greater decline than those with genotype 1 (HCV 0.83 vs. 0.45 log10, p=NS;ALT 47 vs. 22 U/L, p=0.036). Serum IP10 levels declined by a mean of 253 pg/mL after 4 weeks of ribavirin (p<0.001), in correlation with the decreases in HCV RNA and ALT. MIG and MCP1 levels did not change. As expected, serum ISG levels increased after the dosing of PEG-IFN. In untreated patients, there was no change in HCV titers, ALT or serum cytokine levels. Microarray analysis of hepatic gene expression showed that ribavirin alone caused upregulation of ISG RNA levels and down-regulation of genes inhibited by IFN, in a manner similar to Peg-IFN but to a lesser degree. There was a strong positive correlation between the patterns of hepatic gene induction by ribavirin and PEG-IFN (r2=0.24, p<0.001). A 4 week course of ribavirin alone decreased HCV RNA and ALT levels (with a corresponding decrease in serum IP10), possibly through a weak induction of ISGs. These results support the potentiating effect of ribavirin in enhancing the antiviral action of IFN in HCV therapy. S-adenosyl methionine (SAMe) has been shown to enhance interferon signaling in cell culture and animal models by acting as a methyl donor to STAT1, leading to improved STAT1-DNA binding. We performed a clinical study to assess the effect of SAMe on interferon signaling and early viral kinetics in previous non-responders to peginterferon and ribavirin. Previous genotype-1 non-responders were given 2 weeks of peginterferon alfa-2a and ribavirin to establish baseline responses (Course A). After a 1-month washout, patients received SAMe 1600mg daily for 2 weeks, whereupon peginterferon and ribavirin were added for 48 weeks (Course B). Viral kinetics and interferon stimulated gene (ISG) expression in PBMCs were compared between courses. 21 of 24 patients completed both Course A and B, serving as their own controls. HCV RNA decline from 0 to 48 hours (phase 1) was similar with and without SAMe. In contrast, the second phase slope of viral decline was improved with SAMe (Course A 0.110.04 vs Course B 0.270.06 log10IU/mL/week, p=0.009). 11 (53%) patients achieved an early virological response and 10 (48%) had undetectable HCV RNA by week 24. Induction of ISGs in PBMCs was significantly greater in Course B. Cell culture data confirmed that SAMe augmented ISG induction and antiviral effects of interferon by increasing STAT1 methylation, possibly affecting STAT1-DNA binding. The addition of SAMe improves early viral kinetics and ISG induction in interferon non-responders. SAMe holds promise as an adjunct to peginterferon-based therapy in chronic HCV infection. IFN acts by inducing IFN Stimulated Genes (ISGs). Of the hundreds of ISGs, few have been well characterized and shown to be antiviral. In order to identify and characterize genes responsible for the anti-HCV activity of IFN using a high-throughput siRNA approach. 430 genes were identified as induced by IFN (ISGs) or involved in IFN signaling without induction (non-ISG) from microarray studies and extensive literature review. A two-part screen is being conducted to identify genes with activity at different stages of the HCV lifecycle.
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