Interferon (IFN) plays a central role in the innate and adaptive antiviral immune responses. While IFN-alfa is currently approved for treating chronic hepatitis B and hepatitis C, IFN-gamma, in limited studies, has not been shown to be effective for chronic hepatitis B or C. To identify the potential mechanism underlying the differential antiviral effects of IFN-alfa and IFN-gamma, we used cDNA microarray to profile the global transcriptional response to IFN-alfa and IFN-gamma in primary human hepatocytes, the target cell population of hepatitis viruses. Our results reveal distinct patterns of gene expression induced by these two cytokines. Overall, IFN-alfa induces more genes than IFN-gamma at the transcriptional level. Distinct sets of genes were induced by IFN-alfa and IFN-gamma with limited overlaps. IFN-alfa induces gene transcription at an early time point (6 hours) but not a later time point (18 hours), while the effects of IFN-gamma are more prominent at 18 hours than 6 hours, suggesting a delayed transcriptional response to IFN-gamma in the hepatocytes. These findings indicate differential actions of IFN-alfa and IFN-gamma in the context of therapeutic intervention for chronic viral infections in the liver. 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. 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. Furthermore, additional augmentation of gene induction is observed with the transfection of poly (IC) into ribavirin and interferon treated cells when compared to cells treated with these agents separately. Direct upregulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with interferon signal transduction 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-Kappa B pathways in the action of ribavirin. Guanosine blocked ISG induction by ribavirin whereas loxoribine, a guanosine analog, had minimal effect. Additional experiments demonstrated the involvement of a short-lived transcriptional repressor whose activity is inhibited by ribavirin resulting in the upregulation of these antiviral genes. This study suggests that ribavirin, acting via a novel innate mechanism, potentiates the anti-HCV effect of interferon. 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 to peginterferon/interferon and ribavirin were recruited. Patients were given 2 weeks of peginterferon-alfa-2a and ribavirin to establish baseline responses (Course A). Treatment was stopped for a 1-month washout period. Patients then received SAMe 1600 mg daily for 2 weeks, at which point, peginterferon and ribavirin were restarted with SAMe for a planned duration of 48 weeks (Course B). Early viral kinetics and interferon stimulated gene (ISG) expression in PBMC were compared with and without SAMe. Of the 9 patients in the study, all have completed Course A and the first 2 weeks of Course B and 7 have completed 12 weeks of Course B. HCV RNA decline from time 0 to 48 hours was similar between courses (Phase 1 kinetics). There was a significant improvement in the second phase slope of viral decline in 8/9 patients and in the group as a whole with SAMe (A -0.110.07 vs B -0.330.1 log/week, p=0.007). Five of 7 patients, all previous non-responders, have achieved an early virological response (EVR), including 3 who are PCR negative. ISG15 induction in PBMC was statistically significantly greater in 6 of 9 patients and for the group as a whole during Course B than Course A (AUC: A 13324 vs B 18726, p=0.001). ISG15 fold induction was greater at all time points, with the greatest difference seen at 24 hours (A 17154 vs B 350166, p=0.04). A similar pattern was observed with MxA. ISG15 and MxA expression were greater at time 0 in Course B than A, after receiving 2 weeks of SAMe alone. During 2 weeks of SAMe monotherapy, ALT values decreased significantly (mean change 37 U/ml, p=0.004) but HCV RNA levels increased slightly (mean change 0.22 log, p=0.05). SAMe appears to improve early viral kinetics in interferon non-responders and the observation that it leads to greater ISG induction suggests that the effect may be through improved interferon signaling.