Ribavirin enhances interferon-stimulated gene transcription by activation of the interferon-stimulated response element
Authors: Qiuwei Pan, Hugo W. Tilanus, Harry L.A. Janssen, Luc J.W. van der Laan
Journal: Hepatology (2011)

Ribavirin, a synthetic nucleoside analogue, is used in combination with pegylated interferon-α (IFN-α) as the standard of care for the treatment of patients with chronic hepatitis C. The combination of ribavirin significantly improves the sustained virologic response of IFN-α therapy, but the exact mechanism remains enigmatic.1 Although ribavirin monotherapy appears to have only limited clinical efficacy,1, 2 in vitro studies have shown that ribavirin by itself has a remarkable broad antiviral activity, equivalent to IFNs, against a spectrum of RNA and DNA viruses.3 Now, an exciting new study by Thomas et al. in HEPATOLOGY4 shows that ribavirin treatment induces the expression of particular IFN-stimulated genes (ISGs), including IRF7 and IRF9, thereby potentiating the antiviral action of IFN-α in hepatitis C virus (HCV) cell culture models. Because the transcription factors IRF7 and IRF9 are known to be critical for antiviral defenses, including against HCV, the authors conclude that antiviral action of ribavirin alone1, 2 and in particular in combination with IFN-α4 acts via the induction of ISGs. This study supports earlier clinical evidence by the same group that patients receiving ribavirin in addition to IFN-α had a more rapid and higher elevation of the IFN-induced cytokine, IFN-inducible protein 10/chemokine (C-X-C motif) ligand 10.5 The authors put great effort into further unraveling the signal transduction events leading to induction of ISGs and rule out the involvement of nuclear factor κB, IFN-β promoter stimulator 1, and signal transducer and activator of transcription 1 signaling pathways. However, the direct mechanism by which ribavirin induces gene expression of ISGs is not resolved. While studying the antiviral action of inosine-5'-monophosphate dehydrogenase inhibitors,6 we have found new evidence to explain how ribavirin promotes the transcription of a broad range of ISGs. It is known that gene expression of most ISGs are regulated by particular promoter elements, the so-called IFN-stimulated response element (ISRE). IFN stimulation will trigger the binding of particular transcription factors to the ISRE, thereby enhancing the transcription of the ISG genes.7 To mimic this biological process, we used a lentiviral transcriptional reporter system expressing the firefly luciferase gene driven by a promoter containing multiple ISREs (SBI Systems Biosciences, Mountain View, CA). Huh7 cells, the HCV permissive host cell line, were transduced with this vector to create a stable reporter cell line (Fig. 1A). As expected, stimulation with IFN-α resulted in a dose-dependent induction of luciferase activity (Fig. 1B), up to three times the baseline activity. Remarkably, treatment with clinical achievable doses of ribavirin8 also resulted in a dose-dependent induction of ISRE-related luciferase activity (Fig. 1C). For instance, 20 μg/mL of ribavirin increased the ISRE luciferase activity by 45% ± 12% (mean ± SEM, n = 5 [P < 0.05, Mann-Whitney test]) compared with the unstimulated control. No effect on cell viability or control (non-ISRE) luciferase activity was observed (data not shown), suggesting that ribavirin directly augments the ISRE-mediated transcription activity. To address whether ribavirin can potentiate the IFN-induced ISRE transcription activity, we treated the Huh7 reporter cells with a combination of IFN-α and ribavirin. As shown in Fig. 1C, combining IFN (1 or 10 IU/mL) with the lowest dose of ribavirin (0.2 μg/mL) significantly increased the luciferase activity, and combining it with the highest dose (100 μg/mL) enhanced the luciferase activity by 65% ± 17% (mean ± SEM, n = 6 [P < 0.01, Wilcoxon matched pairs test]) compared with IFN treatment alone. Ribavirin enhances ISG transcription by activation of ISRE. (A) A stable reporter cell line containing a luciferase gene under the control of multiple ISREs in the promoter region was used to investigate ISG expression. This cell line was generated by stable transduction of Huh7 cells with a lentiviral transcriptional reporter system. (B) Stimulation with IFN-α for 24 hours resulted in dose-dependent enhancement of luciferase activity in Huh7 cells, up to three to four times the baseline level. (C) Treatment of the Huh7 reporter cells with different doses of ribavirin (2, 20, or 100 μg/mL for 24 hours) resulted in a dose-dependent induction of luciferase activity (triangles) (P < 0.05). When combining ribavirin with 1 IU/mL (squares) or 10 IU/mL (dots) of IFN-α, a significant increase in ISRE-dependent luciferase activity was observed. At the highest dose, ribavirin enhanced luciferase activity by 65% compared with IFN-α alone (P < 0.01). Data are presented as the mean and SEM of relative luciferase activity from three independent experiments (2-3 replicates per condition). Taken together, our findings show that ribavirin potentiates the transcription activity of ISRE and explain the enhanced expression of ISGs when combined with IFN-α.4, 5 Moreover, it is known that ISRE regulates the expression of the IP-10/CXCL10, IRF7, and PKR genes, thereby providing a molecular basis for the observed effect of ribavirin on the expression of these genes.9, 10 Further understanding of the interplay between IFNs and ribavirin could be useful in advancing therapeutic strategies for hepatitis C. Qiuwei Pan*, Hugo W. Tilanus , Harry L.A. Janssen*, Luc J. W. van der Laan , * Department of Gastroenterology & Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands, Department of Surgery and Laboratory of Experimental Transplantation and Intestinal Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands.