Our data suggest that ribavirin may exert its antiviral activity in patients treated with combination therapy by potentiating the effect of IFN action on ISG induction, which results in enhanced antiviral responses. Although we observed the induction of several antiviral genes by ribavirin, IRF7 and IRF9 are of particular interest. Both IRF7 and IRF9 have been shown to be critical for antiviral responses in mice through their roles in endogenous IFN production and signaling.36,37
Furthermore, IRF9 (also known as ISGF3G) has been shown to have direct antiviral activity against HCV in tissue culture (),38
and IRF7 mRNA levels from peripheral blood mononuclear cells have been shown to correlate with responses to antiviral therapy in HCV-infected patients.39
In addition to their direct antiviral effects, IRF7 and IRF9 may be of particular importance because of their role in amplifying the IFN signaling cascade. Improved IFN signaling through the up-regulation of these molecules would have the potential to significantly improve the effects of PEG-IFN treatment.
We have observed that treatment of cells in tissue culture with ribavirin leads to a dose-dependent induction of a subset of ISGs. This observation was unexpected given that the induction of ISGs is a tightly regulated process usually occurring only during viral infections and pathological states that result in the production of IFN.40
Furthermore, ribavirin was also shown to have antiviral effects on JFH1 infection () and on various HCV replicon–containing cells12,41–43
when ribavirin was administered at similar doses used in this study.43
Several studies have demonstrated that ribavirin’s beneficial effect on HCV treatment outcomes is dose-dependent and that higher doses of ribavirin result in higher response rates.44–47
During antiviral therapy, the levels of ribavirin achieved in the serum are 1–4 μ
Correspondingly, we have observed reproducible changes in gene expression and synergistic effects on gene induction by the combination of ribavirin and IFN with doses of ribavirin as low as 1 and 3 μ
g/mL in tissue culture, and these effects were dose-dependent (; Supporting Figs. 1 and 3
Previous pharmacodynamic studies have shown that an extended dosing period (≈4 weeks) is needed to reach steady-state blood concentration.51
Furthermore, the tissue concentration of ribavirin may be substantially higher, because ribavirin tends to concentrate in certain tissues. Indeed, several studies have shown that the intracellular concentration of ribavirin in erythrocytes can reach over 100 times greater than that that found in plasma52,53
and that hepatocytes have specific nucleoside transporters that facilitate uptake of ribavirin from plasma.54
In this study, we used ribavirin at doses up to 100 μ
g/mL in infected or uninfected cells for 24 hours in tissue culture experiments without any observable toxicity (Supporting Fig. 8
and data not shown).
The up-regulation of ISGs by ribavirin may be due to the inhibition of a transcriptional repressor. Other studies have shown that ISGs are under the control of transcriptional repressors, and this may be an increasingly important mechanism for ISG regulation.55
Our data also suggest that this repressor may be a short-lived protein degraded by the 26S proteosome. Consequently, treatment with proteosome inhibitors stabilize this protein, making cells resistant to the effect of ribavirin and cycloheximide on IRF7, IRF9, and ISG15 (). The addition of guanosine to the medium can also block ribavirin-induced gene induction and antiviral activity in a dose-dependent fashion. The ability of guanosine to block ribavirin activity may point to involvement of the IMPDH pathway. IMPDH inhibition by ribavirin could induce guanosine depletion, and guanosine depletion may lead to IRF7/IRF9 induction, which may explain why gene induction can be reversed by supplementation of guanosine to the culture medium. Indeed, IMPDH inhibitors have shown antiviral activity in cell culture models of HCV infection.12,56
However, at this time, no IMPDH inhibitors have obtained U.S. Food and Drug Administration approval for the treatment of hepatitis C, indicating that this may not be the only mechanism by which ribavirin exerts its antiviral activity.57,58
Additional studies will be needed to determine if IMPDH inhibitors can effect ISG induction by IFN in cell culture models of virus infection as well as in patients.
Recognition of ribavirin’s ability to induce ISGs may explain ribavirin’s antiviral activity against numerous viruses and will facilitate the discovery of a class of therapeutic agents that possess more potent antiviral activity by specifically regulating antiviral genes. Importantly, ribavirin’s effect on ISG regulation appears to be IFN-independent (; Supporting Figs. 4B and 6
). Several lines of evidence support the IFN-independent activity of ribavirin. First, we have not observed the full spectrum of ISG induction after ribavarin administration seen following IFN treatment ( and ). Second, RNAi against signaling components, which are critical for IFN production and activity, did not affect ribavirin-stimulated gene induction (). Finally, we did not observe gene induction of either the type I or type III IFNs following ribavirin treatment (Supporting Fig. 6A
). This may be particularly relevant for viruses such as HCV that have developed numerous strategies to circumvent the IFN system.59
This class of molecules, like ribavirin, may thus be able to potentiate the antiviral effects of IFN through synergistic induction of antiviral genes using different molecular mechanisms.
Although it was originally thought that the development of agents specifically targeting steps in the HCV life cycle would soon make IFN and ribavirin obsolete, emerging data suggest that this may not be the case. Because direct antivirals quickly select for resistant strains, it appears that PEG-IFN will likely be required to achieve an optimal response. Importantly, recent trials with HCV protease inhibitors have shown that ribavirin is also needed to prevent viral relapse.60
Therefore, the search for alternative ribavirin-like molecules in potentiating IFN action and for use in combination with specifically targeted antiviral agents remains a promising approach in the therapy of HCV-infected patients.