Reply:
Authors: Emmanuel Thomas, Jake T. Liang
Journal: Hepatology (2011)

We appreciate the interest and comments of Dr. Pan and colleagues regarding our recent article on the role of ribavirin (RBV) in combined therapy for hepatitis C infection.1 We demonstrated the up-regulation of a subset of interferon-stimulated genes (ISGs) after RBV treatment (which had previously been seen in patient studies2, 3) in cell culture and human models of hepatitis C virus infection. Pan et al.'s letter further highlights the ability of RBV to stimulate gene transcription from promoters that are important for antiviral responses. The nucleotide motif responsible for the up-regulation of ISGs after exposure to interferon (IFN) is known as the interferon-stimulated response element (ISRE), and it is composed of the sequence GAAANNGAAACT.4, 5 Pan et al. used an additional cell culture model to explore the effects of RBV on an ISRE-driven luciferase construct in Huh7 cells. Indeed, other groups have used ISRE-driven luciferase constructs to explore RBV's mechanism of action and also have reported stimulation of the ISRE in cell culture models.6 We also performed experiments in cell culture models with a multimeric ISRE-driven luciferase construct (Stratagene, La Jolla, CA), containing a promoter sequence similar to the one used by Pan et al. Figure 1 shows slight stimulation of this ISRE construct after exposure to RBV (mean) in comparison with an unstimulated control; however, this stimulation was much weaker than the stimulation following IFN-α treatment, which was used as a positive control. Effect of RBV on ISRE-driven gene transcription. Huh7.5.1 cells were transiently transfected with an ISRE-driven luciferase construct (1 μg/mL) for 24 hours and were subsequently treated with IFN-α (100 U/mL) or RBV (1, 10, or 100 μg/mL) for 24 hours. As Pan et al. suggested, the stimulation of ISRE promoter elements could be responsible for RBV-induced changes in gene expression. Although this issue is presently being clarified, the role of transcription factors known to be important for ISRE stimulation (e.g., members of the signal transducer and activator of transcription and IFN regulatory factor families)5 will have to be demonstrated. Indeed, we have studied these molecules in RBV-stimulated gene induction and have been unable to show a definitive role for them.1 Furthermore, the selective up-regulation of ISGs by RBV in our article will have to be explained because the stimulation of ISRE motifs should result in a broad up-regulation of ISGs similar to that seen after IFN-α treatment. Current studies should be directed toward a better understanding of the effect of RBV on ISG induction in these and other cell culture models of hepatitis C virus infection. In summary, we thank Pan et al. for their contribution. Additional experiments with RBV will continue to yield useful information pertaining to its mechanism of action. The ultimate goal of these experiments is to spur the generation of novel RBV-like molecules with more potent antiviral activity. Emmanuel Thomas M.D., Ph.D.*, T. Jake Liang M.D.*, * Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.