Regardless of the regimen, HCV RNA can be undetectable in blood for months only to reappear after treatment ends, causing relapse. Ribavirin is a broad-spectrum antiviral drug that reduces relapse when used in combination with interferon and/or with DAAs such as sofosbuvir/ledipasvir. Methods: HCV RNAs were quantified in extracts of human liver and cultured cells. Huh-7.5 cells
replicating Belnacasan supplier Con1/JFH virus were treated with HCV inhibitors, interferon α-2b (IFN; 3 IU/mL 9 IU/mL), ribavirin (25 or 100 μM), and 2′-C-methyl adenosine (2′CMA; 0.22 to 2.2 μM). To allow HCV double-stranded (ds)RNA detection, RNA duplexes were denatured prior to qPCR. RNA was also studied using RNase III (cuts dsRNA), RNase A and RNase T1 (cuts ssRNA), and Northern blotting. Bead array (Illumina) and Western blotting were used to study pathways differentially regulated by IFN compared to IFN/ribavirin. Results: Because relapse is an important clinical problem and ribavirin reduces relapse, we investigated pathways altered by the addition of ribavirin to HCV-infected Huh-7.5 cells treated with IFN. Microarray analysis revealed that IFN-treated
cells had elevated levels of activated PKR, an antiviral protein that binds to and is activated by dsRNA. Ribavirin blocked PKR activation, GSK2118436 concentration suggesting that IFN caused an increase in viral dsRNA (activating PKR) and ribavirin prevented this shift in the viral RNA population. To explore this possibility, RNA from various sources was heated to 106 °C to denature long dsRNA prior to reverse transcription. Using this approach we found that HCV dsRNA is the predominant form of viral RNA in the liver of HCV-infected patients. The abundance of HCV dsRNA correlates with interferon-stimulated gene induction. Northern blotting and ribonuclease digestion showed that IFN increased production of genome-length HCV dsRNA in HCV-infected Huh-7.5 cells and dramatically altered the ratio of HCV plus and minus strands, reducing the level of plus strands while maintaining RG7420 or increasing the level of minus strands
thereby preserving the capacity for progeny plus strand synthesis. This process required de novo production of viral RNA and dsRNA synthesis and was blocked by ribavirin. Conclusions: Our findings demonstrate that HCV can respond to IFN by producing a genome-length viral dsRNA. This dsRNA is a key target of ribavirin. The development of DAAs that target viral dsRNA might improve treatment for HCV and other viruses (DA031095, DK090317). Disclosures: Andrea D. Branch – Grant/Research Support: Kadmon, Gilead, Janssen The following people have nothing to disclose: Arielle L. Klepper, Francis J. Eng, Adeeb Rahman, Brannon Weeks, Ahmed El-Shamy, Erin H. Doyle, M. Isabel Fiel, Gonzalo Carrasco-Avino, Sasan Roayaie, Meena B. Bansal, Margaret R. MacDonald, Thomas D.