VEGF-Induced Endothelial Cell Migration Requires Nox2 and Nox4-Dependent S-Glutathiolation of SERCA

Ethanol and Reactive Species Increase Basal Sequence Heterogeneity of Hepatitis C Virus and Produces Variants With Reduced Susceptibility To Antivirals Jinah Choi, Scott Seronello, Jessica Montanez, Kristen Presleigh, Thu Vo, Seung Bum Park, and Miriam Barlow University of California, Merced Hepatitis C virus exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. in addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism and signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence heterogeneity of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreased intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the HCV database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of hepatitis C virus RNA by 4–18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Tyrosine phosphorylation of Stat1 increased with ethanol and BSO treatments, suggesting a role of redox signaling. Therefore, virus-induced oxidative/nitrosative stress is likely to synergize with ethanol to modulate the basal mutation rate of HCV and contribute to antiviral resistance through positive selection.