UC Merced

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is estimated to have infected 170 million people worldwide. HCV infection can lead to serious liver disease such as cirrhosis, steatosis, and hepatocellular carcinoma. In patients, HCV induces an altered redox status and exhibits a high level of genetic variability, existing as quasispecies. Ethanol consumption has been associated with increased HCV pathogenesis, elevated HCV titer, increased sequence heterogeneity of HCV RNA, and decreased efficacy of antiviral treatment, suggesting multiple modes of interaction between ethanol and HCV. The goal of my studies was to elucidate the mechanisms of these multiple modes of interaction between alcohol, oxidative stress, and hepatitis C. By treating Huh7 human hepatoma cells that naturally express CYP2E1 with physiological relevant concentrations of ethanol or its metabolites, we determined that HCV replication was elevated in the context of the complete virus life cycle. This potentiation of HCV replication was dependent on CYP2E1 activity the host mevalonate pathway, fatty acid synthesis, and an increased NADH/NAD+ ratio. In contrast, reactive oxygen species rapidly suppressed HCV replication as previously shown using HCV replicons. These results suggested that lipid metabolism and alteration of the NADH/NAD+ ratio played an important role in the enhancement of HCV replication by ethanol. Finally, we were able to demonstrate that exposing HCV-replicating cells to ethanol and reactive species elevated both the nucleotide and amino acid substitution rates of HCV RNA, which were accompanied by oxidative RNA damage. The overall dN/dS ratio and percentage of sites undergoing positive selection increased with pronounces changes in the serine/threonine/tyrosine sites. In addition, HCV itself induced oxidative RNA damage that was exacerbated by ethanol and BSO. Agents that decreased the RNA damage also reduced the sequence heterogeneity of the HCV RNA. Conclusions/Significance: Therefore, ethanol potentiates HCV replication through the alteration of lipid metabolism and the NADH/NAD+ ratio, which is likely to contribute to the higher viral titer and pronounced pathogenesis in patients who drink alcohol. Ethanol may also synergize with virus-induced oxidative/nitrosative stress to induce RNA damage and likely amplify the error rate of the HCV replicase in the development of quasispecies, iincreasing the probability of immune evasion and antiviral resistance.