151 - RNA-Dependent RNA Polymerase Activity of HCV NS5B Protein Is Modulated by Glutathionylation

Abstract

Hepatitis C virus (HCV) is a human blood-borne pathogen that causes chronic liver disease. Chronic hepatitis C often leads to development of liver fibrosis, cirrhosis and hepatocellular carcinoma as well as other hepatic and extrahepatic pathologies. HCV genome is presented by a short positive strand RNA encoding ten structural and non-structural proteins. Among these non-structural protein 5B (NS5B) exhibits RNA-dependent RNA-polymerase activity capable of replicating genomic RNA de novo thus acting as a key component of HCV replicase. Investigation of mechanisms of regulation of HCV replication was focused mostly on search for NS5B-interacting viral and cellular proteins. Much less attention was given to metabolic alterations in the infected cells. It was already shown that HCV triggers massive reactive oxygen species (ROS) production, and virus replication can be inhibited by extracellular H 2 O 2 . Our aim was to explore possible redox modifications of NS5B protein as a link between these events. Using antibodies against glutathionylated proteins we showed that the recombinant NS5B protein of 1b or 2a genotypes, expressed in E.coli , contain glytathione residues. Treatment of NS5B with oxidized glutathione increases degree of glutathionylation, whereas reduced glutathione causes opposite effect. Increase of NS5B glutathionylation degree was accompanied by inhibition of its enzymatic activity both in primer-dependent and primer-independent ( de novo ) systems. MALDI MS analysis revealed that glutathionylation of the recombinant protein of 2a genotype occurs at cysteine residues at positions 89, 146, 274, and 279/295, and GSSG treatment affected residues 170, 223, and 521. Construction of a panel of NS5B mutants with substitution of any single cysteine by serine demonstrated that C279S mutant lost its enzymatic activity, whereas other mutant forms retained sensitivity to glutathione and reducing agents. Currently these data are being verified in HCV cellular replicon system. Acknowledgements This study was supported by Russian Science Foundation (grant #14-14-01021).