Kinetic characterization of the inhibition, excision mechanisms, and fidelity of Hepatitis C Virus RNA-dependent RNA polymerase

NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the Hepatitis C Virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs Mericitabine and Sofosbuvir, which get metabolized to the chain terminators 2’-fluoro-2’-C-methylcytidine-5’-triphosphate and 2’fluoro-2’-C-methyluridine-5’-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, work in our lab has shown that NS5B can efficiently remove chain-terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study I use transient-state kinetics to probe the mechanism of inhibition for nucleoside analogs by directly measuring the rates of incorporation, pyrophosphorolysis, and ATP-mediated excision. I find that while CTP and CTP analogs are readily incorporated, they are efficiently excised. However, UTP is highly resistant to excision, and the 2’-C modifications of UTP serve to further inhibit excision. Furthermore, I use these same techniques to measure the in vitro fidelity of NS5B and uncover mechanisms for maintaining fidelity. The data demonstrate that NS5B exhibits a range of fidelity dependent on the nature of the mismatch. I also identified a slow-pyrophosphorolysis mechanism by NS5B used to further increase fidelity by decreasing k [subscript cat] /K [subscript m]. Together, this work offers insight into how current antiviral therapeutics escape excision, and can aid in the development of new antivirals by furthering our understanding of NS5B.