Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs

Brian Villalba,Jiawen Li,Kenneth A Johnson

doi:10.1074/jbc.ra120.013422

Brian Villalba, Jiawen Li + Show 1 more

Open Access

https://doi.org/10.1074/jbc.ra120.013422

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Abstract

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 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, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2'-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.

Highlights

The hepatitis C virus (HCV) infects an estimated 3% of the world’s population, with ;2.4 million people in the United States currently infected [1, 2]
The reactions were measured under single-turnover conditions since the nonstructural protein 5B (NS5B)/9-nt/20-nt elongation complex pauses after the first incorporation because of the absence of the complementary NTP and the dissociation of RNA from the enzyme is exceedingly slow [5]
We set out to understand the kinetic basis for NS5B inhibition by nucleoside analogs and to assess whether they are able to resist ATP-dependent excision

Summary

Introduction

The hepatitis C virus (HCV) infects an estimated 3% of the world’s population, with ;2.4 million people in the United States currently infected [1, 2]. The analogs act as chain terminators by hindering polymerization of the incoming base to prevent further elongation of the nascent RNA strand [11]. These analogs include the prodrugs mericitabine and sofosbuvir, which get metabolized to 29fluoro-29C-methylcytidine-59-triphosphate and 29-fluoro-29Cmethyluridine-59-triphosphate, respectively [7, 12, 13]. WT NS5B has been shown to be ;50-fold more efficient than AZT-resistant forms of HIV RT in removing chain terminators via ATP-mediated excision [14] This raises questions regarding how any chain-terminating nucleoside analog could be effective in treating HCV infections. There is a large discrimination against incorporation of the 29-modified UTP analog compared with unmodified UTP, ATP-dependent excision and pyrophosphorolysis of UTP and its analog are very inefficient

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