Murine Hepatitis Virus Regulation of Nucleotide Selectivity and Fidelity by the RNA-dependent RNA Polymerase and the 3’-5’ Exoribonuclease

Abstract

All positive sense (+)RNA viruses encode RNA-dependent RNA polymerases (RdRps), essential for replication and regulation of the fidelity of nucleotide addition to nascent genomes. The coronavirus (CoV) RdRp is encoded in nonstructural protein 12 (nsp12). CoV replication utilizes a large complex of proteins, many of which may influence replication fidelity. Specifically, nsp14 encodes a proofreading 3'-5' exoribonuclease (ExoN) in nsp14 that when inactivated (nsp14-ExoN(-)) results in decreased replication fidelity with up to a 20-fold increase in mutations across the CoV genome. Nsp12-RdRp and nsp14-ExoN interact during replication; however, the role of nsp12-RdRp in CoV replication fidelity was not known. To test the role of nsp12-RdRp, we identified residues predicted to participate in replication fidelity by homology modeling, minority variants present after treatment with mutagen, and consensus level mutations present after treatment with the mutagen 5-fluorouracil (5-FU). Mutations, at identified residues, were engineered into MHV nsp14-ExoN(+) and nsp14-ExoN(-) isogenic backgrounds. Viable recovered viruses, as well as populations of virus from passage in the presence of mutagen, were tested for sensitivity to the mutagens 5-fluorouracil (5-FU) or 5-azacytidine (5-AZC). Additionally, changes in replication kinetics, RNA synthesis, competitive fitness, reversion, and/or the accumulation of mutations were quantified. These results demonstrate that nsp14-ExoN masks the contributions of nps12-RdRp to the fidelity of genome replication for CoVs. However, fidelity determinants in nsp12-RdRp still affect replication kinetics and fitness. CoVs encode an extensive replication complex with a number of proteins likely involved in fidelity regulation. These results are essential to understanding the core of this fidelity complex.