© 2016, American Society for Microbiology. Positive-sense RNA viruses encode RNA-dependent RNA polymerases (Rd. Rps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (Co. Vs), RNA viruses lack proofreading and thus are dependent on Rd. Rps to control nucleotide selectivity and fidelity. Co. Vs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-Rd. Rp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV Rd. Rps. We modeled the CoV murine hepatitis virus (MHV) nsp12-Rd. Rp structure and superimposed it on solved picornaviral Rd. Rp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-Rd. Rp model structure and then engineered into the MHV genome with [nsp14-ExoN(-)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-Rd. Rp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of Rd. Rp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-Rd. Rp, consistent with its proposed role in a multiprotein replicase-proofreading complex.
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