Mutations in Coronavirus Nonstructural Protein 10 Decrease Virus Replication Fidelity

Coronaviruses (CoVs) are unique in encoding a 3=¡5 = exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication, likely via proofreading. nsp14 associates with the CoV RNA-dependent RNA polymerase (nsp12-RdRp), and nsp14-ExoN activity is enhanced by binding nsp10, a small nonenzymatic protein. However, it is not known whether nsp10 functions in the regulation of CoV replication fidelity. To test this, we engineered single and double alanine sub-stitutionmutations into the genome of murine hepatitis virus (MHV-A59) containing ExoN activity [ExoN()] at positions within nsp10 known to disrupt the nsp10-nsp14 interaction in vitro. We show that an nsp10 mutant, R80A/E82A-ExoN(), was five to ten times more sensitive to treatment with the RNAmutagen 5-fluorouracil (5-FU) than wild-type (WT)-ExoN(), sug-gestive of decreased replication fidelity. This decreased-fidelity phenotype was confirmed using two additional nucleoside ana-logs, 5-azacytidine and ribavirin. R80A/E82A-ExoN() reached a peak titer similar to and demonstrated RNA synthesis kinetics comparable to those seen withWT-ExoN(). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN() relative to MHV-ExoN(), indicating that the decreased-fidelity phenotype of R80A/E82A-ExoN() is linked to the presence of ExoN ac-tivity. Our results demonstrate that nsp10 is important for CoV replication fidelity and support the hypothesis that nsp10 func-tions to regulate nsp14-ExoN activity during virus replication. IMPORTANC