Abstract
ABSTRACTThe coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication.
Highlights
The coronavirus (CoV) RNA genome is the largest among the singlestranded positive-sense RNA viruses
We showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoNinactivating mutations
We first tested whether replication of murine hepatitis virus (MHV)-ExoN(-) P250 was affected by long-term passage by examining replication at two different multiplicities of infection (MOI)
Summary
The coronavirus (CoV) RNA genome is the largest among the singlestranded positive-sense RNA viruses. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. Passage-adapted ExoN(-) mutants demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp and nsp14 These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness. The evolved mutations in MHV-ExoN(-) nsp and nsp, which encodes the RdRp, accounted for only part of the increased nucleoside analogue resistance of MHV-ExoN(-) P250, implicating multiple replicase proteins in adaptation for viral fitness. The results of this study support the proposed link between CoV fidelity and fitness, demonstrate the surprising stability of the ExoN-inactivating substitutions, and identify additional proteins outside nsp and nsp that may contribute to CoV fidelity regulation
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