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Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations.

ABSTRACT: The 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.IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 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.

SUBMITTER: Graepel KW

PROVIDER: S-EPMC5676041 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations.

Graepel Kevin W KW Lu Xiaotao X Case James Brett JB Sexton Nicole R NR Smith Everett Clinton EC Denison Mark R MR

mBio 20171107 6

The 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, u ...[more]

PMID: 29114026

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Project description:The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants.IMPORTANCE Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.

Project description:Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group ?-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.

Dataset Information

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations.

Publications

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations.

Similar Datasets

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