The coronavirus proofreading exoribonuclease mediates extensive viral recombination.
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ABSTRACT:
SUBMITTER: Gribble J
PROVIDER: S-EPMC7846108 | biostudies-literature | 2021 Jan
REPOSITORIES: biostudies-literature
Publications
The coronavirus proofreading exoribonuclease mediates extensive viral recombination.
Gribble Jennifer J Stevens Laura J LJ Agostini Maria L ML Anderson-Daniels Jordan J Chappell James D JD Lu Xiaotao X Pruijssers Andrea J AJ Routh Andrew L AL Denison Mark R MR
PLoS pathogens 20210119 1
Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstra ...[more]
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Project description:High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.
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Project description:The ongoing SARS-CoV-2 pandemic is the third zoonotic coronavirus identified in the last 20 years. Enzootic and epizootic coronaviruses of diverse lineages also pose a significant threat to livestock, as most recently observed for virulent strains of porcine epidemic diarrhea virus (PEDV) and swine acute diarrhea-associated coronavirus (SADS-CoV). Unique to RNA viruses, coronaviruses encode a proofreading exonuclease (ExoN) that lowers point mutation rates to increase the viability of large RNA virus genomes, which comes with the cost of limiting virus adaptation via point mutation. This limitation can be overcome by high rates of recombination that facilitate rapid increases in genetic diversification. To compare the dynamics of recombination between related sequences, we developed an open-source computational workflow (IDPlot) that bundles nucleotide identity, recombination, and phylogenetic analysis into a single pipeline. We analyzed recombination dynamics among three groups of coronaviruses with noteworthy impacts on human health and agriculture: SARSr-CoV, Betacoronavirus-1, and SADSr-CoV. We found that all three groups undergo recombination with highly diverged viruses from undersampled or unsampled lineages, including in typically highly conserved regions of the genome. In several cases, no parental origin of recombinant regions could be found in genetic databases, demonstrating our shallow characterization of coronavirus diversity and expanding the genetic pool that may contribute to future zoonotic events. Our results also illustrate the limitations of current sampling approaches for anticipating zoonotic threats to human and animal health.
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Project description: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.
| S-EPMC5676041 | biostudies-literature
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.
| S-EPMC6798108 | biostudies-literature