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 catalyti...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Kevin W. Graepel, Xiaotao Lu, James Brett Case, Nicole R. Sexton, Everett Clinton Smith, Mark R. Denison
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	RNA virus adaptive evolution competitive fitness coronavirus exoribonuclease plus-strand RNA virus Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/539d7c44a1af46f6b33f4d220f907e6a
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:539d7c44a1af46f6b33f4d220f907e6a
record_format	dspace
spelling	oai:doaj.org-article:539d7c44a1af46f6b33f4d220f907e6a2021-11-15T15:51:56ZProofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations10.1128/mBio.01503-172150-7511https://doaj.org/article/539d7c44a1af46f6b33f4d220f907e6a2017-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01503-17https://doaj.org/toc/2150-7511ABSTRACT 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.Kevin W. GraepelXiaotao LuJames Brett CaseNicole R. SextonEverett Clinton SmithMark R. DenisonAmerican Society for MicrobiologyarticleRNA virusadaptive evolutioncompetitive fitnesscoronavirusexoribonucleaseplus-strand RNA virusMicrobiologyQR1-502ENmBio, Vol 8, Iss 6 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	RNA virus adaptive evolution competitive fitness coronavirus exoribonuclease plus-strand RNA virus Microbiology QR1-502
spellingShingle	RNA virus adaptive evolution competitive fitness coronavirus exoribonuclease plus-strand RNA virus Microbiology QR1-502 Kevin W. Graepel Xiaotao Lu James Brett Case Nicole R. Sexton Everett Clinton Smith Mark R. Denison Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
description	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.
format	article
author	Kevin W. Graepel Xiaotao Lu James Brett Case Nicole R. Sexton Everett Clinton Smith Mark R. Denison
author_facet	Kevin W. Graepel Xiaotao Lu James Brett Case Nicole R. Sexton Everett Clinton Smith Mark R. Denison
author_sort	Kevin W. Graepel
title	Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
title_short	Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
title_full	Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
title_fullStr	Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
title_full_unstemmed	Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
title_sort	proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/539d7c44a1af46f6b33f4d220f907e6a
work_keys_str_mv	AT kevinwgraepel proofreadingdeficientcoronavirusesadaptforincreasedfitnessoverlongtermpassagewithoutreversionofexoribonucleaseinactivatingmutations AT xiaotaolu proofreadingdeficientcoronavirusesadaptforincreasedfitnessoverlongtermpassagewithoutreversionofexoribonucleaseinactivatingmutations AT jamesbrettcase proofreadingdeficientcoronavirusesadaptforincreasedfitnessoverlongtermpassagewithoutreversionofexoribonucleaseinactivatingmutations AT nicolersexton proofreadingdeficientcoronavirusesadaptforincreasedfitnessoverlongtermpassagewithoutreversionofexoribonucleaseinactivatingmutations AT everettclintonsmith proofreadingdeficientcoronavirusesadaptforincreasedfitnessoverlongtermpassagewithoutreversionofexoribonucleaseinactivatingmutations AT markrdenison proofreadingdeficientcoronavirusesadaptforincreasedfitnessoverlongtermpassagewithoutreversionofexoribonucleaseinactivatingmutations
_version_	1718427275566776320

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

Ejemplares similares