SrasV1, PubMed, Curation, bibRecord, 001702

Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Identifieur interne : 001702 ( PubMed/Curation ); précédent : 001701; suivant : 001703

Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Auteurs : Lance D. Eckerle [États-Unis] ; Michelle M. Becker ; Rebecca A. Halpin ; Kelvin Li ; Eli Venter ; Xiaotao Lu ; Sana Scherbakova ; Rachel L. Graham ; Ralph S. Baric ; Timothy B. Stockwell ; David J. Spiro ; Mark R. Denison

Source :

PLoS pathogens [ 1553-7374 ] ; 2010.

RBID : pubmed:20463816

Descripteurs français

KwdFr :
- Animaux, Cellules Vero, Données de séquences moléculaires, Exoribonucleases (génétique), Génie génétique, Génome viral, Modèles génétiques, Mutation, Phénotype, Polymorphisme de nucléotide simple, Protéines virales non structurales (génétique), Réplication virale (génétique), Syndrome respiratoire aigu sévère (virologie), Séquence d'acides aminés, Variation génétique, Virus du SRAS (croissance et développement), Virus du SRAS (enzymologie), Virus du SRAS (génétique), Évolution moléculaire.
MESH :
- croissance et développement : Virus du SRAS.
- enzymologie : Virus du SRAS.
- génétique : Exoribonucleases, Protéines virales non structurales, Réplication virale, Virus du SRAS.
- virologie : Syndrome respiratoire aigu sévère.
- Animaux, Cellules Vero, Données de séquences moléculaires, Génie génétique, Génome viral, Modèles génétiques, Mutation, Phénotype, Polymorphisme de nucléotide simple, Séquence d'acides aminés, Variation génétique, Évolution moléculaire.

English descriptors

KwdEn :
- Amino Acid Sequence, Animals, Chlorocebus aethiops, Evolution, Molecular, Exoribonucleases (genetics), Genetic Engineering, Genetic Variation, Genome, Viral, Models, Genetic, Molecular Sequence Data, Mutation, Phenotype, Polymorphism, Single Nucleotide, SARS Virus (enzymology), SARS Virus (genetics), SARS Virus (growth & development), Severe Acute Respiratory Syndrome (virology), Vero Cells, Viral Nonstructural Proteins (genetics), Virus Replication (genetics).
MESH :
- chemical , genetics : Exoribonucleases, Viral Nonstructural Proteins.
- enzymology : SARS Virus.
- genetics : SARS Virus, Virus Replication.
- growth & development : SARS Virus.
- virology : Severe Acute Respiratory Syndrome.
- Amino Acid Sequence, Animals, Chlorocebus aethiops, Evolution, Molecular, Genetic Engineering, Genetic Variation, Genome, Viral, Models, Genetic, Molecular Sequence Data, Mutation, Phenotype, Polymorphism, Single Nucleotide, Vero Cells.

Abstract

Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

DOI: 10.1371/journal.ppat.1000896
PubMed: 20463816

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Le document en format XML

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<front><div type="abstract" xml:lang="en">Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.</div>
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<Abstract><AbstractText>Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.</AbstractText>
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Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

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