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

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 3...

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Autores principales:	Lance D Eckerle, 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
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/cf6b90cc6aa7402d95e66041305305c6
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spelling	oai:doaj.org-article:cf6b90cc6aa7402d95e66041305305c62021-12-02T20:00:42ZInfidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.1553-73661553-737410.1371/journal.ppat.1000896https://doaj.org/article/cf6b90cc6aa7402d95e66041305305c62010-05-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20463816/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374Most 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.Lance D EckerleMichelle M BeckerRebecca A HalpinKelvin LiEli VenterXiaotao LuSana ScherbakovaRachel L GrahamRalph S BaricTimothy B StockwellDavid J SpiroMark R DenisonPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 6, Iss 5, p e1000896 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Lance D Eckerle 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 Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
description	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.
format	article
author	Lance D Eckerle 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
author_facet	Lance D Eckerle 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
author_sort	Lance D Eckerle
title	Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
title_short	Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
title_full	Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
title_fullStr	Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
title_full_unstemmed	Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
title_sort	infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/cf6b90cc6aa7402d95e66041305305c6
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Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

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