title: Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
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.
authors.affiliation: Department of Pediatrics and Microbiology and Immunology and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Lance D
authors.initials: LD
authors.lastname: Eckerle
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Becker
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Halpin
authors.forename: Kelvin
authors.initials: K
authors.lastname: Li
authors.forename: Eli
authors.initials: E
authors.lastname: Venter
authors.forename: Xiaotao
authors.initials: X
authors.lastname: Lu
authors.forename: Sana
authors.initials: S
authors.lastname: Scherbakova
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Graham
authors.forename: Ralph S
authors.initials: RS
authors.lastname: Baric
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Stockwell
authors.forename: David J
authors.initials: DJ
authors.lastname: Spiro
authors.forename: Mark R
authors.initials: MR
authors.lastname: Denison
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D019143:Evolution, Molecular; D005095:Exoribonucleases / Q000235:genetics*; D005818:Genetic Engineering; D014644:Genetic Variation; D016679:Genome, Viral; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D045169:Severe Acute Respiratory Syndrome / Q000821:virology*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
chemical_list: D017361:Viral Nonstructural Proteins; C525596:nsp14 protein, SARS coronavirus; D005095:Exoribonucleases
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