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

Public Deposited
Analytics

Downloadable Content

Download PDF
Request Version for Screen Reader
Creator
  • Eckerle, Lance D.
    • Other Affiliation: Vanderbilt University Medical Center
  • Becker, Michelle M.
    • Other Affiliation: Vanderbilt University Medical Center
  • Halpin, Rebecca A.
    • Other Affiliation: The J. Craig Venter Institute
  • Li, Kelvin
    • Other Affiliation: The J. Craig Venter Institute
  • Venter, Eli
    • Other Affiliation: The J. Craig Venter Institute
  • Lu, Xiaotao
    • Other Affiliation: Vanderbilt University Medical Center
  • Scherbakova, Sana
    • Other Affiliation: The J. Craig Venter Institute
  • Graham, Rachel L.
    • Affiliation: Gillings School of Global Public Health, Department of Epidemiology
  • Baric, Ralph S.
    • Affiliation: Gillings School of Global Public Health, Department of Epidemiology
  • Stockwell, Timothy B.
    • Other Affiliation: The J. Craig Venter Institute
  • Spiro, David J.
    • Other Affiliation: The J. Craig Venter Institute
  • Denison, Mark R.
    • Other Affiliation: Vanderbilt University Medical Center
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.
Date of publication
Keyword
DOI
Identifier
Resource type
  • Article
Rights statement
  • In Copyright
Journal title
  • PLoS Pathogens
Journal volume
  • 6
Journal issue
  • 5
Language
  • English
ISSN
  • 1553-7366

Relations

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details journal.ppat.1000896.PDF 2020-04-09 Public Download