Single-Amino-Acid Substitutions in Open Reading Frame (ORF) 1b-nsp14 and ORF 2a Proteins of the Coronavirus Mouse Hepatitis Virus Are Attenuating in Mice

Public Deposited
Analytics

Downloadable Content

Download PDF
Request Version for Screen Reader
Creator
  • Sperry, Steven M.
    • Other Affiliation: Vanderbilt University Medical Center
  • Kazi, Lubna
    • Other Affiliation: University of Pennsylvania School of Medicine
  • Graham, Rachel L.
    • Other Affiliation: Vanderbilt University Medical Center
  • Baric, Ralph S.
    • Affiliation: Gillings School of Global Public Health, Department of Epidemiology
  • Weiss, Susan R.
    • Other Affiliation: University of Pennsylvania School of Medicine
  • Denison, Mark R.
    • Other Affiliation: Vanderbilt University Medical Center
Abstract
  • A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The “in vitro-assembled” wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log10 50% lethal dose (LD50) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log10 LD50, and the “corrected” Tyr6398/Leu94 virus had a log10 LD50 identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.
Date of publication
Keyword
DOI
Identifier
Resource type
  • Article
Rights statement
  • In Copyright
Journal title
  • Journal of Virology
Journal volume
  • 79
Journal issue
  • 6
Page start
  • 3391
Page end
  • 3400
Language
  • English
ISSN
  • 0022-538X

Relations

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details 2068-04.pdf 2020-04-06 Public Download