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J. Virol. doi:10.1128/JVI.01296-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

High Fidelity of Murine Hepatitis Virus Replication Is Decreased in nsp14-Exoribonuclease Mutants

Departments of Pediatrics, Microbiology and Immunology, and Biostatistics, and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee 37232

^* To whom correspondence should be addressed. Email: mark.denison{at}vanderbilt.edu.

	Abstract

Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome coronavirus (SARS-CoV) nsp14 has 3'-to-5' exoribonuclease (ExoN) activity in vitro. Here we show that nsp14 ExoN remarkably increases replication fidelity of the coronavirus, murine hepatitis virus (MHV). Substitution of conserved MHV ExoN active-site residues with alanine resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wildtype virus, in absence of changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wildtype MHV was less than the established rates for RNA viruses in general, suggesting that coronaviruses with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.

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