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Journal of Virology, November 2007, p. 12135-12144, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01296-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High Fidelity of Murine Hepatitis Virus Replication Is Decreased in nsp14 Exoribonuclease Mutants{triangledown}

Lance D. Eckerle,1,2,4 Xiaotao Lu,1,4 Steven M. Sperry,1,4,{dagger} Leena Choi,3 and Mark R. Denison1,2,4*

Departments of Pediatrics,1 Microbiology and Immunology,2 Biostatistics,3 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee 372324

Received 13 June 2007/ Accepted 23 August 2007

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 (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3'-to-5' exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs 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.

* Corresponding author. Mailing address: Department of Pediatrics, Vanderbilt University Medical Center, D6217 MCN, 1161 21st Avenue South, Nashville, TN 37232-2581. Phone: (615) 343-9881. Fax: (615) 343-9723. E-mail: mark.denison{at}vanderbilt.edu

{triangledown} Published ahead of print on 5 September 2007.

{dagger} Present address: Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.

Journal of Virology, November 2007, p. 12135-12144, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01296-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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