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Journal of Virology, April 2009, p. 3743-3753, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02203-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Organ-Specific Attenuation of Murine Hepatitis Virus Strain A59 by Replacement of Catalytic Residues in the Putative Viral Cyclic Phosphodiesterase ns2{triangledown}

Jessica K. Roth-Cross,1,{dagger} Helen Stokes,2,{dagger} Guohui Chang,2 Ming Ming Chua,1 Volker Thiel,3 Susan R. Weiss,1*,{ddagger} Alexander E. Gorbalenya,4,{ddagger} and Stuart G. Siddell2*,{ddagger}

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104,1 Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciences, University of Bristol, Bristol, United Kingdom,2 Research Department, Kantonal Hospital St. Gallen, St. Gallen, Switzerland,3 Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands4

Received 17 October 2008/ Accepted 20 January 2009

The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.

* Corresponding author. Mailing address for Stuart G. Siddell: Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. Phone: 0044 117 33 12067. Fax: 0044 117 33 12091. E-mail: stuart.siddell{at}bristol.ac.uk. Mailing address for Susan R. Weiss: Department of Microbiology, University of Pennsylvania School of Medicine, 203A Johnson Pavilion, 36th Street and Hamilton Walk, Philadelphia, PA 19104-6076. Phone: (215) 898-8013. Fax: (215) 573-4858. E-mail: weisssr{at}mail.med.upenn.edu

{triangledown} Published ahead of print on 28 January 2009.

{dagger} J.K.R.-C. and H.S. contributed equally to this work.

{ddagger} S.R.W., A.E.G., and S.G.S. are co-senior authors of the paper.

Journal of Virology, April 2009, p. 3743-3753, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02203-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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