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Journal of Virology, December 2007, p. 13587-13597, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.00547-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biochemical and Genetic Analyses of Murine Hepatitis Virus Nsp15 Endoribonuclease{triangledown}

Hyojeung Kang,1,{dagger} Kanchan Bhardwaj,2,{dagger} Yi Li,2,3 Satheesh Palaninathan,2 James Sacchettini,2 Linda Guarino,2,3 Julian L. Leibowitz,1* and C. Cheng Kao2*

Department of Microbial and Molecular Pathogenesis, Texas A&M University System—HSC,1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843,2 Department of Entomology, Texas A&M University, College Station, Texas 77843-21283

Received 14 March 2007/ Accepted 29 August 2007

The goal of this project was to better define the relationship between the endoribonuclease activity of murine hepatitis virus (MHV) Nsp15 (mNsp15) and its role in virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its severe acute respiratory syndrome coronavirus ortholog. Alanine substitutions at three key residues in the mNsp15 catalytic pocket (H262, H277, and G275) and a double-mutant version (H262P and H277A) generated proteins with greatly reduced but detectable endoribonuclease activities. Furthermore, these mutant proteins demonstrated lower cleavage specificities for uridylate than wild-type (WT) mNsp15. These mutations were successfully incorporated into viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant viruses formed plaques with diameters similar to that of MHV-A59 1000 (WT virus) on several different cell lines. Interestingly, viruses with a mutation at a noncatalytic residue, D324A, could not be recovered despite repeated attempts, and expression of mNsp15 containing the D324A mutation in Escherichia coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6- to 13-fold fewer PFU than those from WT virus. Cells infected with mNsp15 mutant viruses accumulated lesser amounts of plus- and minus-sense subgenomic RNAs and spike protein than WT virus. The expression of mNsp15 in trans by transient transfection partially restored RNA synthesis by vH262A. These results demonstrate that mNsp15 is required for optimal infection by MHV.

* Corresponding author. Mailing address for C. Cheng Kao: Department of Biochemistry and Biophysics, Texas A&M University, 103 Biochemistry/Biophysics Building, 2128 TAMU, College Station, TX 77843-2128. Phone: (979) 458-2235. Fax: (979) 845-9274. E-mail: ckao{at}tamu.edu. Mailing address for Julian L. Leibowitz: Department of Microbial and Molecular Pathogenesis, Texas A&M University System College of Medicine, 407 Reynolds Medical Building, 1114 TAMU, College Station, TX 77843-1114. Phone: (979) 845-7288. Fax: (979) 845-3479. E-mail: jleibowitz{at}tamu.edu

{triangledown} Published ahead of print on 26 September 2007.

{dagger} H.K. and K.B. contributed equally to this work.

Journal of Virology, December 2007, p. 13587-13597, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.00547-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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