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Biochemical and Genetic Analyses of the Mouse Hepatitis Virus Nsp15 Endoribonuclease.

Kang H, Bhardwaj K, Li Y, Palaninathan S, Sacchettini J, Guarino L, Leibowitz JL, Kao CC

Department of Microbial and Molecular Pathogenesis, Texas A&M University System-HSC, College Station, TX 77843, Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, Department of Entomology, Texas A&M University, College Station, TX 77843-2128.

The goal of this project was to better define the relationship between the endoribonuclease activity of Mouse Hepatitis Virus Nsp15 (mNsp15) and its role in virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its SARS coronavirus (SARS-CoV) 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 protein with greatly reduced, but detectable endoribonuclease activity. Furthermore, these mutant proteins demonstrated lower cleavage specificity for uridylate in comparison to Wt mNsp15. These mutations were successfully incorporated into viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant viruses formed plaques of similar diameter 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 E. coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6 to 13 fold fewer plaque forming units when compared to those from Wt virus. Cells infected with viruses mutant for mNsp15 accumulated lesser amounts of plus and minus sense subgenomic RNAs, and spike protein in comparison to 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.

Published 27 September 2007 in J Virol.
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