Biochemical and genetic analyses of murine hepatitis virus Nsp15 endoribonuclease.
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.
Kang
Hyojeung
H
Department of Microbial and Molecular Pathogenesis, Texas A&M University System--HSC, TX 77843-2128, USA.
Bhardwaj
Kanchan
K
Li
Yi
Y
Palaninathan
Satheesh
S
Sacchettini
James
J
Guarino
Linda
L
Leibowitz
Julian L
JL
Kao
C Cheng
CC
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D006517:Murine hepatitis virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D004722:Endoribonucleases