title: Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases.
abstract: Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino-acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. oka-t@nih.go.jp
authors.forename: Tomoichiro
authors.initials: T
authors.lastname: Oka
authors.forename: Kosuke
authors.initials: K
authors.lastname: Murakami
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Katayama
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002139:Caliciviridae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D010447:Peptide Hydrolases