title: Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus.
abstract: The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies. The 3C-like proteinase (3CLpro) of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and characterized. The optimal temperature and pH for its proteolytic activity were 8°C and 7.5, respectively. Na(+) (1000 mM) and K(+) (500 mM) were not inhibitory to its activity but Cu(2+), Zn(2+), PMSF and EDTA were significantly inhibitory. His(39), Asp(64) and Ser(118) residues were identified to form the catalytic triad of PRRSV 3CLpro by a series of site-directed mutagenesis analysis.
authors.affiliation: Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China.
authors.forename: Ao-Tian
authors.initials: AT
authors.lastname: Xu
authors.forename: Yan-Jun
authors.initials: YJ
authors.lastname: Zhou
authors.forename: Guo-Xin
authors.initials: GX
authors.lastname: Li
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Yan
authors.forename: Guang-Zhi
authors.initials: GZ
authors.lastname: Tong
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D020134:Catalytic Domain; D003080:Cold Temperature; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D019316:Porcine respiratory and reproductive syndrome virus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D003546:Cysteine Endopeptidases