title: The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions.
abstract: Porcine reproductive and respiratory syndrome (PRRS) virus nonstructural protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-kappaB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-kappaB activation by interfering with the polyubiquitination process of IkappaBalpha, which subsequently prevents IkappaBalpha degradation. To determine whether the nsp2 protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-kappaB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-kappaB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-kappaB activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.
authors.affiliation: Department of Veterinary and Biomedical Science, South Dakota State University, Brookings, South Dakota 57007-1396, USA.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Sun
authors.forename: Zhenhai
authors.initials: Z
authors.lastname: Chen
authors.forename: Steven R
authors.initials: SR
authors.lastname: Lawson
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007370:Interferon Type I / Q000037:antagonists & inhibitors*; D050296:Microbial Viability; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D019316:Porcine respiratory and reproductive syndrome virus / Q000201:enzymology* / Q000254:growth & development; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
chemical_list: D020672:I-kappa B Proteins; D007370:Interferon Type I; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D025801:Ubiquitin; D017361:Viral Nonstructural Proteins; D000072000:NF-KappaB Inhibitor alpha; D057056:Cysteine Proteases