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-κB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-κB activation by interfering with the polyubiquitination process of IκBα, which subsequently prevents IκBα 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-κB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-κB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-κB 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.