Targeted mutations in a highly conserved motif of the nsp1 β protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus

Non-structural protein 1 beta (nsp1 beta) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a papain-like cysteine protease (PLP beta) domain and has been identified as the main viral protein antagonizing the host innate immune response. In this study, nsp1 beta was determined to suppress the expression of reporter genes as well as to suppress 'self-expression' in transfected cells, and this activity appeared to be associated with its interferon (IFN) antagonist function. To knock down the effect of nsp1 beta on IFN activity, a panel of site-specific mutations in nsp1 beta was analysed. Double mutations K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV) targeting a highly conserved motif of nsp1 beta, GKYLQRRLQ (in bold), impaired the ability of nsp1 beta to suppress IFN-beta and reporter gene expression, as well as to suppress 'self-expression' in vitro. Subsequently, viable recombinant viruses vSD01-08-K130A/R134A and vSD95-21-K124A/R128A, containing double mutations in the GKYLQRRLQ motif were generated using reverse genetics. In comparison with WT viruses, these nsp1 beta mutants showed impaired growth ability in infected cells, but the PLP beta cleavage function was not directly affected. The expression of selected innate immune genes was determined in vSD95-21-K124A/R128A mutant-infected cells. The results consistently showed that gene expression levels of IFN-alpha, IFN-beta and IFN-stimulated gene 15 were upregulated in cells that were infected with the vSD95-21-K124A/R128A compared with that of WT virus. These data suggest that PRRSV nsp1 beta may selectively suppress cellular gene expression, including expression of genes involved in the host innate immune function. Modifying the key residues in the highly conserved GKYLQRRLQ motif could attenuate virus growth and improve the cellular innate immune responses.