title: Mutations in a Highly Conserved Motif of nsp1β Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (nsp1β) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ((123)GKYLQRRLQ(131)) reduced the ability of nsp1β to suppress interferon beta (IFN-β) activation and also impaired nsp1β's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1β mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1β mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1β function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals.
  
  
  OBJECTIVE
  PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1β was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1β function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines.
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Li
authors.affiliation: Food Animal Health Research Program, Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Duan-Liang
authors.initials: DL
authors.lastname: Shyu
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Shang
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Jianfa
authors.initials: J
authors.lastname: Bai
authors.affiliation: Food Animal Health Research Program, Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Kang
authors.initials: K
authors.lastname: Ouyang
authors.affiliation: Food Animal Health Research Program, Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Santosh
authors.initials: S
authors.lastname: Dhakal
authors.affiliation: Food Animal Health Research Program, Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Jagadish
authors.initials: J
authors.lastname: Hiremath
authors.affiliation: Food Animal Health Research Program, Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
authors.forename: Basavaraj
authors.initials: B
authors.lastname: Binjawadagi
authors.affiliation: Food Animal Health Research Program, Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA gourapura.1@osu.edu yfang@vet.k-state.edu.
authors.forename: Gourapura J
authors.initials: GJ
authors.lastname: Renukaradhya
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA gourapura.1@osu.edu yfang@vet.k-state.edu.
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D054884:Host-Pathogen Interactions; D057131:Immune Evasion; D007113:Immunity, Innate; D016899:Interferon-beta / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology*; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D017361:Viral Nonstructural Proteins; D016899:Interferon-beta