Amino acid residues in the non-structural protein 1 of porcine reproductive and respiratory syndrome virus involved in down-regulation of TNF-α expression in vitro and attenuation in vivo

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses tumor necrosis factor-alpha (TNF-α) production at both transcriptional and post-transcriptional levels by its non-structural proteins 1α and 1Β (Nsp1α and Nsp1Β). To identify the amino acid residues responsible for this activity, we generated several alanine substitution mutants of Nsp1α and Nsp1Β. Examination of the mutant proteins revealed that Nsp1α residues Gly90, Asn91, Arg97, Arg100 and Arg124 were necessary for TNF-α promoter suppression, whereas several amino acids spanning the entire Nsp1Β were found to be required for this activity. Two mutant viruses, with mutations at Nsp1α Gly90 or Nsp1Β residues 70-74, generated from infectious cDNA clones, exhibited attenuated viral replication in vitro and TNF-α was found to be up regulated in infected macrophages. In infected pigs, the Nsp1Β mutant virus was attenuated in growth. These studies provide insights into how PRRSV evades the effector mechanisms of innate immunity during infection.