Identification of three site mutations in nonstructural protein 1β, glycoprotein 3 and glycoprotein 5 that correlate with increased interferon α resistance of porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically significant pathogen that has been recognized for its genetic variation, rapid evolution, and immune suppression. Type I interferons (IFNs) play an important role in host defense against viral infection by inducing many antiviral effectors, which might be a selective pressure driving viral evolution towards IFN resistance. To investigate the IFN resistance-related variation of PRRSV genome under IFN selective pressure and explore the molecular mechanism of IFN sensitivity changes, PRRSV strain JXwn06 was serially propagated in porcine pulmonary alveolar macrophages (PAMs) with IFNα treatment for 45 passages and 3 rounds of purification. Four mutant strains named JX-αP51n (n = 1, 2, 3 and 4) with reduced IFNα sensitivity were selected; the strains showed a 100-fold higher titer than the passaging-control strain JX-P51 in IFNα-treated PAMs. IFNα-resistant strains were found to antagonize the IFNα-activated JAK-STAT signaling pathway to a greater extent than the nonresistant strain by down-regulating the expression level of IFNα-activated pJAK1 through interfering with phosphatase. Furthermore, the PRRSV genetic variations interacting with IFNα were identified by full genomic sequencing and alignment. Among these mutations, amino acid substitutions in nsp1β (E87 G), GP3 (F143 L) and GP5 (Y136 H) were found to correlate with increased IFNα resistance by enhancing the suppression effect on pJAK1, which could be further increased if these three substitution sites were combined. These findings provide some novel evidence for understanding PRRSV genetic variation under host selective pressure and viral evolution strategies to evade the host innate immune response.