Study On The Mechanisms Of Which Porcine Reproductive And Respiratory Syndrome Virus Evades Type Ⅰ IFN Responses

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen, causing severe diseases in pigs and great economic losses to the swine industry worldwide. In 2006, a highly pathogenic PRRSV (HP-PRRSV) strain associated with porcine high fever syndrome (PHFS) was reported in China. It is characterized by high fever, high morbidity, and high mortality. Previous studies have reported that PRRSV infection inhibits host immune response. One of the important reasons is the alteration of cytokine expression caused by PRRSV infection. Type I IFNs (IFN-I) are important antiviral cytokines that represent one of the first lines of defense against virus infection. The critical role of IFN-I is not only to interfere with virus life cycle infection by inducing hundreds of interferon stimulated genes (ISGs) production but also to facilitate viral clearance by promoting the adaptive immune system. However, PRRSV infection triggers poor type I IFN responses. In order to further understand PRRSV pathogenesis, we have studied how HP-PRRSV evades host innate immunity, especially type I IFNs.In the present study, we demonstrate that infection with the HP-PRRSV strain JXwn06 antagonizes IFNβ and IFNa expressions induced by poly(I:C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). The production of ISGs is also inhibited in PRRSV-infected PAMs. Subsequently, we show that the inhibition of poly(I:C)-induced IFNβ production by PRRSV is dependent on the blocking of NF-κB and IRF3 signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrate that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppresses IFNp expression. Moreover, we verify that nsp4 reduces IκBα phosphorylation and p65 nuclear translocation, and inhibits NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF and IKKβ. These data suggest that nsp4 might target IKK signaling molecules. We then investigate the effect of nsp4 on these molecules, including IKKa, IKKβ or NEMO. Results show that nsp4 targets the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolish its ability to cleave NEMO and inhibit IFNβ production.In addition, we investigated the mechanism of HP-PRRSV inhibiting IRF3 signaling pathway. Results show that HP-PRRSV nsp4 mediates the cleavage of virus-induced signaling adaptor (VISA) and dislodges it from the mitochondria, thus blocking IRF3 and NF-κB signaling pathways, and impairing RLR antiviral signaling. Interestingly, compared to nsp4 of the typical PRRSV strain CH-la, nsp4 of the HP-PRRSV strain JXwn06 is more efficiently to suppress IFNβ, IRFs and NF-κB luciferase activities induced by poly(I:C). Further study reveals that infection of PAMs by typical PRRSV strain CH-la does not down-regulate VISA expression, and CH-la nsp4 has no effect on VISA. These results partially explain why there exist differences in pathogenicity between HP-PRRSV and typical PRRSV strains.Taken together, our findings reveal that the viral protein nsp4 is a virulence factor that blocks signaling transductions to impair IFN production by mediating the cleavage of NEMO and VISA, a novel strategy used by HP-PRRSV to escape type I IFN responses. These data might have implications for us to futher understand HP-PRRSV pathogenesis.