Molecular Mechanism For The Differential Inhibition Of IFN-? Expression By Different Pathogenic PRRSV Strains

Porcine reproductive and respiratory syndrome (PRRS), which is characterized by reproductive disorders of pregnant sows and respiratory disease of growing pigs, is a viral disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and has brought enormous economic losses to global swine industry. Porcine alveolar macrophages (PAMs) is the main target cell of PRRSV, which is of strain diversity and complex pathogenic mechanism and is able to lead to immunosuppression against innate immune response during viral infection. To explore the mechanism for the PRRSV-mediated inhibition of innate immunity in target cells, the molecular basis for IFN-? expression in PAMs during PRRSV infection was focused in this study. The effect on IFN-? expression regulated by different pathogenic type 2 PRRSV strains were firstly discussed, and then the cellular microRNA-mediated modulation for IFN-P protein expression and the viral proteins correlated different pathogenic PRRSV- mediated differential inhibition of IFN-? expression, were investigated.The mRNA transcriptions of molecules associated with innate immunity and PRRSV receptors of different pathogenic PRRSV-infected PAMs in vitro were investigated by Real-time PCR. The results showed that mRNA transcriptions of type I interferon, ISGs, most cytokines and chemokines, and PoSn,were upregulated. At the same time, the mRNA transcriptions of type I interferon, ISGs, IL-6, IL-7,IL-10, IL-15, CXCL10 and PoSn in the JXwn06-infected PAMs were higher than that in the HB-1/3.9-infected PAMs, but the mRNA transcriptions of IL-la, IL-?, IL-23p19, CCL3, CCL20,CXCL2, CXCL8 and MIF in the JXwn06-infected PAMs were lower than that in the HB-1/3.9-infected PAMs. The mRNA transcriptions and protein expressions of type I interferon of different pathogenic PRRSV-infected PAMs in vitro were investigated by Real-time PCR and quantitative ELISA,respectively. The results showed that the mRNA transcriptions of type I interferon were upregulated but the protein expressions of type I interferon were downregulated, and the regulation were in a virus dose-dependent manner. Meanwhile, the mRNA transcriptions of type I interferon in the JXwn06-infected PAMs were higher than that in the HB-1/3.9-infected PAMs, but the protein expressions of type I interferon in the JXwn06-infected PAMs were lower than that in the HB-1/3.9-infected PAMs. These results indicate that there is a PRRSV-mediated post-transcriptional inhibition of type I interferon protein expression in its natural host cells, and that inhibition by HP-PRRSV is stronger.The regulation of porcine IFN-? protein expression by porcine miRNAs, including miRs -26a, -34a,-145, and let-7b,were analyzed. The results of dual firefly/Renilla luciferase reporter assay showed that porcine miRs -26a, -34a, -145, and let-7b, could directly target sequences within the porcine IFN-?3'UTR,and their recognition elements in the IFN-? 3'UTR were located in 9-31 bp,27-47 bp, 12-32 bp and 160-181 bp, respectively. The data of transfection of miRNA mimics and inhibitors suggested that miRNA mimics inhibited the IFN-P protein secretion of PAMs in a dose-dependent manner, and miRNA antagonists relieved native miRNA inhibition of IFN-? secretion in a dose-dependent manner too. Poly I:C treatment of PAMs induced IFN-? protein production and that increased subsequently the expression of four miRNAs, which showed that there was a negative feedback mechanism in IFN-?protein-mediated modulating the expression of miRNAs. These results indicate that porcine miRs -26a,-34a, -145, and let-7b, can inhibit IFN-P protein expression in porcine primary PAMs by directly targeting sequences within the porcine IFN-P 3'UTR.The expression of miRNAs in different pathogenic PRRSV-infected PAMs in vitro were investigated by Real-time PCR. The results showed that miRs -26a, -34a, -145, and let-7b, were upregulated, and the expression level of miRNAs in the JXwn06-infected PAMs were higher than that in the HB-1/3.9-infected PAMs. The data of transfection of miRNA inhibitors suggested that miRNA antagonists relieved native miRNA-mediated inhibition of IFN-? secretion in the PRRSV-infected PAMs. Based on the miRNA sensor dual firefly/Renilla luciferase reporter assay, the viral proteins inducing miRs -26a, -34a, -145, and let-7b, were mainly NSP1?, NSP1? and NSP2, and that causing significant differences of the upregulation of miRNAs between HP-PRRSV strain and low pathogenic PRRSV strain were mainly NSP1? and NSP2. By using the Lentiviral packaging technology, LvJnl(3,LvJn2, LvHnl(3 and LvHn2 were packaged and rescued. The results of lentivirus-mediated inhibition of IFN-? protein expressionindicated that LvJnlp, LvJn2, LvHn1? and LvHn2 suppressed IFN-? secretion significantly, and the inhibitons of IFN-P by LvJn1? and LvJn2 were stronger than that by LvHn1? and LvHn2, respectively. This suggests that NSP1? and NSP2 are the main viral proteins that cause significant differences of the inhibition of IFN-P between HP-PRRSV strain and low pathogenic PRRSV strain.Alignment of amino acids of NSP1?3 and NSP2 among 28 highly pathogenic viruses and 10 low pathogenic viruses indicated that there were 2 amino acid mutations in NSP1? and 14 amino acid mutations in NSP2 were conserved between HP-PRRSV strains and low pathogenic PRRSV strains.Furthermore, 6 mutant viruses with NSP1? and NSP2 site mutations (designated as RvJMn1?97L104N,RvJMn2, RvJMn1?97L104N+n2, RvHMn1?97S104Y, RvHMn2 and RvHMn11?97S104Y+n2,respectively) were generated based on the full-length cDNA clones of JXwn06 and HB-1/3.9. Based on the miRNA sensor dual firefly/Renilla luciferase reporter assay, the conserved amino acid mutations in NSP1? were related to the significant differences of the expression of miRs -26a, -34a, -145 in PRRSV-infected PAMs between HP-PRRSV strain and low pathogenic PRRSV strain, and the conserved amino acid mutations in NSP2 were related to the significant difference of the expression of let-7b in PRRSV-infected PAMs between HP-PRRSV strain and low pathogenic PRRSV strain. The mutant virus-mediated inhibition of IFN-?indicated that the inhibition of IFN-? by RvHMn1?97S104Y+n2 was stronger than that of RvJMn1?97L104N+n2. There was no significant difference between RvJMn1?97L104N and RvHMn1?97S104Y, so was between RvJMn2 and RvHMn2. These data suggest that conservative amino acid mutations of NSP1?+NSP2 are related to the significant difference of the inhibition of IFN-? between different pathogenic PRRSV strains.In short, our results indicate that: (i) PRRSV infection suppresses the protein expression of IFN-?in PAMs by post-transcriptional control, and HP-PRRSV has stronger effect on the down-regulation of IFN-P protein expression; (ii) Porcine miRNAs, including miRs -26a, -34a, -145, and let-7b, can inhibit porcine IFN-? protein expression by targeting sequences within the IFN-P 3'UTR; (iii) miRs -26a, -34a,-145, and let-7b, were upregulated in PRRSV-infected PAMs in vitro, and the expression level of miRNAs in the JXwn06-infected PAMs were higher than that in the HB-1/3.9-infected PAMs; (iiii) The viral proteins inducing miRs -26a, -34a, -145, and let-7b, were mainly NSP1?, NSP1? and NSP2, and that causing significant differences of the upregulation of miRNAs and the inhibition of IFN-? between HP-PRRSV strain and low pathogenic PRRSV strain were mainly NSP1? and NSP2; (iiiii)Conservative amino acid mutations of NSP1?+NSP2 are related to the significant difference of the inhibition of IFN-? by different pathogenic PRRSV strains. All these works contribute to understand the molecular mechanism of the PRRSV-mediated inhibition of porcine IFN-? in PAMs, and give more scientific evidences to explore the molecular mechanism for PRRSV-mediated inhibition of innate immunity in target cells.