Suppression Of Interferon Beta By Nonstructural Protein4of Porcine Respiratory And Reproductive Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is the primary disease against the pig industry. PRRSV encoded13non-structural proteins, in which NSP1, NSP2, NSP4, NSP11are able to inhibit the expression of type I interferon. PRRSV NSP4, a molecular weight of about21kDa, encoded204amino acids. NSP4has a3C-like serine protease activity (3C-like the serine proteinase3CLSP), which is an important protease PRRSV proliferation process. So far, PRRSV NSP4inhibition mechanism of type I interferon remains unknown. The purpose of this study wants to know how NSP4downregulate IFN-(3transcription. Application of dual luciferase reporting system, confocal analysis and site-directed mutagenesis techniques to different the IFN-β inhibition of NSP4from different pathogenic virus. On the other hand, application of proteomics technology to analysis NSP4and MARC-145cell interaction. This study provides data and information for further understanding of PRRSV molecular pathogenesis.In order to compare the IFN-β, IRF3and NF-κB initiate transcription of NSP4from different pathogenic strain, dual luciferase reporter experiments and laser confocal technology were performed. The results showed that NSP4from low pathogenic strain of HB-1/3.9has weaker transcription suppression than that of highly pathogenic strain JXwn06. There are two conservative amino acid differences between different pathogenic PRRSV NSP4proteins among GenBank recording45strains of PRRSV NSP4protein sequences, in its76th and155th amino acid sites at V76A/P, K155A/T. So the site-directed mutagenesis technique was used to generate mutated plasmids. The results showed T155K mutation can significantly reduce the transcription of IFN-β, which suggested that the155amino acid is the key site to affect its suppression.In order to analysis the mechanism of IFN-β suppression, dual luciferase reporter system analysis was used. The results show that both IRF3and NF-κB pathways were inhibited, but the inhibition of NF-κB pathway activation is more obvious. Confocal laser scanning and the Western Blot results show that nuclear localization and phosphorylation of NF-κB p65protein were not affected, which suggested that NF-κB pathway may influenced in the nucleus. Using site-directed mutagenesis techniques, plasmids of pCMV-NSP4-delet24and pCMV-NLS-NSP4were constructed, which missing nuclear localization signal (Aal45-168) or inserted SV40large T antigen localization signal (PKKKRKV).Confocal laser and dual luciferase reporter experimental results showed that the after missing nuclear localization signal, NSP4lost the nuclear localization character, and has no effect on IFN-P transcription; after nuclear localization signal of SV40is added, both nuclear localization and inhibition were raised. It can be inferred that nuclear distribution of NSP4and the suppression of IFN-β transcription in the nucleus were positively correlated. These results indicate that NSP4lowered the level of IFN-β transcription in the nucleus by inhibiting NF-κB pathway.On the other hand, the molecular mechanism was analysis by protein interaction method. Application of AP-MS (affinity purified and identified by mass spectrometry MS) techniques were screened to nine host proteins can interact with NSP4, two protein seem regulated to interferon transcription. The results showed the IFN-β transcriptional level can be lowered when silenced NONO protein expression. But NONO protein plays no function in the process of NSP4downregulate IFN-β transcription.In addition,2D DiGE (2-dimensional fluorescence difference gel electrophoresis) was use to analysis proteomic differences between cellines expressed different proteins. The results showed that compared to the GFP expressed MARC-145cells, HB-1/3.9NSP4expressed MARC-145cells has total of33protein spots differentially expressed; JXwn06NSP4expressed MARC-145cells total of27protein spots occurred differences expressed. MARC-145cells expressed different the pathogenic PRRSV NSP4proteins presented a total of six spots differentially expressed. These results suggested that NSP4protein has effect on MARC-145proteomic, and there are different affect between NSP4from different pathogenic strains.In summary, the results show that, the PRRSV NSP4play its inhibition of IFN-β transcription function mainly by inhibiting the activation of NF-κB pathway in the nucleus. At the same time the PRRSV NSP4the155amino acids related to the inhibition of IFN-β transcription efficiency. The results revealed for the first time the PRRSV NSP4inhibition of IFN-β transcription mechanism and supplied a molecular basis for PRRSV in-depth study of the molecular pathogenesis of clues.