Preliminary Study On The Anti-PRRSV Effect And Molecular Mechanism Of Porcine Mx1 Protein

Porcine reproductive and respiratory syndrome（PRRS）is a serious viral infectious disease in the swine industry,which causes serious reproductive disorders and respiratory diseases in pigs.The disease is caused by Blue ears virus（PRRSV）,which is characterized by reproductive disturbance,respiratory symptoms and high mortality in piglets and adult pigs.The disease was first discovered in North America in 1987 and spreaded worldwide,causing great economic losses to swine industry.At present,there is no effective treatment for this disease,and prevention is the main method.Studying the host cell anti-PRRSV mechanism could provide new ideas for the prevention and control of PRRS and the development of new effective vaccines.The results of the proteomic iTRAQ performed by the members of our laboratory indicated that the expression of Mx1 protein in peripheral blood mononuclear dendritic cells（MoDCs）in pigs was significantly up-regulated over a long period of time after infection with highly pathogenic PRRSV.So,does the Mx1 protein have anti-PRRSV function?What is the mechanism?To this end,I mainly carried out the research in the following aspects:1.Anti-PRRSV activity of porcine Mx1 proteinI constructed eukaryotic expression plasmids of Mx1 protein and its segmented protein.Through the TCID₅₀ assay,I proved that the porcine Mx1 protein could significantly reduce the titer of PRRSV,indicating that the Mx1 protein has a significant inhibitory effect on the propagation of PRRSV.The G domain of Mx1 protein plays a major antiviral role.The results of real-time PCR assay further proved that Mx1 protein could significantly inhibit the replication of PRRSV genomic RNA in MARC-145 cells,thereby inhibiting the propagation of the virus,in which the G domain played a major role.2.The G domain of Mx1 protein interacts with the C terminal domain of PRRSV nucleocapsid protein NIn order to explore the relationship between Mx1 protein and PRRSV proteins,I constructed expression vectors of all structural proteins and non-structural proteins of PRRSV.Based on the Renilla Luciferase Assay（RLA）experiment,I found that N protein interacts with Mx1 and G domain of Mx1 protein.These results were subsequently confirmed by coimmunoprecipitation assays（Co-IP）.It was further demonstrated that the G domain of Mx1interacts with the C terminal domain of PRRSV N protein by Co-IP.3.The G domain of Mx1 protein and the C-terminal domain of N protein are co-localized in the cytoplasmThe eukaryotic plasmids expressing Mx1,mutated Mx1 or G domain of Mx1 protein was co-transfected with eukaryotic plasmids expressing PRRSV N protein,N terminal of PRRSV N,C terminal of PRRSV N or NLS mutation.The results of indirect immunofluorescence assay（IFA）indicated that Mx1 co-localizes with N protein in the cytoplasm and G domain of Mx1also co-localizes with C terminal of PRRSV N.In summary,this study revealed for the first time the molecular mechanism of Mx1 protein inhibiting propagation of PRRSV.Mx1 interacts with the C-terminal domain of N protein in the cytoplasm through its G domain and binds to the viral capsid protein N,thereby inhibiting the synthesis of PRRSV genome RNA and reducing the titer of the virus.The results of this study expand the diversity of antiviral molecular mechanism of Mx1 protein,and provide new ideas and methods for prevention and control of PRRSV infection.