Study On The Molecular Mechanisms Of Wild Type Mouse Mx1 And Its Mutants Inhibiting Classical Swine Fever Virus Replication

The interferonIinduced Mx proteins ofv ertebrates are dynamin-like GTPases and have antiviral activities.Classical swine fever(CSF)is a highly contagious infectious disease caused by classical swine fever virus(CSFV),which caus ing severe economic losses in the global porcine industry.Oue previous researches indicated that porcine Mx 1(poMx1)protein inhibited CSFV replication by targeting on NS5B protein.In this research,we investigated the effect of wild type mouse Mx1 protein(mmMx1)and its mutants on the replication of CSFV in porcine kidney cells(PK-15).Several mmMx1 mutants were constructed based on the previous studies.The results showed that the subcellular localization,GTPase activity and oligomeric state of Mx proteins were closely related to the function of mmMxl.Studys on the mmMx1 mutant R614E demonstrate that it interacts with the 1-33 amino acids in the N-terminus of the CSFV Core protein directly,which indirectly affects the polymerase activity of NS5B.The expression of wild type mmMx1 increases mRNA expression levels of several interferon stimulated genes(ISGs)in PK-15 cells infected with CSFV,especially ISG15,level of mRNA and protein of which increasing simultaneously.In further researches,we find that ISG15 has the ability to inhibit CSFV replication.This mechanism of ISG15 might be related to interferon regulatory factor 3(IRF3).Through the study of the mechanism of different species of Mx proteins inhibitioning CSFV,the common features of different Mx proteins and the pathogenic mechanism of the virus may be demonstrated clearly.At the same time,it provides ideas for designing antiviral drugs for new drug targets in the virus life cycle and developing vaccines.Eventually,it will enhance the ability to prevent and treat CSFV effectively.1.The construct of mouse Mx1 proteinandit mutants along with the identification of their functionsIn order to explore the effect of point mutations in different domains of mouse Mx1 protein on its antiviral activity,this study designed specific primers based on the point mutations constructed by the company and the seven reported mmMx 1 mutants ofmmMx1(K49A?G83R?A222V?A516V?G540E?R614Eand ?L4),wild-type mmMx2 and poMx 1 were constructed into pcDNA3.0 and pEGFP-C 1 plasmids making recombinant vectors.Theresults of Confocal microscope assays showed that the mutant R614E localized in the cytoplasm and the remaining mutants still localized in the nucleus.Mutants(K49Aand ?L4)lost stable status of oligomerization and meanwhile K49A lost activity of GTPase.After 24 or 48 hours of infection,results of Confocal microscope?Western blotting?RT-qPCRand TCID50 assays showed that mmMx 1(wild type?G83R and R614E)remained the activity inhibiting CSFV,other mutants and mmMx2 losing.All results demonstrate that subcellular location?GTPase activity and oligomerization status have a significant influence on antiviral activity of mmMx 1.2.Study on the molecular mechanism of mouse Mx 1 mutant R614E against CSFV replicationIn order to further explore the molecular mechanism of mmMx 1 protein mutant R614E inhibiting CSFV,this study designed specific primers based on the sequence of CSFV nucleocapsid protein Core,non-structural proteins NS2,NS3,NS4B,NS5A and NS5B.These protein was constructed into the p3×FLAG-CMV-7.1 vector.Confocal microscope?Co-IP and GST pull-down assays showed that mmMx1 mutant R614E localized in the cytoplasm interacted with the CSFV nucleocapsid protein(Core),thereby inhibiting viral replication.Results of RdRp demonstrated that R614E undermined the interaction between Core and NS5B,accordingly impairing the RdRp activity of NS5B.BiFc assays found that the first 33 amino acids ofN-terminal of Core protein were the target of mmMx1 mutant R614E.Compared with previous results,which poMx1 targeting on NS5B directly,mmMx1 mutant R614E interacts with NS5B and inhibits CSFV replication indirectly.3.The study on molecular mechanism of wild type mouse Mx1 against CSFV replicationTo make a further research on the of antiviral mechanism of mmMx1,wild type mmMx1 and several mutants were transfected into PK-15 cells.RT-qPCR and Western blotting assays results showed upregulation of ISG genes(ISG15?ISG49?ISG54 and ISG56)induced by wild type mmMx1 and mutant G83R,especially ISG15.Deep research found that upregulation of I SG15 was dependent on the phosphorylation of IRF3,having nothing to do with STAT1.On the other hand,results of RT-qPCR and Western blotting assays showed that ISG15 also had the activity of inhibiting the replication of CSFV,which laying a certain foundation for further exploring the antiCSFV mechanism of wild type mmMx1.