The Research Of Mechanism About Bovine Mx1 Inhibiting The Replication Of Foot-and-mouth Disease Virus

Foot-and-mouth disease(FMD) is a highly contagious and economically important agent of cloven-hoofed animals, affecting cattle, swine, and sheep. Because of more serotypes and rapid mutations of the virus, it is difficult to prevent and control FMD only with vaccine.Need to explore multiple means for control of FMD, including natural immunity.Innate immunity, especially the anti-virus genes, exerts an important barrier function in preventing virus infections. After invading host cells, viruses can induce the generation of interferon(IFN). Type I interferons(I-IFN) and type III interferons(III-IFN) play an important role in innate immunity against viral infections, whichcan be induced directly in response to viral infection and trigger the different processes including the Myxovirus-resistant(Mx) proteins. Previous studies have shown that Mx is with broad-spectum anti-viral effects, it could inhibit RNA viruses and DNA viruses. Mx is very important in the field of natural immune research. But the effect of bovine Mx1 protein resist against FMDV is not clear. In the present study, bovine Mx1 protein resisted against FMDV with overexpression and RNAi technology, In addition, the molecular mechanisms of bovine Mx1 FMDV resistance was explored through bovine Mx1 effected to FMDV genomic RNA replication and viral protein. The mainly results as follow:1. Establish the overexpression bovine Mx1 of cell lines BHK21-Mx1. Bovine Mx1 expressed in the cytoplasm by immunofluorescence, and found the activity of resist against FMDV.Bovine Mx1 gene was obtained by RT-PCR, and to construct eukaryotic expression vector pcDNA3.1-Mx1 with Flag tag. The transient expression of Flag-Mx1 was high after pcDNA3.1-Mx1 transfected 293 T cells.Then pcDNA3.1-Mx1 and pcDNA3.1 were transfected to BHK-21 cells susceptible to FMDV. The overexpression bovine Mx1 clone cells were selected using G418 resistance culture and PCR indetify and Western blot. The overexpression bovine Mx1 cell line named BHK21-Mx1 and control cell line BHK21-3.1were established after generation more than 60 d, and BHK21-Mx1 remained Mx1 high expression. Bovine Mx1 expressed in cytoplasm using Flag monoclonal antibody immunofluorescence staining. The overexpression of bovine Mx1 cell line BHK21-Mx1 could resist the infection of 100 TCID50 FMDV in 48 h.FMDV titration of BHK21-Mx1 cells were 10 fold lower than BHK21-3.1 cells post infection. Thus, bovine Mx1 overexpression could decrease the replication of FMDV in vitro.2. The bovine primary fetal trachea epithelial cells, which was sensitive to FMDV, was used as a naturally susceptible host cells model, it was confirmed that bovine Mx1 could inhibit viral replication after comparison between bovine primary fetal trachea epithelial cells normally expressing or silencing bovineMx1 gene.The sequences of these of shRNA oligonucleotides targeting to bovine Mx1 were designed and synthesized. These oliog-nucleotides were annealed to each other and ligated into H1 lentivirus vector,followed by DNA sequence confirmation. The lentiviral shRNAs vectors and pcDNA3.1-Mx1 were co-transfected into 293 T cells. After 48 h, transient expression of the Flag-Mx1 fusion proteins were determined by Western blot. The eliminated effect of bovine Mx1 expression of RNAi-Mx1-A was highest.Then, vector RNAi-Mx1-A was transfected into the bovine fetal primary trachea epithelia cells. All bovine fetal primary trachea epithelia cells expressed eGFP after screened by FACS based on shRNA recombinant H1 lentivirus encoding eGFP, and were confirmed by Western blot. The stable silencing bovine Mx1 of the bovine fetal primary trachea epithelia cells was named BPTE-si Mx1, and the control cell was named BPTE-Lac Z. The optimal induction dose of α-IFN was 1×104IU/mL, the optimal induction time was 6 h,which could induce the highest expression level of bovine Mx1 expression using real time RT-PCR. Afterα-IFN inducing, BPTE-siMx1 and BPTE-Lac Z cells were inoculated with FMDV at multiplicity of infection of 100 TCID50 per well, and the viral titer of infected cells were determined. By TCID50 Assay, FMDV titration of BPTE-siMx1 cells were significant higher than BPTE-LacZ cells post infection, respectively. Thus,bovine Mx1 silencing could increase the replication of FMDV in vitro.3. The strand-specific real-time fluorescence quantitative RT-PCR(ssqRT-PCR) of FMDV were established. The viral genomic RNA of FMDV infected cells were subjected to the strand specific RT-qPCR process. The result showed that bovine Mx1 protein could inhibit the synthesis of the genomic RNA of FMDV.The genetic sequences of conservative was determined by comparing FMDV genome sequence. The tagged reverse transcription primer(RT-primer) contained a non-viral tag sequences were designed and synthesized. The primers were used to amplificated the viral RNA negative strand and viral RNA positive strand, respectively. The ssqRT-PCR of FMDV were established, which the ssqRT-PCR method had good sensitivity, specificity and repeatability. The copy number of the positive strand RNA of the infected BHK21-Mx1 cells were 20-, 34- and 22-fold higher than the cotrol cells at 8 h, 16 h and 24 h post infection,respectively.The copy number of the negative strand RNA of BHK21-Mx1 cells were 12-, 19- and 17-fold higher than the infected BPTE-LacZ cells at 8 h, 16 h and 24 h post infection, respectively.The copy number of the positive strand RNA of the infected BPTE-siMx1 cells were 29-, 47- and 14-fold higher than the cotrol cells at 8 h, 16 h and 24 h post infection, respectively.The copy number of the negative strand RNA of BPTE-siMx1 cells were 24-, 32- and 14-fold higher than the infected BPTE-Lac Z cells at 8 h, 16 h and 24 h post infection, respectively. The result showed that bovine Mx1 protein could inhibit the synthesis of the genomic RNA of FMDV.4. FMDV P1, P2 and P3 proteins were prokaryotic expressed and purified, the rabbit antiserum wereperpetrated of the viral proteins. The 2C protein of FMDV decreased in bovine Mx1 effecting by Western blot.The bovine Mx1 could affect to N-terminus of FMDV 2C protein to resistive against FMDV affection with GST pull-down and immune co-precipitation method.The pET32 a prokaryotic expression vector expressed FMDV P1, P2 and P3 protin were constructed,respectively. The viral proteins were purified and were perpetrated to rabbit polyclonal antibody. 2C protein of FMDV expression was significant decreased in overexpression bovine Mx1 cells BHK21-Mx1 than the cotrol cells BHK21-3.1 post infection of FMDV. The pGEX prokaryotic expression vector expressed FMDV 2C and bovine Mx1, and pcDNA3.1-2C vector were constructed, repectively. The bovine Mx1 could combine with FMDV 2C protein with GST pull-down. The pcDNA3.1-2C and pcDNA3.1-Mx1 were co-transfected into293 T cells. The bovine Mx1 could affect to FMDV 2C protein to resistive against FMDV affection with GST pull-down an immune co-precipitation method. The bovine Mx1 could combine to N-terminus of FMDV 2C protein using FMDV 2C protein deletion mutants.