Inhibition Of Signalling Pathway Of Ivterferon-Beta By 3C Protein Of Encephalomyocarditis Virus

Encephalomyocarditis virus (EMCV) is considered as a relatively important pathogen which can cause encephalitis, myocarditis and myocardinal inflammation in piglets. It will also bring about reproductive disorder to sows and silent subclinical infection in adult swine worldwide. It can infect human and animals, having the potential of reducing amphixenosis of natural focus. However, its pathogenic mechanism remains unclear.3C protein is one of EMCV nonstructral proteins, which functions as proteinase to cleave viral polyprotein and host protein as well, playing an important role in virulence. In this research, a eukaryotie expression plasmid of EMCV 3C was constructed and identified. The changes of host molecules participated in interferon-beta signaling pathway were found after the transfection of the plasmid into cell in vitro. It provides a firm foundation for clarifying the pathgenic mechanism of EMCV. The contents of research are listed as below:1. Construction and identification of eukaryotie expression plasmid of EMCV 3CThe 3C gene of EMCV NJ08 was obtained by RT-PCR, and cloned into eukaryotie expression plasmid pVAX1. The validity of recombinant eukaryotie expression plasmid pVAX-3C was identified by double enzyme digestion and sequencing. With the help of lipofetamine 2000, the plasmid was transfected into HeLa cells, and immunofluorecence and western blot were performed. Results showed that the recombinant 3C protein was correctly expressed, whose molecular weight was in line with expectation. It provides a useful tool for further functional study of 3C protein.2. Effect of EMCV 3C on the molecules in IFN-β signaling pathwayIn this study, pVAXl-3C was transfected into HeLa cells and luciferase assay was performed. Results showed that in cells transfected with pVAXl-3C, expressions of luciferase that were driven by IFN-β, IRF3 and PRDII promoter were remarkably decreased when compared with those of control group, indicating that 3C protein could inhibit the production of IFN-β.Results of real time-PCR showed that relative expressions of ISG15, ISG56, Mxl, OAS and PKR in pVAXl-3C-transfected cells were reduced in comparison with those in cells transfected with pVAXl or GST eukaryotic expression plasmid. The results of luciferase activity driven by ISRE promoter showed that pVAXl-3C might restraint the down-stream signaling pathway of IFN. Western blot showed that total protein of STAT1 and STAT2 remained the same as before, as well as the total protein of phosphorylated STATs, illustrating that 3C protein would not lead to the degradation of STATs and the phosphorylated STATs. Immunoprecipitation results showed that 3C protein did not contribute to heterotrimerization of STAT1, STAT2 and IRF9. It demonstrated that EMCV 3C protein could inhibit IFN signaling pathway, including its production and exertion of its antiviral function, therefore antagonizing host innate immunology.