Inhibition Of Signalling Pathway Of Interferon-Beta By 2c Protein Of Encephalomyocarditis Virus

Encephalomyocarditis(EMCV)can cause encephalitis and myocarditis in pigs and many kinds of mammals,which has important public health significance.EMCV 2C nonstructural protein is involved in virus-induced autophagy,but it is unclear whether it has the ability to antagonize the innate immune response.In this study,we constructed the eukaryotic expression plasmid of all the non structural proteins of EMCV,and observed the changes of IFN-beta signaling pathway in vitro,which enriched the pathogenic mechanism of EMCV.In addition,a recombinant EMCV rNJ08/Cap strain capable of expressing PCV2 ORF2 gene was constructed by reverse genetic technique,which laid a theoretical foundation for exploring the suitable sites for the expression of foreign genes in EMCV live virus vectors.The contents of research are listed as below:1.Cloning and expression of important molecules of type I interferon signal pathwayIn this study,the primers were designed for RT-PCR amplification these genes include MDA-5,RIG I,VISA,IRF3,P65 and IKKp.The PCR product was cloned into the eukaryotic expression vector pCAGGS.The recombinant plasmids were successfully obtained after double enzyme digestion and sequencing.With the help of lipofetamine 3000,the plasmid was transfected into 293A cells,and western blot were performed.Results showed that these proteins were correctly expressed,whose molecular weight were in line with expectation.n order to explore the relationship between the non-structural protein The mechanism of host innate immune response laid an important foundation.2.Effect of EMCV 2C on molecules in interferon-beta signaling pathwayIn this study,all the non-structural protein gene of EMCV NJ08 were obtained by PCR and colned into eukaryotic expression plasmid,which were named pCAGGS-YY-Flag(YY:2A,2B,2C,3A,3C,3D).These plasmids were transfected into 293A cells respectively,and the double luciferase assay were performed.Results showed that 2C protein could inhibit the activity of IFN-promoter.Results of real-time PCR showed that the relative expressions of MDA5,RIG,IFN-,MX1,ISG56,ISG15 and OAS in pCAGGS-2C-flag transfected cells were reduced in compared with those in cells transfected with pCAGGS eukaryotic expression plasmid.The results of localization experiments showed that 2C protein inhibited IFN-β production by interacting with MDA5 or RIG I,and this inhibitory effect was closely related to MDA5.Co-immunoprecipitation assays result indicated that 2C protein interacted with MDA5 in 293A cells.By constructing and expressing of 2C truncations and mutant,we found that 2C N-terminal 1-31 amino acids mediated the inhibitory activity.This study enriched the mechanism of EMCV escape from innate immune response.3.Construction and identification of a recombinant EMCV expressing ORF2 of porcine circovirus type 2In this study,fused PCV2 ORF2 gene at the region of amino acid 6 and 7 in the leader protein gene of EMCV NJ08 strain and generated recombinant EMCV rNJ08/Cap containing Cap gene.It had the special molecular marker.Western Blot and IFA results showed that the recombinant virus successfully expressed Cap protein.The growth characteristics and the plaque size of the recombinant virus rNJ08/Cap were similar to those of parent virus NJ08.It indicated that EMCV could be used as a viral vector to express foreign genes.It made an important foundation for studying on the EMCV and PCV2 vaccine in the future.