Effect Of Encephalomyocarditis Virus HB10 3C Protein On The Signaling Pathway Of Type â…  Interferon

Encephalomyocarditis vims (EMCV) is a positive sense single-stranded RNA vims and it belongs to Picornaviridae. EMCV is the major pathogen for porcine myocarditis, and a widely spread natural foci humans and animals comorbidity pathogen. EMCV can cause myocarditis, encephalitis or inflammations around the heart, neurological disorders, reproductive disorders and diabetes, which leads huge economic losses to the swine industry worldwide. The vims has occurred in the farms in our country at present, but its condition is not very clear, so there is no suitable way for tracking and controlling EMCV infection. EMCV 3C protease has one cysteine nucleophilic attacking active site and it is involved in almost all the late process of EMCV vims protein hydrolysis Its prokaryotic expression products 3C protein had good immunogenicity. In this study,3C gene isolated from EMCV HB10 was cloned to make prokaryotic expression vector, and then 3C prokaryotic expression protein and the EMCV 3C protein monoclonal antibodies were obtained, which laid the foundation for the epidemiology and molecular diagnostics of this virus. Previous studies showed that EMCV infection can induce the degradation of pattern recognition receptor RIG-I, but it still can activate the type I interferon signaling pathway through another pattern recognition receptor MDA5. However, there are reports showed that EMCV 3C protein can inhibit the activation of IFN β signaling pathway. So it is necessary to clarify the influence of EMCV HB10 on IFN β signaling pathway.Firstly, the EMCV HB10 strain was inoculated on BHK-21 cells. Cell pseudopodia, round shrink, collapse, and fall off occurred 24 h later. Specific primers of EMCV 3C were used to amplify gene from cytopathic cells by RT-PCR and we got a fragment of 615 bp, the result was consistent with the expected EMCV 3C fragment length, and then weinserted it into pET-28a vector to construct a recombinant vector which was called plasmid pET-28a-3C. Secondly, we transformed the pET-28a-3C into BL21 and cultivated 12 h until the OD value reached 0.5 to 0.6,1 M IPTG induced 4 h, then used nickel column to purify His-3C protein. The first immunization of purified recombinant His-2A protein with Freund’s complete adjuvant in 1:1 (V/V) ratio emulsion after emulsification immunized BALB/c mice, and then twice emulsifying the same proportion with incomplete Freund’s adjuvant, and then immunized mice. After three consecutive immunization bloods sample was detected by ELISA titer, take BALB/c immunization spleen cells with myeloma cells SP2/0 fusion mouse when potency reaches a certain value. Finally, screened positive hybridoma cells with purified His-3C protein coated ELISA plates, got three strains of stable hybridoma subclones after three times subcloning, which was named 7H5,4F4 and 2G3. By using ELISA,3C monoclonal antibody western blot analysis and IFA can be well prepared to identify specific protein and EMCV 3C whole virus.To explore the impact of EMCV 3C protein on type I interferon signaling pathway, this study identified that EMCV HB10 could not induce activation of type I interferon signaling pathway by dual luciferase reporter gene system and western blot firstly, and then the preliminary results were confirmed by quantitative fluorescence, ELISA, and VSV-GFP reporting systems. The EMCV-HB10 was found to inhibit poly (I:C)-mediated type Ⅰ interferon signaling pathway, To clarify this mechanism, the plasmids expressing protein encoded by EMCV and IFN (3 reporter gene plasmids were co-transfected into HEK 293T cells and the results showed that EMCV 3C protein plays a key role. Then, we constructed EMCV 3C plasmids and EMCV 3C double mutant plasmids and dual luciferase reporter gene system and western blot experiments showed EMCV 3C could inhibit the activation of type I interferon signaling pathway and inhibit IRF3 phosphorylation. In fact, studies have shown that, EMCV-3C inhibited the activation of type I interferon signaling pathway. Base on the theory, poly (I:C) and SeV were used to induce type I interferon, but when EMCV 3C protein expressed, the production of type I interferon was significantly decreased. But through phosphorylation we found that this kind of inhibitory effects depend on its protease activity. Finally confocal laser experiment observed the situation in the case of EMCV-3C IRF3 presence into core confocal, which showed EMCV-3C can be held hostage by TBK1 and IKKs and so due to the nuclear IRF3, but after EMCV-3C double mutant of this hostage recovery effect, and in the case of EMCV whole virus infection, we carried out EMCV experiment and found phosphorylation can inhibit the phosphorylation of Statl action. Experimental results showed that EMCV 3C protein can inhibit type I interferon, and may affect a certain key point in JNK pathway of type 1 interferon signaling pathway downstream.