Molecular Mechanism Of IFN-? Expression Induced By TGEV M Protein And Its Functional Domain

Transmissible gastroenteritis virus(TGEV)is an enveloped,single-stranded positive-sense RNA virus that can cause transmissible gastroenteritis(TGE).TGE is an acute,highly contagious,intestinal disease in piglets.Since 2010,TGE has been occurred in many provinces of China.It caused huge economic losses to the pig industry,and restricted the healthy and rapid development of large-scale pig farms in China.Studying of the pathogenic mechanism of TGEV will help to prevent and control TGE.Studies have reported that TGEV infection could induce the production of type I interferon(IFN).IFN and its downstream interferon-stimulators(ISGs)are effective in inhibiting viral infection and proliferation in vivo,and they are important part of host antiviral infection.Many viral infections antagonize the production of IFN,and TGEV can induce the production of type I IFN after infection of cells.However,there are few reports on the specific molecular mechanism of TGEV until now.This study used the TGEV HE-1 strain to passage in IPEC-J2 cells.When blindly passed to the eighth generation,cells showed stable and typical cytopathic effect(CPE).TGEV HE-1 strain was confirmed by RT-PCR that it could adapt in IPEC-J2 cells.Under the fluorescence microscope,it was observed that the green fluorescent signal became stronger as the in oculated virus titer increased.Therefore,the TGEV HE-1 strain infected IPEC-J2 cells model could be used to study the molecular mechanism of TGEV-induced IFN-? production.IFN-? expression was up-regulated in a time-and dose-dependent manner detected by real-time RT-PCR and dual luciferase assay systems after TGEV infection of IPEC-J2 cells.At the same time,TGEV infection activated IRF-3 and NF-?B promoter activities,and the expression level of IRF-3 promoter was significantly higher than that of NF-?B.Western blot and laser confocal results showed that TGEV infection induced phosphorylation of IRF-3,and nuclear transfer of IRF-3.This indicates that up-regulated of IFN-? expression by TGEV infection may be primarily dependent on the regulation of IRF-3 transcription factors.The S,E,M,and N gene of TGEV were amplified by RT-PCR and linked them to the p CMV-HA-N eukaryotic expression vector to construct recombinant plasmids.All proteins were successfully expressed in the transfected IPEC-J2 cells.Dual luciferase assay showed that TGEV S,N and E could induce small amount of IFN-? expression,but M protein was the most important activator for IFN expression.After TGEV M gene si RNA was transfected into IPEC-J2 cells,the expression of IFN-? was significantly down-regulated,further demonstrating that TGEV M protein can induce IFN-? production.Western blot and laser confocal assay showed that M protein activated IFN-? by inducing phosphorylation and nuclear translocation of IRF-3.Five recombinant plasmids were constructed to express different designed TGEV M truncated genes.The dual luciferase assay confirmed that the key functional domain of M protein to activate IFN-? expression located at 1-88 aa.In conclusion,this study demonstrated that the TGEV isolate HE-1 strain could adapt in IPEC-J2 cells.IFN-? production induced by TGEV infection in IPEC-J2 cells is dependent on IRF-3 transcription factor regulation,and in a time-and dose-dependent manner.TGEV M protein is a key structural protein that induces IFN-? production and activates IFN-? signaling pathway by inducing phosphorylation and nuclear transfer of IRF-3 too.The key functional domain of the TGEV M protein to activate IFN-? signaling pathway is located at 1~88 aa.This study laid the foundation for elucidating the pathogenesis of TGEV,and provided a theoretical basis for the research of targeted drugs and the effective prevention and control of TGE.