Screening And Functional Domain Study Of TGEV Proteins Activating NF-?B Signaling Pathway

The pathogen of transmissible gastroenteritis(TGE)is transmissible gastroenteritis virus(TGEV),with clinical symptoms typified by lethal watery diarrhea and severe dehydration in piglets.Due to mortality can reach 100% in infected with TGEV piglets less than 2-week old,this disease always causes high economic losses once outbreaks occur and poses a threat to swine industry worldwide.Previous research has demonstrated that virulent TGEV caused severe pathological changes in small intestine,as well as atrophy of the villi in jejunum and ileum.In addition,TGEV also could up-regulated IL-6?IL-8?TNF-??IFN-? produced by mesenteric lymph node cells and dendritic cells.Thus,TGEV-mediated significant inflammatory response is possibly associated with the pathogenic mechanism during TGEV infection in host cells.NF-?B is an inducible transcription factor involved in promoting inflammatory response,as well as the regulation of cell proliferation and survival.The pathogenesis of TGEV is strongly associated with inflammation,in which NF-?B plays a pivotal role.Nevertheless,there is only limited information on the mechanisms of TGEV pathogenesis and TGEV-mediated inflammatory response.Firstly,we investigated the activation of NF-?B induced by TGEV using a luciferase reporter assay.The result showed that the NF-?B activation was more obvious with the increase of TGEV infection dose and extension of the infectious time of TGEV,but UV-TGEV have no change compared with the control.In addition,nuclear translocation of p65 induced by TGEV infection,which further confirmed that TGEV infection activated NF-?B signaling pathway,was detected by fluorescence microscope.In conclusion,TGEV activates NF-?B dependent on its replication and in a dose-dependent manner.Given that many viruses encode proteins that activate or modulate NF-?B signaling pathways for their own advantage,we used NF-?B related luciferase reporter assay to identify the key TGEV protein(s)involved in NF-?B activation.Expression plasmids of TH-98 strains TGEV proteins(Nsp1,Nsp2,Nsp3,Nsp4,Nsp5,Nsp6,Nsp7,Nsp8,Nsp9,Nsp10,Nsp11+Nsp12,Nsp13,Nsp14,Nsp15,Nsp16,ORF3 a,ORF3b,ORF7 and the gene of S,E,N)were constructed and their expression was verified by transfection in ST cells as well as detection by western blot and IFA.Finally,the key TGEV protein(s)activating NF-?B signaling pathway was screened using a luciferase reporter assay,which revealed that Nsp1,Nsp2,Nsp3,Nsp6,Nsp14,ORF7 induced NF-?B activation,but Nsp2 was the most obvious one.To investigate the effect on p65,I?B? of Nsp2 by western blot and IFA,the result showed that overexpression of Nsp2 induced phosphorylation of p65,I?B? degradation and nuclear translocation of p65.Therefore,Nsp2 activeNF-?B signaling pathway by Classical NF-?B pathway.In addition,expression plasmids of Nsp2 gene fragments were constructed and their expression was verified by transfection in ST cells as well as detection by IFA.Using a luciferase reporter assay,the key function domain responsible for NF-?B activation was located in the 1-120 amino acid in the TGEV region of Nsp2.In this study,we confirmed that TGEV could activates NF-?B dependent on its replication and in a dose-dependent manner.Nsp2 of TGEV was screened which was able to active NF-?B signaling pathway obviously,as well as we confirmed that the 1-120 amino acid in the TGEV region of Nsp2 was the key function domain responsible for NF-?B activation,which provide a theoretical basis for understanding molecular mechanism of TGEV pathogenicity and researching of antiviral drug against special target.