| Since the Atypical Porcine Pestivirus(APPV)was first reported in the US in2015,it had been prevalent in many countries worldwide,which posed a serious threat to the healthy development of global pig industry.APPV is a newly discovered pestivirus in recent years,and its pathogenesis and immunosuppression mechanism are still unclear.Therefore,effective prevention and control measures are still lacking.We isolated APPV Yunnan strain(APPV-YN)and identified Mut,a key gene of atypical clinical symptoms,and found that Mut significantly affected the expression of inflammatory genes regulated by NF-κB transcription factor in host cells.It was speculated that the protein encoded by Mut might be a new immune regulatory protein.Inhibition of NF-κB activity regulates host immunity and pathological damage,but the specific regulatory molecular mechanism remains unclear.In order to investigate the biological characteristics of APPV-YN Mut and its effect on the transcriptional activity of NF-κB signaling pathway in host cells.In this study,APPV-YN Mut was used to construct eukaryotic expression plasmid,lentiviral recombinant plasmid and stable Mut expression cell line.Biological characteristics of APPV-YN Mut were analyzed by bioinformatics software.TNF-α was used to simulate infection to detect the nuclear localization of NF-κB P65 protein and the effect of Mut on the transcriptional activity of NF-κB.The following results were obtained:(1)Eukaryotic expression and biological characteristics of APPV-YN Mut.The eukaryotic expression plasmids p CMV-Mut and p EGFP-N1 Mut constructed by APPV-YN Mut and the lentiviral recombinant plasmids p CDH-CMV-Mut were stably expressed in SVEC cells with the expression protein size of 13 k Da.Physical and chemical properties predicted that Mut was a stable hydrophilic protein with a molecular weight of about 13 k Da.Secondary and tertiary structure analysis showed that Mut protein had no transmembrane helical structure and no signal peptide.The results of subcellular localization showed that the protein was mainly located in the cytoplasm,and the fluorescence subcellular localization was consistent with the predicted results.Protein phosphorylation sites and functional kinase prediction results showed that the protein had 8 threonine phosphorylation sites,8 serine phosphorylation sites,1 tyrosine phosphorylation site,It can act on protein kinase C(PKC),mitogen activated protein kinase(P38MAPK),protein kinase A(PKA),epidermal growth factor receptor(EGFR),cyclin dependent protein kinase 5(CDK5)and other related kinases and protein receptors.(2)Effect of APPV-YN Mut on the transcriptional activity of NF-κB.The eukaryotic expression plasmids p CMV-Mut and p EGFP-N1-Mu constructed by APPV-YN Mut and the lentiviral recombinant plasmids p CDH-CMV-Mut were stably expressed in SVEC cells with the expression protein size of 13 k Da and subcellular localization in the cytoplasm,which was consistent with the predicted results.The results of CCK8,dual luciferase reporter gene and EMSA assay showed that overexpression of Mut protein affected cell proliferation under TNF-α,inhibited TNF-α-mediated NF-κB transcription activity(****P < 0.0001),and reduced the binding ability of transcription factor NF-κB to target sequences.The inhibitory effect was related to the expression level of Mut protein.Indirect immunofluorescence results showed that Mut protein colocalized with NF-κB P65 in both the nucleus and cytoplasm,reducing the amount of P65 into the nucleus(****P < 0.0001)and prolongs the activation time after stimulation.The results of Western Blot and q-RTPCR showed that the cells expressing Mut protein significantly inhibited the phosphorylation and degradation of IκB-α(****P < 0.0001),and the amount of PP65 protein was significantly lower than that of the control cells(****P < 0.0001).The transcription levels of Rel A,NFKBIA,CHUK and IKBKB genes were significantly down-regulated(****P < 0.0001).In summary,the results of this study suggest that APPV-YN Mut affects the activation of NF-κB pathway by inhibiting protein signals associated with the typical NF-κB pathway.The APPV-YN Mut protein targets the phosphorylation and degradation of IκBα to prevent the release of P65 into the nucleus and phosphorylation modification,inhibit the activation of downstream NF-κB-dependent genes,and significantly reduce the expression of inflammatory genes.This study provided the research basis for elucidating the pathogenesis of APPV-YN Mut in atypical porcine pestivirus and the molecular mechanism of atypical clinical symptoms,and provided a new idea for the atypical APPV and immune escape mechanism. |
