Molecular Mechanism Of The Inhibition Of NF-?B Signaling Pathway By Ankyrin Repeat Proteins Of Orf Virus

Objective: Orf virus(ORFV)is a pathogen which can cause an acute,highly contagious disease——contagious ecthyma in goats and sheep,which can also infect humans and cause highly infectious zoonoses.ORFV is a major member of chordopoxvirinae parapoxvirus in the family Poxviridae.Most poxviruses have evolved a unique ability to regulate host immune responses against the NF-?B signaling pathway in order to ensure their normal replication in host cells.The anchor protein,which is composed of multiple copies of the ankyrin repeat(ANK)motifs,as the largest family of poxvirus proteins,plays a crucial part in the virus evading the host immune response.At present,there are many studies on the regulation of host NF-?B signaling pathway by poxvirus ankyrin repeat proteins,but less research on the interaction between Parapoxvirus ankyrin repeat proteins and NF-?B signaling pathway.In order to systematically investigate the regulation mechanism of NF-?B signaling pathway by five anchor proteins(ORF008,ORF123,ORF126,ORF128 and ORF129)encoded by ORFV of Parapoxvirus,firstly,to verify whether the five ankyrin repeat proteins encoded by ORFV can be correctly expressed in 293 T cells,and to elucidate the regulating effect of ankyrin repeat proteins on host nuclear factor kappa B(NF-?B)signaling pathway;secondly,to elucidate the regulation mechanism of ORF128 encoded by ORFV on NF-?B signaling pathway;in the meanwhile,the ORFV-?ORF123 recombinant virus was initially constructed to lay a foundation for further study on the biological function of ORFV encoding anchor proteins in viruses.Methods: 1.Western-blot was used to detect the expression of ORFV ankyrin repeat proteins which were constructed into eukaryotic expression vector pCMV-tag2 B in 293 T cells;a dual luciferase reporter assay system was used to analyze the regulating effect of NF-?B signaling pathway by five ORFV ankyrin repeat proteins.2.The effect of ORF128 protein on the nuclear translocation of NF-?B-p65 and the phosphorylation of I?B? protein(p-I?B?)were detected by Western-blot.3.ORF123-deleted recombinant plasmid pSP72-ORF123 LR and transfer vector pORF123LR-Eg carrying EGFP marker gene and gpt resistance gene were constructed according to the sequence of orf virus(ORFV).The homologous recombination between the pORF123LR-Eg transfer vector and the ORFV genome was performed in OT cells,and then the recombinant mutant virus ORFV?ORF123 was selected using EGFP as screening marker under fluorescent microscope.Results: 1.Ankyrin repeat proteins recombinant plasmids constructed into eukaryotic expression vector pCMV-tag2 B can be correctly expressed in 293 T cells verified by Western-blot method.When the prorein loadings of five ankyrins all are 20?g,ORF123 followed by ORF128 had the highest protein expression in 293 T cells.All five ankyrin repeat proteins could inhibit the luciferase activity of NF-?B transcription factor reporter vector;ORF123 followed by ORF128 had the most significant inhibitory effect on NF-?B signaling pathway,and the inhibitory effect was dose-dependent.2.Western-blot assay showed that the nuclear translocation of NF-?Bp65 protein was significantly inhibited in the ORF128 transfected group compared with the control group of vector pCMV-tag2B;the detection of phosphorylation level of I?B?(NF-?B-p65 protein inhibitor)upstream of the signaling pathway showed that,phosphorylation of I?B? was not affected by the increase of stimulation time of TNF-?,while the degradation of p-I?B? was significantly inhibited in the ORF128 transfection group.3.The results both of double enzyme digestion and sequencing showed that the pSP72-ORF123 LR recombinant plasmid and pORF123LR-Eg transfer vector were correctly constructed;fluorescence microscopy showed that green fluorescence was observed only in the experimental group transfected with the transfer vector two hours after the poisoning,compared with the blank control group,the single poison control group,and the separate transfection vector control group.Conclusion: ORFV ankyrin repeat proteins can be expressed correctly in eukaryotic cells and inhibit the NF-?B signaling pathway of host cell.2.ORF128 protein inhibits the activation of NF-?B signaling pathway by blocking the nuclear translocation of NF-?B-p65 and the degradation of p-I?B?.3.The construction of ORFV?ORF123 recombinant virus preliminaryly laid a foundation for further screening and obtaining stable genetic ORFV?ORF123 recombinant virus.