Research On The Activation Of Host Cell NF-κB Regulated By Poxvirus Ankyrin Protein From Orf Virus

Objective: NF-κB pathway plays important role in the process of antiviral responses such as innate immune responses and inflammatory,it will be actived in virus infection.Studies show that many of ankyrin proteins encoded by poxvirus can inhibit NF-κB activation and escape the host immune response.To illuminate the mechanism of Orf virus(ORFV)encodes ankyrin protein regulating of NF-κB activation in host cells.Perliminary to explore the process of ORFV regulating host immune response and the mechanism of ORFV entering host cell.Methods : Firstly,preliminary prediction of the structure of ORFV ankyrin protein and analysis of its biological function by bioinformatics method;secondly,construction of its eukaryotic expression vectors were transected in 293 T cells,analysis to screen out regulating NF-κB activation that has a significant effection of the ankyrin protein by dual-luciferase reporter assay,then verified the molecules biological characteristics by using real-time semi-quantitative RT-PCR and cell location experiments;finally,using western blotting to analyze selected molecules effect on the nuclear translocation of NF-κB p65 and the degradation of IκBα or the level of phosphorylation IκBα in 293 T cells.Results:(1)Biological information analysis showed that ORFV encoding five kinds of ANK/F-box protein has similar characteristics: contain 5~9 ANK motif in the N-terminal and the sequence of amino acids at the key sites the F-box-like domain of C-terminal was the same as the cell F-box protein.In ORF126 from 155~177 amino acid region is transmembrane region,and in ORF128 after 433 amino acid the "PRRARRL" is the nuclear localization sequence(NLS).(2)RT-PCR results showed that the five kinds of ankyrin proteins were expressed in the early stage of virus infection,cellular localization experiment showed that ORF123 localized in the cytoplasm,ORF126 localized in the cell mitochondria and ORF128 nuclear localization is mediated by ORF128 ANK and NLS.(3)Dual luciferase reporter assay showed that ORF123 and ORF008 can significantly inhibit NF-κB(P<0.01)and ORF128 also can blocked the expression of NF-κB reporter luciferase activity(P<0.05).(4)Western-blot results showed that ORF008,ORF123 can prevent IκBα protein phosphorylation degradation process of the activation of NF-κB induced by TNF-α and can inhibit the nuclear translocation of NF-κB p65 protein.Conclusions:(1)ORFV encodes ankyrin protein ORF008 and ORF123 can significantly inhibit the NF-κB signaling pathway activation process induced by TNF-α.Mechanism of ORF008 and ORF123 action that is can prevent the protein phosphorylation degradation process and block the activation of NF-κB.(2)ORFV encodes the five ankyrin began to express in the early stage of viral infect susceptible cells,then interfere with cellular antiviral response or inflammation by inhibiting activation of NF-κB signaling pathway or other ways to facilitate invading cells and replicating in the host cell.