Molecular Mechanisms Of Regulation Of NF-κB Signaling Pathway By Orf Virus Encoding Protein:ORFV024

Contagious ecthyma, commonly known as Orf but also called contagious pustular dermatitis, is an urgent contagion caused by the Orf virus (ORFV) which infects goats and sheep. It always presents as a community disease and it has a low incidence rate among young sheep, although lambs from3-6months of age may be especially susceptible. The typical symptoms of Orf are erythema, papule and lanata on the lips.This disease has spread globally, especially in the north west and east of China, and it is one of main epidemic diseases among sheep.ORFV is a double-stranded DNA viruse, a member of the capripoxvirus genus of the poxvirdae. The whole length of ORFV is about135kb, it is rich in G+C contain (63%-64%), concludes about131predicted gene, among which middle sites of the viral genome is highly conserved. It plays an important role in virus replication and morphogenesis. The left and riggt-end regions changed greatly, more likely play its role in pathogenicity and immumoregulation.Orf virus (ORFV) is recognized as a strong host immune function. Poxvirus’s immunomodulatory effects on the host regulatory by encoding a large number of protein to achieve immune or inflammatory response in the host immune pathways. NF-κB signaling pathway is an important target for the virus to the host immune modulator. This pathway plays a key role in the pathogenesis of viral infection of host. Our previous experiments found that, ORFV024is an early gene and spotty distribution in the cytoplasm. ORFV024affects the NF-κB signaling pathway by inhibiting and its protein can inhibit the its activation.Although ORFV024is located in the middle sites of the viral genome, it has low homology with the others. ORFV has a great deal of sequence variation between the different strains of orf virus and other poxviruses. When defect strain OV-IA82A024and wild strain OV-IA82are used to infect OFTu, that ORFV024had no effect onvirus replication, but apparently it can decrease cytopathic effect. ORFV024inhibits the increase of cytokine sregulated by the signaling pathways (IL-6, IL-8). Further study indicates that ORFV024affects the NF-κB signaling pathway by inhibiting the phosphorylation of the IKKs complex. The NF-κB signaling pathway participates in the innate and acquired immune responses. However, in natural hosts, ORFV024does not affect the virulent characteristics. ORFV024might regulate the immunity of the host by interacting with the host cell proteins. Therefore, our purpose of this research is to discover and validate the interaction of NF-kappaB signaling pathway and ORFV024encoded protein, further clarify the molecular mechanisms of ORFV024regulation of NF-κB signaling pathway.In order to ensure the progress of experimental studies, we must first solve the problem of mass culture orf virus. ORFV is able to grow in cattle and sheep’s skin cells, kidney cells or calves and lambs’s testicular cells, which can form cytopathic effect. To establish the native primary cell culture and long term subculture, the fetal turbinate tissues were isolated from a4month ovine fetus and the cultivation of primary ovine fetal turbinate cells (OFTu) was performed. The first is the sheep embryo turbinate cells in primary culture. In the abdomen and uterine surgery pregnant sheep, remove and keep the spare embryos. Take sheep embryos turbinate with sterile methods. Digest with trypsin, diluted to5×105cells/ml of cell inoculums,100mm dish were seeded10ml liquid inoculated cells, put it into CO2incubator. Use the inverted microscope to observe the phase of the cell growth and the morphological changes.Observing its dynamic growth, we found that the cells were attached and showed a fibroblast-like morphology. After72h, cells grow normally. The double time of the OFTu is about36h based on the growth curve. Typical cytopathic effect (CPE) is observed at24h after infected with ORFV-NA in the OFTu. Immunofluorescence signal can be observed at12h in OFTu cells which infected with GFP-expressed ORFV. After12h of the adaptation period, the replication of the virus began to accelerate in12-24h, and then slow down in the time during36-72h. Using those cells, a cDNA library was constructed to study the interaction between ORFV proteins and host proteins by Yeast Two-hybrid System.Screening of host interacting proteins with ORFV024we select yeast two-hybrid system. The Yeast Two-hybrid System is widely used in the research of the interaction of proteins. Using those cells, a cDNA library was constructed. Extraction OFTu RNA, reverse transcription cDNA and synthetic single-stranded cDNA than double-stranded cDNA by PCR. cDNA and linear pGADT-Rec vector were cotransformed into yeast Y187, Spread them in SD/-Leu flat, after4-5days to collect yeast clones into nutritionally adequate liquid medium, packed and frozen for use. We constructed a0.57ML, cell density of7.5x107/ml, library titter of8.3x106cfu/ml of OFTU cDNA library. We construct bait plasmid vector PGBKT7-ORFV024, screened OFTu cells cDNA library and searched for the interactions between ORFV024and host proteins. After the self-activation and toxicity identification, expression in yeast cells, and then fused with OFTu library. We obtained61positive clones and identified11host proteins that interact with ORFV024. These were screened using the DNA sequencing technique. We discovered that IGFBP6(insulin-like growth factor binding protein6) and LAGE3(1antigen family, member3) are connected with the NF-κB signaling pathway.Further research by locating, Pull down experiments, Co-IP experiments on the interaction of ORFV-024and IGFBP6or LAGE3were identified. Using the molecular cloning techniques, we construct eukaryotic expression plasmid, the pEGFP-N1/IGFBP6and pEGFP-N1/LAGE3with GFP fusion, the pCDNA3.0-Flag/IGFBP6and pCDNA3.0-Flag/LAGE3with Flag tag, then transfected the OFTu, IGFBP6and LAGE3have a same distribution with ORFV024in the cells by observing the location, the IGFBP6were diffusely distributed in the cytoplasm, and express lower. The LAGE3were distributed in all part of the cell. The constructed pCDNA3.0-Flag/IGFBP6and pCDNA3.0-Flag/LAGE3was transfected OFTu cells together with pEGFP-N1/OFRV024. Preliminary analysis of the interaction between IGFBP6, LAGE3and ORFV024suggested that they have a spatial overlap.To confirm the The Yeast Two-hybrid results, the His experiments were carried out. A expression vector pET32a/ORFV024was constructed, The His-tagged ORFV024recombinant protein was purified and used to pull down with IGFBP6-GFP and LAGE3-GFP, IGFBP6-Flag and LAGE3-Flag separately. The results show that ORFV024has an interaction with LAGE3, but the interaction with IGFBP6was not detected. In addition, the pcDNA3.0/LAGE3and pcDNA3.0/IGFBP6plasmids were constructed. And IGFBP6-Flag or LAGE3-Flag fusion proteins were expressed. The results indicate that the physical protein interaction was observed between LAGE3-Flag and ORFV024-His. However, the interaction between IGFBP6-Flag and ORF V024-His was not observed.To further confirm the interaction between ORFV024and LAGE3or IGFBP6, the Co-Immune precipitation experiment was performed. The pEGFP-N1/IGFBP6and pEGFP-N1/LAGE3were transfected into293T cells together with pCMVTag-4A/ORFV024, using the anti-GFP antibody to pull down and anti-Flag to do Western Blot, the results shows that the interaction between ORFV024and LAGE3was confirmed, ORFV024and IGFBP6was not because of the very low level of IGFBP6expression. Therefore, the interaction between ORFV024and IGFBP6need further verification.In Conclusion,1) the technique with the characterization of isolation efficiency, fast propagation, simple operation and long term subculture, and the cultivation of primary ovine fetal turbinate cells (OFTu) was established.2) The OFTu cDNA library was constructed for the study of the protein interaction between the ORFV024and host proteins.3) ORFV024can physically interact with LAGE3protein followed by inhibition of NF-κB signaling pathway. The dysregulation of NF-κB signaling pathway may induce inflammatory diseases and tumor progression. Elucidation of the molecular mechanisms employed by parapoxviruses to modulate the NF-κB signaling pathway may contribute to developing potential therapeutic-valuable drugs for tumor and inflammation.