The Function Of Orf Virus-encoded 120 Protein And Regulation Mechanism On NF-?B Signaling Pathway

Contagious pustular dermatitis,also known as contagious ecthyma and generally referred to as Orf,is a highly contagious viral disease caused by the Orf virus(ORFV).ORFV primarily infects sheep and goats,and herders and workers in related occupations are also often infected,so ORFV is also an important zoonotic disease,posing a serious threat to sheep farming and human health.The ORFV genome is large and encodes numerous proteins.The ORFV120 gene is located at the end of the viral genome,with no clear matches to other poxviruses,and its function is unknown.Given that the terminal region of ORFV genome encodes many genes related to virulence,pathogenicity and immune regulation,it is speculated that ORFV120 gene may encode a factor related to pathogenesis and virulence.To clarify the function of ORFV120 gene,OV-SY17 was used as the parental strain in this study,and the ORFV120 gene was knocked out by homologous recombination technology,resulting in the successful construction of the OV-SY17?120 gene deletion strain carrying GFP protein.with p UC18 as the backbone plasmid,the red fluorescent protein RFP-labeled OV-SY17-RV120 reversal virus was constructed.The biological properties of ORFV120 gene OV-SY17?120 and OV-SY17-RV120 strains were investigated,and it was discovered that there was no significant difference in the cytopathic changes and viral titer.The results of the native animal regression experiment revealed that typical mouth sore lesions were appeared in lambs inoculated with OV-SY17 and OV-SY17-RV120 strain on the third day after infection.The lambs inoculated with the OV-SY17?120 strain,on the other hand,only showed light erythema at the early stage,which subsided later.The ORFV120 gene,according to the above conclusion,is an important virulence gene encoded by the virus,but deletion of the ORFV120 gene has no effect on the viral normal replication and proliferation.To determine the cellular localization and expression characteristics of ORFV120protein were further examined.Laser confocal observation revealed that ORFV120protein was mainly distributed in the cytoplasm of infected host cells in a punctate pattern,and occasionally in the nucleus.The expression of ORFV120 gene was detected by RT-PCR and real-time fluorescence quantitative PCR with the addition of Ara C,which is a commonly used as a viral late DNA synthesis inhibitor,and the results showed that the expression of ORFV120 gene could be detected at the early stage of viral infection(0.5 h),and confirmed that ORFV120 gene was a virus-encoded early-late gene.Based on the above results,transcriptome sequencing technology was used to detect the effect of the ORFV120 gene on the regulation of intracellular m RNA transcription based on the above results.The analysis results showed that some m RNAs related to the NF-?B signaling pathway,such as NF-?BIA,ICAM1,IL-8,and PTGS2,were significantly down-regulated infected with OV-SY17?120 strain.The results suggest that ORFV120 protein has a regulatory role on NF-?B signaling pathway.As a class of transcription factors involved in the regulation of the host innate immune response,NF-?B plays an important role in mediating inflammation,immunity,cell proliferation,differentiation and apoptosis.ORFV is recognized to have potent immunomodulatory functions that are thought to be related,at least in part,to its ability to express a range of immunomodulatory factors that target host inflammatory pathways,and several of the viral encoded proteins(ORF002,ORF024,ORF073,ORF119,ORF121)have now been shown to block,and disrupt the host innate immune by inhibiting the NF-?B signaling pathway for viral replication.It is thus clear that the NF-?B pathway is one of the important targets of ORFV for immune regulation of the host.However,the exact regulatory role and mechanism of ORFV120 protein on NF-?B signaling pathway needs to be studied in depth.Next,the dual-luciferase reporter system was firstly used to detect the effect of ORFV120 protein on the transcriptional activity of NF-?B-p65.The results showed that the transcriptional activity of NF-?B-p65 infected with OV-SY17?120 strain was lower than OV-SY17 and OV-SY17-RV120 strains,indicating that ORFV120 protein can promote the transcription of NF-?B-p65.In addition,the phosphorylation levels of IKK?/?complex,I?B?and p65 at different time points with OV-SY17,OV-SY17?120and OV-SY17-RV120 strains infection was detected.The protein results showed that OV-SY17?120 regulated the phosphorylation of IKK?/?complex,I?B?and p65 at lower levels than that of OV-SY17 and OV-SY17-RV120 strains.However,OV-SY17and OV-SY17-RV120 strains have the same activation level of NF-?B signaling pathway.The nucleation level of p65 in OV-SY17?120-infected cells is also lower than that in OV-SY17 and OV-SY17-RV120 strains by the confocal microscopy detection.Overexpression of ORFV120 protein can release p65 through phosphorylation of IKK?/?complex and I?B?,so that it can enter the nucleus after phosphorylation.In addition,p65 could be detected in the nucleus,indicating that ORFV120 protein can activate the NF-?B signaling pathway.However,in the late stage of OV-SY17 infection,the degree of activation of the NF-?B signaling pathway was inhibited.This may be related to the evolution of other NF-?B pathway inhibitors by ORFV at the later stage of infection.The results suggest that the activation of NF-?B signaling pathway by OV-SY17 strain at the early stage of infection may depend on the ORFV120 protein.Through the above experiments,this study confirmed that ORFV120 protein can activate the NF-?B signaling pathway,but the activation mechanism is still unclear.To elucidate the detailed mechanism of 120 protein regulation of NF-?B signaling pathway,the host cell proteins interacting with ORFV120 protein were screened using yeast two-hybrid-membrane system assay,and the reciprocal protein was identified as Ras-GTPase-activating protein(GAP)-binding protein 1(G3BP1),a key component of mammalian cell stress granules(SGs).With co-immunoprecipitation and colocalization,it verified that ORFV120 can interact with G3BP1.To determine the binding sites for the interaction between G3BP1 and ORFV120 protein and to construct the corresponding truncates of G3BP1,immunoprecipitation assay showed that G3BP1^NTF2 and G3BP1^{Pxx P} domains were the key domains binding with ORFV120.To clarify whether ORFV120-mediated NF-?B activation is related to G3BP1,it was found that the expression level of G3BP1 protein increased and then decreased during OV-SY17 infection.OV-SY17?120 strain infection has significantly lower regulation level of G3BP1 protein expression than OV-SY17 and OV-SY17-RV120 strains.However,OV-SY17 and OV-SY17-RV120 strains had similar regulation levels of G3BP1 protein expression.In the case of overexpression of ORFV120 protein,the expression level of G3BP1 protein was significantly increased,which indicated that ORFV120 protein could promote the expression of G3BP1 protein.With using G3BP1 specific si RNA and then overexpressing ORFV120 protein,the result showed that the phosphorylation levels of IKK?/?Complex,I?B?and p65 were significantly decreased after interfering with G3BP1 protein expression.With interference with G3BP1 and OV-SY17 or OV-SY17?120 re-infection,the results showed that in the presence of the ORFV120 gene,the phosphorylation levels of IKK?/?complex,I?B?and p65 were also significantly decreased after interfering with G3BP1 protein expression.Thus,it was confirmed that G3BP1 was involved in the activation of NF-?B signaling pathway by ORFV120protein.According to the relationship between ORFV120 and G3BP1.The results of double luciferase showed that ORFV120 positively regulated the activation of NF-?B signaling pathway through the full length of G3BP1 or G3BP1^RRM+RGG domain.In conclusion,this study systematically carried out functional studies targeting ORFV120 protein and confirmed that ORFV120 protein is a virulence gene that can positively regulate NF-?B immunoregulatory signaling pathway.This study focuses on revealing the molecular mechanism of ORFV120 protein activation of NF-?B signaling pathway.ORFV120 protein can bind to G3BP1^NTF2and G3BP1^{Pxx P}domains of G3BP1protein,and positively regulate the activation of NF-?B signaling pathway through G3BP1 full-length or G3BP1^RRM+RGG domain.This study not only enriches the data of functional studies on ORFV unknown function genes,but also fills the gap in the functional studies of ORFV120 protein.The study reveals the regulation mechanism of ORFV120 protein on NF-?B signaling pathway,which lays the foundation for the comprehensive elucidation of ORFV immune escape mechanism and provides important ideas for ORFV vaccine design and antiviral drug design.