Research On Bovine Herpesvirus Type 1 BICP27 Protein Involved In Regulating The Expression Of Interferon And Its Transcription Factor

Infectious bovine rhinotracheitis(IBR)is an important infectious disease of cattle.The clinical symptoms are mainly respiratory symptoms,accompanied by conjunctivitis,mastitis,abortion and other symptoms.The pathogen is infectious bovine rhinotracheitis virus(IBRV),namely bovine herpesvirus 1(BHV-1).BHV-1 infection can form immunosuppression,resulting in secondary mixed infection of multiple pathogens in cattle,resulting in an increase in mortality.The immediate early(IE)genes bICP0,bICP4 and bICP27 encoded by BHV-1 are important factors that cause immunosuppression.Among them,bICP27 is an early viral protein that shuttles between the cytoplasm and the nucleus and has a wide range of gene expression regulation.Studies have shown that herpes virus regulates the production of interferon(IFN)through IE gene,inhibits its antiviral effect,and promotes viral replication.Therefore,this paper aims to study the regulation of BHV-1 bICP27 gene on IFN and its transcription factors,and provide research basis and reference materials for further elucidating the molecular mechanism of BHV-1 using bICP27 protein to cause immunosuppression.The main research contents are as follows.1.Construction of BHV-1 bICP27 gene deletion strainThe transfer vector prPgP carrying green fluorescent protein(GFP)was constructed on the basis of plasmid pVAX1.The upstream and downstream homologous arms of BHV-1 bICP27 gene were inserted into the corresponding multiple cloning sites of prPgP to obtain the recombinant transfer vector p△bICP27rPgP.The recombinant transfer vector was transfected into MDBK cells infected with BHV1,and the bICP27 gene was knocked out by homologous recombination.The BHV-1 bICP27 deletion strain was obtained by GFP labeling,cytopathic effect(CPE)screening and PCR identification,and named rBHV-1/△bICP27.2.The effect of bICP27protein on IFN and its transcription factor mRNA levelsMDBK cells were transfected or infected with plasmid pBIND-bICP27,recombinant virus rBHV1/△ bICP27,rBHV-1/△ bICP27+pBIND-bICP27,and parental virus BHV-1,respectively.Cells infected with bovine viral diarrhea virus(BVDV)were used as positive controls and untreated cells were used as negative controls.The mRNA levels of IFN-Ⅰ,IFN-Ⅲ,interferon regulatory factor(IRF)and NF-κB were detected by qPCR after 48 h.The results showed that the mRNA levels of IFN-Ⅰ,IFN-Ⅲ,NF-κB and IRF 1/3/7 induced by bICP27 gene deletion strain rBHV-1/△bICP27 were significantly higher than those of the parent virus,while the IKKβ level was opposite.MDBK cells were infected with rBHV-1/△ bICP27 and BHV-1,respectively,and cells were collected at different time points within 24 h to detect mRNA levels of IFNs.The results verified that the mRNA levels of IFN-α,IFN-β2,IFN-λ and IRF1 continued to rise with the increase of time after rBHV-1/△bICP27 infection.IFN-β1,IFN-β3,NFκB,IRF3 and IRF7 reached the peak at 6-8 h,then decreased,and increased again at 24 h,showing different timing characteristics.3.Study on the interaction between bICP27 protein and IRF 1/3/7The interaction between bICP27 protein and IRF was studied by CheckMateTM mammalian twohybrid system.The bICP27 gene and bovine IRF1/3/7 gene were amplified by PCR to construct pBIND-bICP27,pACT-IRF1,pACT-IRF3 and pACT-IRF7 eukaryotic expression plasmids.The constructed plasmid was transfected into HEK 293T cells,and the luciferase activity was detected by the dual luciferase reporter system detection kit.The results confirmed that pBIND-bICP27 and pACTIRF1/3/7 transfection did not enhance the expression level of the reporter gene.The results showed that BHV-1 bICP27 protein could inhibit the mRNA transcription levels of IFN-α,IFN-β1,IFN-β2,IFN-β3,IFN-λ,NF-κB,IRF1,IRF3 and IRF7,but this inhibitory effect may not be produced by the interaction between bICP27 and IRF1,IRF3 and IRF7.The results provide a reference for revealing the immunosuppressive mechanism of BHV-1.