Identification And Bioinformatics Analysis Of Differential Expression Proteins Of BDV-P/BDV-N Interaction With Host

Background:In 1885,a disease that caused the death of a large number of horses were endemic in Borna town,Sachsen,Germany,named Borna disease(Borna disease,BD).Borna disease has been shown to be caused by a neurotropic virus called the Borna disease virus.Natural infection of BDV has now been found in a wide variety of vertebrates,suggesting that the host range of this virus probably includes all warm-blooded animals.BDV persistently infects the CNS of many animal species and causes behavioral disturbances,such as anxiety,aggression,hyperactivity,abnormal behavior,and cognitive deficits.In a large epidemiological study of mental patients(especially schizophrenia,depression and bipolar disorder)in the past twenty years,it has been found that BDV-antibodies and nucleic acids can be detected in the cerebrospinal fluid and blood of some patients with mental disorders,suggesting that BDV infection may be related to human mental illness.Natural and experimental BDV infections can result in severeimmune-mediated neurological disease and central nervous system inflammation.How BDV infects and how to cause the disease is still unclear,especially the initial target of BDV in the host.The current literature has studied the mechanism of phosphoprotein(P),nucleoprotein(N),glycoprotein(G),and non-glycosylated protein(X)in BDV.Among them,P protein and N protein play a major role in pathogenesis.It is also the most studied at present,especially the study of phosphoprotein.Objective:In this study,lentiviral vectors were constructed for P24 and P40 plasmids of BDV,and primary neurons were infected with lentivirus.Different proteins that bind to the BDV-P/N protein were screened,and it was expected to further identify the key protein that binds to the BDV-P/N protein and explore its pathogenic effect.Methods:1.SH-SY5 Y cells were successfully transfected after plasmid amplification,and normal expression of P24 and P40 proteins was verified by Western-Bolt.2.Lentivirus packaging and titer determinations were performed and primary hippocampal neurons were infected.Western blot was used to detect the expression of P24 and P40.3.Lentivirus was used to extract proteins from SD fetal rat hippocampus for co-immunoprecipitation experiments.The experimentswere divided into BDV-P(P24)group,BDV-N(P40)group,and NC group.The antibodies were P24 antibody,P40 antibody,rabbit IgG antibody.Co-immunoprecipitation complexes were analyzed by LC-MS/MS mass spectrometry.4.The results of mass spectrometry identification of protein complexes compared with the control group yielded differential proteins,and bioinformatics(Pathway and GO)analysis was used to predict differential proteins.5.Verify the above predicted differential proteins.Results1.SH-SY5 Y cells were successfully transfected with plasmid-amplified sequencing sequences and Western-Bolt was used to verify the normal expression of P24 and P40 proteins.2.Overexpression of the lentiviral vector was successfully constructed and the titer of 1 × 10 9 TU/ml was determined.The primary cell was successfully infected and expressed BDV-P(P24)and BDV-N(P40).3.The results of mass spectrometry analysis of co-immunoprecipitation complexes yielded differential proteins,and 453 candidate proteins that interacted with BDV-P were screened by venny analysis,;259 screened to interact with BDV-N.4.Based on bioinformatics analysis,it was predicted that acyl-CoA b inding protein(ACBP)binds to BDV-P24,predicting acyl-CoA b indingprotein(ACBP)and BDV-P24 binding,RT-qPCR experiments ACBP and involved GABA-?1,GABA-?2,GABA-?3 further validation,ACBP,GABA-?1,GABA-?2 upregulation(P <0.05).There was no significant difference in GABA-?3(P>0.05).Conclusion:1.In our study,the lentiviral vector of phosphoprotein P24 and nucleoprotein P40 of BDV was constructed and infected cells successfully,providing a more powerful technical method for the basic research of BDV.2.The differential protein results can be used for subsequent experiments.We have screened 453 candidate proteins that interact with BDV-P and 259 proteins that interact with BDV-N.These data are in-depth the clues to study the pathogenic mechanism and mode of action of BDV-P/N binding to the host.We hoped that the proteins associated with BDV and human psychiatric disorders can be screened to further explore the pathogenesis of BDV.3.We speculate that acyl-CoA binding protein(ACBP)binds to BDV-P24 and may participate in the process of anxiety induced by BDV infection in the host.4.Combined with bioinformatics analysis and literature reports,we hypothesized that the first: histamine H1/H2 receptor-mediated signal transduction pathway of BDV-P protein may be the key to anxiety and memory impairment after BDV infection.Second,the relationship betweenSlit/Robo axon guidance molecules of BDV-P protein and tumors may inhibit glioma invasion after infection with BDV.