Detection Of Host Protein FKBP12 Response In Infectious Bursal Disease Virus Infection And Its Antiviral Effect

Infectious bursal disease(IBD),an immunosuppressive disease,is caused by nfectious bursal disease virus(IBDV).IBDVs targete at the bursa of the central immune organ of chickens,destroy the B lymphocytes of the core immune cells,not only induce the death of chickens,but also cause severe immunosuppression in surviving chickens,which results in compromised immunity and caused serious co-infection and secondary infections,seriously endangering the healthy development of poultry industry.IBDV is an icosahedral virus with a double-segmental,double-stranded RNA genome(segments A and B).Segment B encodes VP1 protein which has RNA dependent RNA polymerase activity(RdRp).Segment A contains two overlapped Open reading frames(ORF),the small one encods VP5,and the large one encods a Polyprotein(PP)precursor pvp2-vp4-vp3,which can be released by VP4 trans-enzymatic hydrolysis to VP3 and pVP2 that is further matured into VP2.Host innate immunity plays an important regulatory role in IBDV infection.As an important member of the FKBPs family,FK506 binding protein 12(FKBP12)has a variety of physiological functions including peptidyl-proly cis-trans isomerases(PPIase)activity,and is also involved in the regulation of several human viruses.Preliminary iTRAQ test data in our laboratory suggested that the host FKBP12 was significantly mobilized during IBDV infection.In this study,a fluorescence quantitative RT-PCR detection method of host FKBP12 was established.Multiple IBDV reference strains with different virulence were used,and SPF chicken and DT40 cells were used as infection models to study the host FKBP12 response to IBDV infection from both in vivo and in vitro.The results showed that FKBP12 is down-regulated after vvIBDV infection and is up-regulated after attenuated IBDV infection.This suggests that FKBP12 plays an important regulatory role in IBDV infection.In order to confirm this hypothesis,this study used overexpression and siRNA interference technology to up-regulate and down-regulate the expression of FKBP12,respectively.And evaluating the effect of FKBP12 on IBDV replication from the perspectives of gene transcription level,protein expression level and virus titer.The results showed that up-regulated expression of FKBP12 inhibited IBDV replication and down-regulated expression promoted virus replication.This results prove that FKBP12 is a host limiting factor for IBDV infection.In order to explore the mechanism of host FKBP12 as the host restrictive factor of IBDV,this study further confirmed the interaction between FKBP12 and IBDV VP4 protein through GST pull down and Laser confocal test.In view of the PPIase activity and serine protease activity of these two interacting proteins,we speculate that FKBP12 may inhibit IBDV replication by affecting VP4 enzyme activity.Based on the research model of IBDV polyprotein(PP)by VP4 trans-enzymatic hydrolysis,we found that overexpression of FKBP12 inhibited the trans-enzymatic hydrolysis of viral PP protein by IBDV VP4,which was specifically reflected in the down-regulation of VP2 and VP3 derived from PP enzymatic hydrolysis.This results indicated that host FKBP12 inhibited the VP4 transenzymatic hydrolysis activity of IBDV.In this study,the effects of host protein FKBP12 on IBDV replication were detected from threeaspects: gene transcription,protein expression and virus titer.FKBP12 was identified as a new host limiting factor for IBDV.The FKBP12 fluorescence quantitative RT-PCR assay was established to detect the response of FKBP12 to IBDV infection.Host restriction factor FKBP12 is downregulated in IBDV virulent infection.The interaction between host FKBP12 and IBDV VP4 protein was proved,and it was preliminarily revealed that FKBP12 could play the role of host limiting factor by inhibiting the protease activity of VP4 trans-enzymatic hydrolysis virus polyprotein.This study is of great significance to explore new prevention and control agents and new strategies for IBD.