The Mechanism Of Bovine Herpesvirus Type ? Immediate Early Protein BICP0 Negatively Regulating TRAF6

Bovine herpesvirus I?BHV-1?,also known as bovine infectious rhinotracheitis virus?IBRV?,is the causative agent of bovine infectious rhinotracheitis?IBR?.As a virulence protein of BHV-1,BICP0 can be continuously express throughout the infection period of BHV-1.A large number of studies have shown that the expression of BICP0 cause resuscitation and massive proliferation of BHV-1.TRAF6 is a key linker molecule in multiple innate immune pathways of the host,whic h mediates NF-?B signaling pathway?classic pathway and non-canonical pathway?,MAPK pathway?JNK1/2,ERK1/2,p38?,and IRFs pathway?IRF3,IRF4,IRF5 and IRF7?and the PI3K pathway.TRAF6 plays a crucial role in the host's inflammatory response and natura l antiviral response.This study investigated the structure and mechanism of the interaction between BICP0 and TRAF6.The immune evasion mechanism of BHV-1 using BICP0 was further studied.It can provide the oretical support and material basis for the prevention and treatment of BHIV-1.The main research contents are as follows:1.Amplification of BICP0 gene and construction of recombinant baculovirus RE-BICP0-FLAGIn this study,PCR primer pairs were designed based on the sequence information of the BHV-1.1 Cooper strain?USDA NVSL challenge 97-11?in Gen Bank?Gen Bank accession number:JX898220?.The genome template of PCR was prepared by laboratory-preserved BHV-1 strain.In this study,the gene fragment of BICP0 was successfully obtained and the p EASY-Blunt Simple-BICP0 recombinant plasmid was constructed.The recombinant baculovirus RE-BICP0-FLAG was subsequently successfully constructed by using the commercial Multi Bac Mam TM system.As an effective foreign gene delivery system,recombinant baculovirus RE-BICP0-FLAG successfully solved the problem of difficult transfection of MDBK cells,which laid a material foundation for the independent study of the function of BICP0 by delivering BICP0gene in MDBK cells.In addition,this study introduced the Flag tag sequence at the carboxy terminus of BICP0,which simplified the identification of BICP0 and laid the foundation for the subsequent research.2.Application of recombinant baculovirus RE-BICP0-FLAGThe virus titer of the P3 generation RE-BICP0-FLAG was 2.7×10⁶ TCID₅₀/100?L as measured by the Reed-Muench method.Used the gradient infection assay and Western Blot method,the optimal dose of RE-BICP0-FLAG transduced MDBK cells was determined to be1×10⁶ TCID₅₀ to 2×10⁶ TCID₅₀.Subsequently,quantitative PCR analysis of RE-BICP0-FLAG-transduced MDBK found that BICP0 can reduce the transcription levels of IL-6,IL-8,ISG15 and ISG56,suggested that BICP0 BICP0 may inhibit the inflammatory pathway and antiviral pathway of MDBK cells.Western Blot identification showed that BICP0 caused the decrease of TRAF6 at the protein level.By used of the proteasome inhibitor MG132 and the lysosomal inhibitor chloroquine,respectively,this study found that the decrease of TRAF6 caused by the BICP0 in MDBK cells was degraded via the ubiquitin-proteasome pathway.The successful application of RE-BICP0-FLAG had confirmed that the BICP0 can achieve immune evasion by hydrolyzing the host's innate immune-related molecule,TRAF6,without the involvement of other viral proteins.3.Effect of BICP0 on TRAF6 during transient transfectionTo further investigated the mechanism of BICP0 on TRAF6,several recombinant expression vectors of BICP0 were constructed:pc DNA3.1-BICP0-Flag,pc DNA3.1-?BICP0-Myc and pc DNA3.1-BICP0-Flag?13A/51A?).On the other hand,the bovine TRAF6 gene was amplified by using c DNA from bovine liver,and TRAF6 related recombinant expression vectors were successfully constructed:pc DNA3.1-TRAF6-HA and pc DNA3.1-C71A-HA.These plasmids were delivered to HEK293T cells by transient transfection,and the activation of NF-?B promoter and IFN?promoter was evaluated by double luciferase activity assay.The results showed that BICP0can inhibit TRAF6 activates of the NF-?B promoter and the IFN?promoter.On the other hand,Western Blot analysis showed that the BICP0 transiently expressed in HEK293T also played a role in degrading TRAF6,and this effect was inhibited by BICP0 si RNA.Further identification results indicated that the RING finger domain of the amino terminus of BICP0 ha d an E3 ubiquitin ligase activity essential,while the carboxy terminus?357-637 aa?was not required for its function.4.The modification type of TRAF6 by BICP0The TRAF6-HA protein transiently expressed in HEK293T cells was isolated and purified from whole cell lysates by immunoprecipitation.The TRAF6-HA protein was identified by Western Blot.The detection of rabbit anti-HA antibody showed that the BICP0 had a modification effect on TRAF6.The modification was confirmed to be an ubiquitination modification by identification using a rabbit anti-Ub antibody.Finally,through the rabbit anti-K48-Ubiquitin antibody and rabbit anti-K63-Ubiquitin antibody,which can specifically recognize the ubiquitin chain type,it was clarified that BICP0 can promote the ubiquitin chain modification of TRAF6 by K48 linkage.5.Identification of key amino acids in BICP0The interaction between BICP0 and TRAF6 was identified by immunoprecipitation method.The results showed that there were protein-to-protein binding and only 1-536aa of the amino terminus of BICP0 participated in the above-mentioned effects.Subsequently,the mutants of the BICP0"346-PAERQY-351"motif were constructed for subsequent identification.The Western Blot results indicated that the"346-PAERQY-351"motif of the BICP0 was the binding site of its interaction with the TRAF6,and Tyrosine at position 351?Y351?was a key amino acid.Indirect immunofluorescence results indicated that the BICP0 colocalized with the TRAF6 in the nucleus,whereas the Y351A mutant failed to observe this phenomenon.The detection of dual luciferase activity showed that the inhibition of the TR351 gene activation promoter by the Y351A mutant also decreased.6.BICP0 inhibits the activation of IRF7 by reduces TRAF6 levelsBy detecting the dual luciferase activity,it was found that the BICP0 can inhibit the co-activation of the IFN?promoter by TRAF6 and IRF7.Western Blot results showed that BICP0inhibited the K63-linked ubiquitination of IRF7 by interfering with TRAF6.In this study,we preliminarily demonstrated that BICP0 can exert immune evasion function by negatively regulating TRAF6.The mechanism of interaction between BICP0 and TRAF6 was studied in depth and the structural basis of binding between proteins was found.This study provides theoretical support for the immune evasion mecha nism of BHV-1.The research results can provide theoretical basis and technical support for the development of new vaccines,and provide material basis and technology for further research on BHV-1 evading host inherent antiviral immune system.