Annexin A2 Promotes The Release Of Virus By Regulating The Cell Membrane Location Of BEFV Matrix Protein

Bovine epidemic fever virus（BEFV）is the pathogen of bovine epidemic fever（BEF）,which causes high fever,dyspnea,decrease of milk production,decrease of fertility and other clinical symptoms in diseased cattle,causing huge losses to cattle industry and seriously affecting economic benefits.In 2014,the Ministry of agriculture has listed bovine epidemic fever as one of the most important bovine infectious diseases.At present,the prevention and control of the disease is mainly based on vaccine immunity,but the effect of prevention and control is not ideal.Therefore,in-depth study on the molecular mechanism of BEFV replication,especially the mechanism of interaction between host proteins and viral proteins to regulate viral replication,is expected to find new drug targets and provide new ideas for the development of antiviral drugs.Annexin A2（AnxA2）is a Ca²⁺-dependent phospholipid binding protein,which is involved in the regulation of endocytosis,exocytosis,membrane domain formation,cell proliferation and so on.Its N-terminal domain can affect the location and function of the protein in the cell.The C-terminal domain is the basis for AnxA2 protein to bind with other proteins and play a role.Although AnxA2 regulates the replication of African swine fever virus,it has not been reported that AnxA2 participates in the replication of BEFV.The C-terminal domain,known as the functional region of the AnxA2 protein,is the basis of the interaction between the AnxA2 protein and other proteins and plays a role in cell life activities or virus proliferation.However,it has not been reported that AnxA2participated in BEFV replication.BEFV matrix protein（M）,a lipid bilayer membrane protein encoded by M gene,is distributed between the envelope and nucleocapsid of the virus.The M gene function of rabies virus and vesicular stomatitis virus,which belong to the same family as BEFV,has been reported more,but the research of BEFV M protein has not been reported.In this paper,the relationship between AnxA2 and M gene in BEFV replication was studied as follows:（1）The expression of AnxA2 and its role in the replication of BEFV were detected.The results showed that the expression of AnxA2 mRNA and protein was up-regulated,especially when the virus was infected for 48 hours.To construct the Lentivirus Expression Vector pLVX-AnxA2-Flag-IRES-Puro of AnxA2,and established a stable cell line of over expression of AnxA2 gene,inoculated 0.1 MOI BEFV,detected TCID₅₀,and found that over expression of AnxA2 can promote the release of BEFV compared with control.We designed and synthesized the siRNA targeting to inhibit AnxA2,and transfected BHK-21 cells to silence the AnxA2 gene,and detected TCID₅₀0 that the titer of BEFV decreased significantly compared with control.The above results showed that AnxA2 promoted the release of BEFV.（2）The interaction between AnxA2 and M protein was detected and the binding site was located.The interaction between matrix protein M of BEFV and AnxA2 protein was revealed by GST pull-down and immunoprecipitation.According to the prediction of the website software,the AnxA2 protein is divided into two structural domains,and it was found that the second domain（AnxA2-C2,186-340 aa）of the AnxA2 protein interacts with the M protein.In order to further locate the binding domain of M protein and AnxA2 protein,AnxA2 was divided into five domains.It was found that M protein bound to the fifth domain of AnxA2 protein（AnxA2-V,268-334 aa）by immunoprecipitation experiment.（3）To explore the molecular mechanism of the interaction between AnxA2 and M protein to promote the replication of BEFV.The total virus titer and the virus titer in the supernatant of cell culture were detected.It was found that the overexpression of M protein significantly promoted the release of BEFV.It was found that the AnxA2 protein promoted the localization of M protein on the cell membrane by plasma membrane separation experiments.Cells were transfected with AnxA2-△V and inoculated with0.1 MOI BEFV to detect virus titer,the titer of BEFV in the supernatant of the cells was significantly lower than that of the control cells.The above results showed that AnxA2promoted the localization of M protein on cell membrane through the binding of V domain and M protein,and then promoted the release of BEFV particles.Starting from the relationship between AnxA2 and BEFV replication,the interaction between AnxA2 protein and BEFV M protein was taken as the starting point in this study.The role of AnxA2 gene in the process of BEFV replication was first discovered,and the molecular mechanism of AnxA2 protein promoting the release of BEFV through its 268-334aa domain and M protein binding was revealed,which provided clues for the development of antiviral drugs.