The Screening Of Host Cellular Proteins Interacting With Porcine Epidemic Diarrhea Virus M Protein

Porcine epidemic diarrhea is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus.It is characterized by vomiting,diarrhea and loss of appetite.All age pigs are susceptible and bring huge losses to the pig industry.PEDV encodes structural proteins S,E,M,N and 16 non-structural proteins.M protein is the most abundant protein in the envelope protein of PEDV virus.It participates in the assembly of the virus and plays an important role in evading the innate immunity of the host.However,it is not clear whether the host protein directly interacts with M protein and whether M protein has other biological characteristics.Therefore,the screening of these host proteins is helpful to understand the pathogenesis of viruses,and to identify targets that affect viral replication,assembly and interaction with the host.It has a significant impact on the development of new antiviral drugs and vaccines.The contents and results of this study are as follows:(1)Screening of Host Proteins Interacting with PEDV M1~stt method:Recombinant plasmid was transfected into the IPEC-J2 cells to screen the protein interacting with PEDV M protein.The recombinant plasmid containing M-APEX2 and APEX2 genes was constructed and transfected into IPEC-J2 cells.The recombinant plasmid containing M-HA gene was labeled with ascorbic acid peroxidase(APEX2)by substrate catalysis.The recombinant plasmid was transfected into IPEC-J2 cells and screened with HA labeled monoclonal antibody for immunoprecipitation(IP).Comparing the screening results of the two methods,based on the analysis of the number of peptide segments,40 endogenous proteins interacting with M protein were identified in the experimental group compared with the control group.They participated in different metabolic pathways.S100 calcium-binding protein A11(S100A11),protein coding gene(SLC9A3R1),Claudin 4(CLDN4),Probable ATP-dependent RNA helicase(DDX58)and Peptidyl-prolyl cis-trans isomerase D(PPID)were used as candidate proteins for validation.Cyclin-dependent kinase-like 2(CDKL2)in control group was selected as negative control.2~edd method:Infection with attenuated PEDV DR13 strain screened proteins interacting with PEDV M in African green monkey kidney(Vero)cells.The virus infected cells with MOI=0.1 and was screened by IP using M monoclonal antibody.After mass spectrometry analysis,62 endogenous proteins interacting with M protein were identified,which were closely related to protein synthesis,metabolism,cell signaling pathway transduction and so on.Cell division cycle 42(CDC42)was selected as the target protein,eukaryotic translation initiation factor 3 subunit L(eIF3L)and G protein binding protein(RAB11A)were selected as candidate proteins for validation,recombinant H2A histone family,member Y(H2AFY),was selected as negative control.(2)Co-IP validation of interaction between PEDV M and host protein:Recombinant plasmids with Flag tag and S100A11,SLC9A3R1,CLDN4,DDX58,PPID,CDKL2(negative control),CDC42,eIF3L,RAB11A,H2AFY(negative control)genes were constructed respectively,and recombinant plasmids with HA tag and M gene were also constructed and co-transfected into Hela cells.Co-IP was performed with Flag monoclonal antibody and Western blot with HA labeled monoclonal antibody.Except negative control,M protein bands were detected.The results confirmed that S100A11,SLC9A3R1,CLDN4,DDX58,PPID,CDC42,eIF3L,RAB11A protein interacted with PEDV M protein in cells.(3)Co-localization validation of the interaction between PEDV M and host protein:The recombinant plasmids with Flag tags and S100A11,SLC9A3R1,CLDN4,DDX58,PPID,CDKL2(negative control),CDC42,eIF3L,RAB11A,H2AFY(negative control)genes were co-transfected into Hela cells with recombinant plasmids with HA tags and M genes.Using HA and Flag labeled monoclonal antibodies(diluted at 1:50)as antibodies,488 goat anti-mouse(diluted at 1:200)and647 goat anti-rabbit(diluted at 7:100)were used as second antibodies for immuno-fluorescence(IFA).The results showed that host proteins S100A11,SLC9A3R1,CLDN4,DX58,PPID,CDC42,eIF3L,RAB11A and AIFM1 were co-localized with M proteins except control group.By screening and validating the host proteins interacting with PEDV M protein,we concluded that the identified this study identified that PEDV M protein could interact with S100A11,SLC9A3R1,CLDN4,DDX58,PPID,CDC42,eIF3L and RAB11A in host cells,and identified other host proteins that might interact with M protein.The study is to provide an important theoretical basis for the study of the interaction between PEDV and host cell proteins.