Screening And Identification Of Proteins Interacting With Bovine Viral Diarrhea Virus NS4B

Bovine viral diarrhea-mucosal disease(BVD-MD)is a severely harmful infectious disease caused by Bovine viral diarrhea virus(BVDV).It can be transmitted through respiratory tract,gastrointestinal tract and placenta.The pathogen has been detected in 88 countries around the world.Studies have shown that BVDV NS4 B is an important part of replicase,has membrane topology and immunogenicity,and plays a role in inducing autophagosomes in vitro.The interaction between NS4 B protein and anti-apoptotic protein FHC can enhance virus replication,and play an active role in the process of virus anti-apoptosis by regulating the accumulation of reactive oxygen species(ROS).Therefore,this study focused on NS4 B,screened and identified the host proteins interacting with NS4 B,which provided a new basis for exploring the function of NS4 B protein.The main results are as follows:(1)14 host proteins interacting with BVDV NS4 B were screened by yeast two hybrid(Y2H).The bait plasmid p GBKT7-NS4 B was constructed,and it was identified that the NS4 B protein has no self-activating activity and no toxicity to yeast cells;Using the MDBK-c DNA library constructed in the laboratory and the bait strain culture medium for hybridization,14 host proteins that interacting with the NS4 B protein were screened out and the protein interaction network diagram was drawn;(2)We selected 9 proteins from 14 interacting proteins to perform yeast reverse hybridization experiments to further verify the interaction relationship with NS4 B protein.Firstly,recombinant prey plasmids AD-AKIP1?AD-LY6E?AD-RABAC1?AD-RPS20?AD-SYNGR2?ADTIMM17B?AD-LGALS1?AD-RPL31?AD-D18.3 were constructed;Secondly,the constructed prey plasmid and bait plasmid p GBKT7-4B were co-transferred into Y2 H yeast competent cells to perform the yeast recovery hybridization experiment,which proved that the NS4 B protein can respectively interact with AKIP1,LY6 E,RABAC1,RPS20,SYNGR2,TIMM17 B,LGALS1,RPL31,MHC class I molecular protein;(3)We selected 4 proteins from 9 interacting proteins to perform Co-immunoprecipitation(Co-IP)and subcellular co-localization experiments to further confirm the protein interaction.Firstly,the eukaryotic expression vector pc DNA3.1(+)-HA-NS4 B,pc DNA3.1(+)-3×FlagSYNGR2,pc DNA3.1(+)-3×Flag-RABAC1,pc DNA3.1(+)-3×Flag-D18.3 and pc DNA3.1(+)-3×Flag-AKIP1 were constructed,and the expression of the fusion protein in HEK293 T cells was verified;Secondly,they were transfected into HEK293 T cells,under the laser confocal microscope,it was observed that NS4 B protein with SYNGR2,RABACI,AKIP1 and MHC Class I molecule protein were co-localized in the cells respectively;The results of Co-IP showed that NS4 B interacted with SYNGR2 and RABACI respectively.In summary,this study obtained the interaction of BVDV NS4 B with SYNGR2 and RABACI proteins through Y2 H,Co-IP and subcellular colocalization experiments,which can provide new scientific data for the preliminary exploration of the function of BVDV NS4 B protein.