Preparation Of Polyclonal Antibody Against Encephalomyocarditis Virus 3AB Protein And Screening And Verification Of The Interaction Between 3AB Protein And Host Proteins

Encephalomyocarditis virus?EMCV?is a non-enveloped single-stranded positive chain RNA virus,which belongs to cardiovirus genus in the family picornavirus.EMCV can infect pigs,mouse,elephants and orangutans.Pigs infected with EMCV will lead to abortion,stillbirth and other reproductive disorders,while piglets infected with EMCV will lead to sudden death,which greatly reduce the survival rate of piglets and bring huge losses to the pig industry.In addition,EMCV can infect human beings,although the incidence is low.However,it brings great risks to public health and safety.In previous studies,EMCV is often used as a model virus to study diabetes,mellitus viral myocarditis,toll like receptors and intracellular receptors.Emcv genome encodes 13 viral proteins,of which 3A protein is about 9.9 KDa and3B protein is about 2.3 KDa.EMCV 3A and 3B protein plays important roles during EMCV replication.In the process of virus replication,3A protein is located on the endoplasmic reticulum,combines with and recruites phosphatidylinositol 4-kinase???PI4K???to catalyze the formation of a microenvironment rich in phosphatidylinositol4 phosphate?PI4P?lipids which is viral replication organelles,which helps viral genome replication by promoting oxysterol-binding protein?OSBP?to transport cholesterol to viral replication organelles.EMCV 3B protein is covalently linked to genomic RNA.At the start of viral RNA replication,3B protein acts as a primer protein.Only the genome RNA of the offspring linked to 3B protein can be packed into the capsid during EMCV assembly.During virus replication,host proteins often regulate viral replication by interacting with viral proteins.However,there are few reports on the interaction network between 3AB protein and host proteins.In order to further study the function of 3AB protein,the mouse polyclonal antibody against 3AB protein was prepared.And the titer of the mouse polyclonal was detected by ELISA method.Western blotting and indirect immunofluorescence?IFA?were used to analyze the specific effect of recognition EMCV 3AB protein.The results showed that the polyclonal antibody of against EMCV 3AB had significant higher titer.The titers of the five antibodies were 1:256000,1:256000,1:256000,1:128000,1:256000 respectively.All of them could specifically identify the overexpressed 3AB protein and the 3AB protein encoded by EMCV in BHK-21 cells.Yeast two-hybrid?Y2H?systemis based on the characteristics of eukaryotic transcription factor.The Y2H system can be used to screen the binding partners of the known protein and identify the interaction between protein-protein in a simple,fast and high-throughput way.To further study the function of EMCV 3AB protein,EMCV 3AB was used as“bait”and Y2H system was used to screen binding proteins of EMCV 3AB in this study.Six host proteins interacting with EMCV 3AB protein were identified,including RFC5,RPS20,RPL11,LRWD1,PIH1D1 and LGALS1.The results were verified by Y2H retest.We found that 3AB protein interacted with RFC5,RPS20,RPL11,LRWD1 and LGALS1 but not PIH1D1.Then,the total RNA were extracted of mouse peritoneal macrophages and RT-PCR was performed to amplify the cDNAs of RFC5,RPS20,RPL11,LRWD1,PIH1D1 and LGALS1 genes.These genes were further cloned into pCAGGS-Flag?pFlag?vector.The interactions between EMCV 3AB protein and host proteins were verified by co-immunoprecipitation.The results showed that RFC5,RPS20,RPL11,LRWD1,PIH1D1 interacted with 3AB protein.However,the interaction between LGALS1 and 3AB protein was not detectable.RFC5,RPS20,RPL11,LRWD1 and PIH1D1 are colocated with 3AB protein.Finally,we investigated the effect of RFC5,RPS20,RPL11,LRWD1 and PIH1D1 on the replication of EMCV.BHK-21 cells transfected with plasmids expressing RFC5,RPS20,RPL11,LRWD1 and PIH1D1 were infected with EMCV for 12 h or 24 h and genomic copies and titer of EMCV were detected by qPCR and TCID₅₀.The results showed that overexpressed RFC5,RPS20,RPL11,LRWD1 and PIH1D1 neither affected the genomic copy nor the titer of EMCV.It has been reported that RFC5,PIH1D1,RPS20,LGALS1 and RPL11are involved in apoptosis and tumorigenesis.Of note,EMCV can induce apoptosis and necrosis of acinar cells in tissues,suggesting that the interaction between these proteins and EMCV 3AB proteins may be involved in other cellular processes.In conclusion,the polyclonal antibody of EMCV 3AB protein was successfully prepared,which provided a tool to further study the function of 3AB protein.And six proteins interacting with EMCV 3AB protein were screened and identified,which lays a foundation for further research on the interaction network between EMCV 3AB protein and these host proteins.