Identification Of The Interaction Between Encephalomyocarditis Virus VP1 Protein And Host Protein RPS20

Encephalomyocarditis virus(EMCV)is one of the newly emerging zoonotic pathogen in China.It has a wide of host range,including humans,so it possesses a great threat to the public health.Animals infected with EMCV have the main features of encephalitis,myocarditis,and pericarditis.Piglets infected with EMCV leads to acute death and extensive pathological damage to parenchymal organs while pregnant sows infected with EMCV infection causes miscarriage,stillbirths,weak fetuses,and mummified fetuses.EMCV is a good model virus for the study of diabetes and viral myocarditis,which also be widely used in the studies of Toll-like and innate immune.EMCV belongs to the picornavirus family,a genus of cardioviruses.Its genome encodes four structural proteins VP4,VP3,VP2,and VP1.It has been reported that VP1 contains an important neutralizing epitope.After immunized with VP1 protein,mice can produce polyclonal antibodies with neutralizing activity to inhibit EMCV infection and replication in mice.VP1 is also associated with the virion topology,receptor adsorption,and virus shelling.VP1 protein is the main component of virion that forms the binding site with viral receptors,which plays an important role in virus adsorption and invasion.To further study the function of VP1 protein,we prepared recombinant protein His-VP1 and immunized BALB/c mice.We obtained anti-VP1 polyclonal antibody and two VP1 monoclonal antibody hybridoma cell lines,designated as 1D3 and 6E4.ELISA,Western blotting and IFA assay were used to test the antibodies and we found that the polyclonal antibody and monoclonal antibodies can recognize VP1 protein.To further identify the EMCV VP1 protein binding proteins,yeast two-hybrid system was used to screen the HBK21 cDNA yeast expression library by using VP1 as “bait”.RPS20 was identified as a novel EMCV VP1 binding protein.The interaction between RPS20 and VP1 protein was confirmed by co-immunoprecipitation(Co-IP).The localization of VP1 protein and RPS20 was observed in BHK-21 cells infected with EMCV by laser confocal microscopy.To test the effect of RPS20 on EMCV replication,HKE293 T cells were transfected a plasmid expressing RPS20,and then infected with EMCV HB10.The cells were collected at 12 h or 24 h post infection,respectively.The genomic copies of EMCV were detected by qRT-PCR.We found that RPS20 had no effect on the EMCV replication.In conclusion,we prepared anti-EMCV VP1 polyclonal antibody and monoclonal antibodies,and RPS20 was identified as VP1 binding partner although RPS20 has no effect on the EMCV replication.