Identification Of Proteins Interacting With PEDV E Protein In PK-15 And Their Effects On Viral Release

Porcine epidemic diarrhea(PED)is a contagious disease of digestive tract caused by porcine epidemic diarrhea virus(PEDV),which has high mortality rate of newborn piglets,and PED has caused serious economic losses to the pig industry around the world.Because the the pathogenesis of PED and interaction between PEDV and host have not been clear,there are still conundrums in prevention and treatment of PED.E protein is the smallest structural protein of PEDV,and the expression of the protein is also low on the membrane of the virus.Studies have shown that E proteins of most coronavirus can self-assemble into pentamer with ion channel(IC)activity,but whether the E protein of PEDV has the same characteristics has not been reported yet.The E protein plays an important role in viral assembly in ER-Golgi intermediate compartment(ERGIC),and is involved in viral intracellular transport and budding process,and moreover,is related to the pathogenesis of PED,but the specific mechanism is still not clear.Studying the interaction proteins between E protein and host cells is of great significance to further explore the function of E protein and the process of PEDV release.In order to study the role of E protein in the pathogenesis of PEDV,E gene was cloned with different tags(His,MBP,eGFP)into eukaryotic expression vector pcDNA3 respectively.Sequencing showed that the E gene of PEDV strain CV777 used in this paper had seven bases mutations and tow amino acids mutations compared with the original strain.Homology analysis of E gene and E protein sequences showed that this virus had higher homology with PEDV strain SQ2014 and PEDV strain JS2008.The gene level was 99.1% and 98.7% respectively,and the protein level was 97.4% and 96.1% respectively.In addition,through the conservative analysis of amino acid sequences and the prediction of transmembrane region of the E protein of other coronavirus,it is speculated that the E protein of PEDV used in this paper may also form pentamer.Three recombinant plasmids were transfected into host cell(PK-15)respectively,and the total protein was extracted and detected by western boltting,it showed that E protein can express in PK-15 cells with MBP tag,and detected specific bands at about 49.9 kD and 99.8 kD.In order to obtain a large number of E protein expressed in PK-15,cell lines expressing MBP protein or MBP-E protein were constructed by recombinant lentivirus.Purification of the target protein by MBP-pull down,western boltting showed that the target protein mainly exists in amylose resin before eluting,and detected specific bands at about 49.9 kD,99.8 kD and 249.5 kD which further demonstrated that E protein may self-assemble into pentamer.Silver staining showed that there were different bands in MBP-E samples compared with MBP samples.Mass spectrometry showed that there are 42 interaction proteins of E protein in PK-15 cell.In view of E protein involved in viral assembly in ERGIC,and involved in viral intracellular transport and release process,we knockdown proteins participating in the intracellular transport(MYH9,SEC22 B and Jup),molecular chaperones located in the endoplasmic reticulum(HSPA5 and CANX),and a 14-3-3 protein(YWHAB)by RNA interference(RNAi),and polyclonal cell lines were constructed by shRNA recombinant lentivirus.Real-time quantitative PCR(qPCR)showed that expression of genes were significantly decreased except HSPA5.Then PEDV was inoculated into polyclonal cell lines(MYH9,SEC22 B,Jup,CANX and YWHAB),and the cells and supernatant were collected at 12 h and 24 h respectively.The viral copies in supernatant were detected by absolute real time quantitative PCR,and the intracellular ?-actin protein abundance was used for data normalization.Absolute real time quantitative PCR showed that viral copies were decreased obviously compared with negative control,which indicated that intracellular transport,endoplasmic reticulum stress(ERS)and the 14-3-3 protein family may play an important role in the process of viral release.This paper first identified the interaction proteins beteween E protein and PK-15 cell,and studied the effect of interaction proteins which involved in intracellular transport,ERS and a 14-3-3 protein on viral release,providing a theoretical basis for the further study of the structure and function of E protein and the release process of PEDV,moreover,providing new ideas for the prevention and treatment of PED.