Identification And Function Analysis Of Proteins Interacting With Porcine Epidemic Diarrhea Virus Nsp12

The Porcine Epidemic Diarrhea Virus(PEDV)isoneofthemost economically important pathogens that threaten the development of swine industry.It mainly infects suckling piglets,causing diarrhea,vomiting,dehydration and death of pigs,and leadsto huge economic losses to the pig industry.The disease first occurred in 1971 and after that spread widely.Since2006,due to the gene mutation,PEDV variants have emerged and/or re-emergedworldwide,includingSouth Asia,East Europe and North America.Swine small intestinal epithelium cell(IECs)is known as the target cells of Porcine Epidemic Diarrhea Virus(PEDV),but little is known about the interaction of PEDV and the proteins of IECs.In this study the total RNA of IECs were first extracted,reverse-transcribed into the corresponding complementary DNA(cDNA)by the SMART technology,and co-transfected into the Y187 component cells with pGADT7-Rec and carrier DNA.Then,the target library was screened by using the selective medium(SD/-Leu)and its quAntity and quality were carefully assessed using titration and PCR.The result showed that the titer of our newly constructed IECs-cDNA library was 1.3×10~8pfu/mL;the length of the inserted cDNA ranged from 250 to 25000 bp in all randomly selected 35 colonies.It suggests our IECs-cDNA library has a good polymorphism and could serve as a solid basis for our further research on the interaction of PEDV and its host cell proteins.PEDV non-structural protein 12(Nsp12)is an RNA-dependent RNA polymerase(RdRp),which is the key replicase for RNA virus replication and proliferation.In order to study the interactions between Nsp12 and proteins of IECs,a bait plasmid containing the Nsp12 gene was constructed and transfected into the Y2HGold for further analysis.It was found that Nsp12 has neither toxicity nor autoactivation ability.Using yeast two-hybrid system,we screened 9 blue colonies,and the cross-validation assay showed four of them were positive colonies containing prey genes that can activate the expression of reporter genes together with Nsp12,i.e.,RING finger Protein7(RNF7),Ribosomal Protein S20(RpS20),galectin-1(Lgals1)and Dipeptidyl pepdiase8(DPP8),all of which have ever been reported to affect the replication of PEDV.Among them,RNF7 is an E3 ubiquitin ligase that functions in the ubiquitin-proteasome pathway in eukaryotic cells.In order to further analyze the interaction between Nsp12 and RNF7,we constructed two plasmids-pCAGGS-Nsp12-FLAG and pCAGGS-RNF7 and the Co-IP validation between Nsp12 and RNF7 confirmed that Nsp12 can co-immunoprecipitate with RNF7.And theconfocal experiment demonstrated that Nsp12 and RNF7co-located in cytoplasm and nucleus.To investigate whether RNF7 could affect the replication and proliferation of PEDV,PEDV isolate FJzz1/2011 was used to infect the Vero cells over-expressing RNF7 and was further tittered;meanwhile,the expression level of endogenous RNF7 was assayed after the infection of the same isolate.The result showed that overexpression of RNF7 can promote the replication of PEDV strain FJzz1/2011,and the infection of PEDV could increase the expression level of endogenous RNF7.In this study,an IECs-cDNA library was constructed,and the library was used to screen and identify the protein RNF7 which couldinteract with PEDV Nsp12.The overexpression of RNF7 can affect the replication of PEDV.These results can serve a foundation of the pathogenic mechanism of PEDV infection and replication.