Screening And Identification Of PEDV NSP5 Host Interacting Proteins

Porcine epidemic diarrhea(PED)is currently one of the main diseases that endanger the world's pig industry.The disease is an acute,highly infectious intestinal disease characterized by vomiting,diarrhea,dehydration,and anorexia,causing severe diarrhea and high mortality in piglets.The pathogen of the disease is the porcine epidemic diarrhea virus(PEDV).It is a single-stranded positive-stranded RNA virus.The PEDV genome is about 28000 nt in length,and contains a total of 7 open reading frames(ORF),namely ORF1a,ORF1b,S,ORF3,E,M,and N genes.Among them,the ORF1a and ORF1b genes at the N-terminal are responsible for the encoding of the two replicase polyprotein precursors(pp1a and pp1ab).These two replicase polyprotein precursors are subsequently cleaved and processed by related enzymes,eventually forming 16 non-structures protein(nsp).PEDV nsp5 is an hydrolytic enzyme,also known as3C-like protease(3CLpro).It has a conserved functional domain and catalytic residue site at the C-terminus.3CLpro plays a vital role in the processing of coronavirus polyproteins.During the replication and production of a new generation of progeny viruses,its hydrolase function is utilized to cleave the replicase polyprotein,and finally 12 non-structural proteins(nsp5?nsp16)are formed.In addition,nsp5 is also a key interferon antagonist protein,which exists in a variety of virus families.The screening and characterization of its host-interacting proteins has implications for elucidating the its biological functions and pathogenesis of PEDV.In this experiment,the E.coli expression system was used to express PEDV nsp5 in vitro.In order to obtain water-soluble nsp5,the induction conditions of the two recombinant expression plasmids pGEX-6P-1-nsp5 and pET-30a-nsp5 were optimized,and finally the highly expressed water-soluble GST-nsp5 protein was obtained.The high-purity nsp5 protein was obtained by tag affinity purification technique,and then the purified nsp5 protein mixed adjuvant was used to immunize healthy New Zealand white rabbits,and finally a rabbit polyclonal antibody that c ould effectively recognize nsp5 was prepared.ELISA method detects its titer can reach 1:10~5.Western blot and IFA test results show that the prepared polyclonal antibody has good specificity,which not only recognize the HA-nsp5 produced by pCMV-HA-N-nsp5 transfected cells,but also react with nsp5 protein in PEDV-infected cells.The GST pull-down method was used to screen the host IPEC-J2 cell target proteins that interact with PEDV nsp5 protein in vitro.After the pull-down product was identified by mass spectrometry and exclusion of the irrelevant protein pulled-down by the GST tag protein,a total of43 proteins that might interact with nsp5 were obtained.A preliminary bioinformatics analysis was performed on these 43 proteins.GO analysis found that these 43 protei ns mainly involved in the biochemical process of intracellular translation and large ribosomal subunit assembly.They mainly distributed in the nucleus,membrane system and the ribosomal subunit,the function of the screened proteins was mainly to bind RNA and act as the main component of ribosomes.KEGG pathway analysis showed that these 43 proteins were mainly concentrated in the ribosome signaling pathway.Finally,two proteins,RPL11 and PSMD7,were selected to verify their biological functions.Indirect immunofluorescence test showed that RPL11 and PSMD7 could co-localize with PEDV nsp5 protein.The immunoprecipitation results showed that RPL11 and PSMD7 also co-precipitated with PEDV nsp5 protein.In summary,the PEDV nsp5 protein can interact with the two kinds of proteins of host cells,RPL11 and PSMD7,and might participate in the virus replication in infected cells,and play important biological functions in the process of virus pathogenesis.