The Study On The Structure And Biofunction Of PDCoV Endoribonuclease Nsp15

PDCoV?Porcine deltacoronavirus?belongs to?coronavirus which has gradually become an attracting pathogen due to its potential danger in swine industry.It mainly causes severe vomit,diarrhea and dehydration in new-born piglets and finally leads to death.No efficient medicine or drugs against PDCoV have been invented yet.PDCoV endoribonuclease?mature nonstructural protein-15,nsp15?is under nidovirus endoribonuclease family and can excise the uridylate site on single or double-strand RNAs which plays vital role in virus pathogenicity.In this research,we firstly found,verified and illustrated that PDCoV nsp15 was active as a dimer with its underlying mechanism,which varied much from other hexameric nsp15in SARS-CoV and PEDV.Our finding provided novel insight into the evolution of nidovirus endoribonuclease also the basis of designing the drugs and vaccine against PDCoV,or even the coronavirus.The research mainly contains the following aspects:1.The verification of dimeric formation of PDCoV nsp15The E.coli system of prokaryotic expression,Ni-NTA and chromatography were used to obtain PDCoV nsp15?H234A?,SARS-CoV nsp15?H234A?and PEDV nsp15?H241A?mutant proteins.Later on,SEC and AUC methods were used to analyze the oligomeric formation among these mutant proteins.The results show that PDCoV nsp15 is a dimer and monomer whereas SARS-CoV nsp15 and PEDV nsp15 is a hexamer.2.The verification of important regions on PDCoV nsp15 which determine its dimeric formationThe amino acid sequence alignment and the three-dimensional structure alignment were used to find out the supposing regions.Later on,the mutants of PDCoV nsp15_1N-27N?and nsp15_1S-27I,SARS-CoV nsp15_1N-27N and nsp15_259Q-279F were obtained.The SEC and AUC results show that PDCoV nsp15_1N-27N?has more monomers,PDCoV nsp15_1S-27IS-27I remains almost the same as that of PDCoV nsp15?H234A?,SARS-CoV nsp15_1N-27NN-27N remains almost the same as that of SARS-CoV nsp15?H234A?whereas the SARS-CoV nsp15_259Q-279F59Q-279F has changed into a dimer and monomer from its original hexamer.3.The biochemical experiment to further verify the enzymatic activity in PDCoV nps15mutant proteinsThe catalytic and RNA binding sites on PDCoV and SARS-CoV nsp15 were mutated into alanine.The RNA substrate was synthesized and FRET experiment was used to analyze their catalytic activity.The results show that wild-type PDCoV nsp15 owns the highest enzymatic activity,then the D276A,1N-27N?,H219A and K269A,the H234A has the lowest one.In SARS-CoV mutants,nsp15_259Q-279F has the highest one,then the nsp15_1S-27I?and nsp15_1N-27N,H234A owns the lowest one.4.The proposing evolutionary process of nidovirus endoribonucleaseThe three-dimensional structural alignment of XendoU,PRRSV nsp11,SARS-CoV nsp15 and PDCoV nsp15 shows that Active Loop,Supporting Loop and catalytic sites are common existence in these four endoribonucleases.XendoU is a monomer because its own Supporting loop and other loops give the support to Active Loop whereas other ones need to form higher oligomers and be supported by other monomer to stabilize their Active Loops.The supposing evolutionary hypothesis is that nidovirus obtained its endoribonuclease from XendoU of eukaryotic cell and it became a dimer in PRRSV nsp11through multiple times of gene mutation and then higher oligomer was formed in SARS-CoV nsp15,the dimeric formation of PDCoV nsp15 as an intermediate is the best verification of this hypothesis.