The Structure And Function Research Of Porcine Delta Coronavirus Nsp3 Multi-Functional Domains

Coronavirus(CoV)has many members and can infect a variety of animals and humans,posing a serious threat to animal husbandry and human health.Among them,the coronaviruses that can infect pigs mainly from ?-CoV and ?-CoV genus,porcine transmissible gastroenteritis virus,porcine epidemic diarrhea virus and swine acute diarrhea syndrome coronavirus belong to ?-CoV genus,Porcine delta coronavirus belongs to ?-CoV genus.Nonstructural protein 3(nsp3)is the largest nonstructural protein encoded by the coronavirus genome.It participates in the formation of viral replication and transcription complexes(RTC)and contains multiple functional domains.Macrodomain(Macro),Ubiquitin-like domain 2(Ubl2)and Papain-like protease(PLpro)are three conserved functional domains of nsp3.Adenosine diphosphate ribose(ADPr)is involved in post-translational modification of viruses;PLpro is involved in the hydrolytic release of nsp1/2-nsp4 and immune evasion of viruses,so it is one of the important targets of coronavirus antiviral drugs.This study uses PDCoV nsp3 as the research object,revealing the structural basis of the PDCoV nsp3 multifunctional region Mac-Ubl2-PLpro involved in virus replication and immune evasion,laying a foundation for the research of coronavirus replication mechanism and antiviral drugs.The specific contents are as follows:1.The cloning construction,protein expression and purification,crystal screening and optimization of the PDCoV nsp3 multifunctional domain Macro-Ubl2-PLproIn this study,using bioinformatics analysis technology,sequence comparison analysis and structural simulation prediction of PDCoV nsp3,the functional domains of Macro and Ubl2-PLpro were initially determined respectively.In order to harvest the purified Macro and Ubl2-PLpro proteins,a variety of vector cloning and truncation construction were attempted,the protein expression and purification conditions were explored.After crystallization screening and optimization,we merely obtained the crystals of Mac and Ubl2-PLpro with a resolution 6.0 (?) and 3.7 (?).Although we have not successfully obtained the crystal structures of Macro and Ubl2-PLpro,we tried to combine the three functional domains and successfully expressed the multifunctional domain protein Macro-Ubl2-PLpro.After crystal screening and optimization,the coronavirus nsp3 multifunctional domain protein crystal with a resolution of 2.5 (?) were obtained for the first.2.Depicting the three-dimensional structure of the PDCoV nsp3 multifunctional domain Macro-Ubl2-PLproThe crystal structure of the multifunctional domain Macro-Ubl2-PLpro protein of PDCoV nsp3 from ?-CoV genus was depicted for the first time.In the asymmetric unit of Macro-Ubl2-PLpro,two of the subunits form the head-to-tail homodimer with an interaction interface between Macro and PLpro,however,it exists as a monomer in solution.The Macro-Ubl2-PLpro monomer structure presents a malleable linear structure,including three conserved domains: namely Macro,Ubl2 and PLpro domain.At the flanking N-terminus of the crystal structure,Macro adopts a conserved three-layered ?-?-? sandwich fold;following Macro is Ubl2 which contains three ?-stands and one ?-helix,while the structural superpositions show that the torsion direction of PDCoV Ubl2 is obviously shifted;the thumb,palm and finger domains form the catalytic core of PLpro,which has an extended right-hand architecture.The sequence alignment and structural homology indicated that PDCoV PLpro poses the same conserved amino acid sites Cys260,His398 and Asp412 as other coronavirus PLpro,indicating that it may have the same biological functions.3.In vitro biochemical establishment of PDCoV nsp3 multifunctional domain Macro-Ubl2-PLproBased on the fluorescence resonance energy transfer technology(FRET),the fluorescent substrate DABCYL-PGFKAGSDELFI-(E-EDANS)-amide,Ubiquitin-AMC and ISG15-AMC were used to explore the biological function of the PDCoV Macro-Ubl2-PLpro(wild-type,truncations and mutants).The results show that PDCoV PLpro has the protease activity of hydrolyzed peptides,deubiquitinating(DUB)activity and de ISGylating activity,and the enzyme activity is significantly reduced after Ubl2 truncation,and after Macro truncation its DUB activity and de ISGylating enzyme activity decreased significantly,indicating that these three functional domains work together.Based on the analysis of amino acid sequence and structure,a series of mutants were designed,two active sites Cys260 and His398 are determined by vitro enzyme activity experiments,and the loop of 409-413 amino acids(aa)is key motif for regulating the enzyme activity of PDCoV PLpro.In summary,this study provides theoretical basis for understanding the immune evasion and replication mechanism of the coronavirus,and it also provides new ideas for the research of new anti-coronavirus drugs at the same time.