Structural And Biochemical Characterization Of SADS-CoV Papain Like Protease 2

From October 2016 to May 2017,the outbreak of swine acute diarrhea syndrome coronavirus(SADS-Co V)in southern China had caused the death of more than 20,000 piglets,and it had recurred in the subsequent 2018 and 2019 spring,resulted in a large number of piglets died and serious economic losses.The outbreak of SARS coronavirus accelerated the research of coronavirus by scientists,but so far,no antiviral drugs that can effectively inhibit coronavirus have been developed successfully.Antibodies developed against the spike protein of coronavirus are also limited in application due to the high mutability of the spike protein.Under the background of this research,the investigation of inhibitors against highly conserved non-structural proteins in coronaviruses has become a new target for the development of antiviral drugs.Coronavirus encodes polyprotein pp1 a and pp1 ab,which are cleaved into 14-16 nonstructural proteins under the action of non-structural protein PLPs and main protease to participate in the formation of coronavirus replication complex.PLP2 is a papain-like protease that will recognize and cleave specifically the N-terminal sequence of polyprotein pp1 a to form mature nsp1,nsp2,and nsp3.In vitro studies on SARS-Co V PLpro have shown that PLpro has deubiquitination and de ISGylating activity,which is speculated to help coronavirus to escape the immune response of the host after the infection.Both Ub and ISG15 are important signaling components of the host’s innate immune response to viral infections,which can be negatively regulated by the viral DUB and de ISGylating enzymes,so they can be used as targets for the development of new antiviral drugs.In this paper,SADS-Co V PLP2 was selected as the research object,and it was found that it has low sequence homology with PLpro of SARS-Co V or MERS-Co V.The structural biology of SADS-Co V PLP2 was studied by methods of protein crystallography and biochemistry,to explore the molecular mechanism of its structure and function,and to screen the inhibitors based on the structural and biochemical data.We expressed and purified the SADS-Co V PLP2 through the prokaryotic expression system,and obtained the protein crystals with high quality.The three-dimensional structure of the PLP2 protein was determined by X-ray diffraction with the resolution of 1.72 (?).After comparison with the structure of SARS-Co V PLpro,it is found that there are some significant differences in the structure of SADS-Co V PLP2 and SARSCo V PLpro.Based on the analysis of modeling with Ubiquitin-PLP2 complex,it was found that the active sites of SADS-Co V PLP2 were C101,H256 and D269 catalytic triad and other surrounding amino acid residues to help Ub to enter the active site correctly.Enzymatic characterization of PLP2 was carried out by in vitro enzymatic experiments,that is,PLP2 can effectively cleave nsp1-AMC,ubiquitin-AMC,ISG15-AMC and peptide-AMC.The key active site residues related to substrate binding and catalysis were determined by structural analysis,site-directed mutagenesis and enzyme kinetics,and the Michaelis constant Km and kcat and the activity changes after mutation of the amino acid residues at the active site were determined,to verify the results of structural analysis.Finally,we obtained three inhibitors with significant inhibitory effects through inhibitor screening and measured IC50.The detailed description of SADS-Co V PLP2 is crucial for understanding the mechanism of function of the enzyme in the coronavirus replicase complex and its impact on the host immune response,and also provides an important theoretical basis for subsequent research and drug development.