The Structure And Function Research Of PRRSV Non-structural Protein Nsp2 PL2 Domain

Porcine reproductive and respiratory syndrome virus(PRRSV)gives rise to reproductive failure in sows and respiratory distress in young pigs,which would lead to prevalent and troubling disease for the swine industryThe PRRSV-encoded large non-structural protein,nsp2,is the most rapidly mutated protein among the genomes of individual strains,and its amino-terminal PL2 core domain is highly conserved among members of the family Arterivirus.PL2 protease can inhibit the interferon signaling pathway through its deubiquitinase activity,affecting the innate immune response to the virus.The PL2 protease possessed both trans-and cis-cleavage activities,which can cleave the downstream nsp2/3 junction site,playing an important role in the regulation of viral transcription and replication.Presumably it has a triple catalytic active site(Cys55-Asp91-His124)and three additional conserved cysteine residues(Cys111,Cys142,and Cys147)which may play an important role in enzyme activity.The analysis of the 3D structure of PL2 can provide a fine structural basis for the understanding of the complex functions of nsp2.In this study,the PL2 domain of the non-structural protein nsp2 and the ubiquitin molecule closely related to its modification were selected to construct the different fusion proteins.The crystal structure is analyzed in order to provide a structural basis for revealing its understanding of the mechanism of virus replication and pathogenicity.The main findings are as follows:(1)Different Nsp2 PL2 ubiquitin-linked protein expression characteristics and crystal preparation: In order to understand the role of PL2 protease in DUB activity and ubiquitination of cellular processes,we constructed a Strep tag-purified prokaryotic expression plasmid containing PL2 and Ub molecule linkages,to understand the expression and purification characteristics of different proteins.The PL2 protein was purified by Strep affinity,ion exchange and gel filtration chromatography step by step after it was expressed in E.coli.The purity of 95% protein was concentrated to about 5 mg/mL to screen crystallization conditions.The effects of different purification processes,reducing agents,and crystallization kits on the formation of crystals were investigated.After optimization of the crystallization conditions,the oil droplets were crystallized at 16℃ for 24-36 h,and PL2 protein diamond crystals was observed at 20% PEG 3350 and 0.2 M magnesium formate.In the crystallization buffer of 30% PEG 400,sodium cacodylate PH6.5,0.2 M lithium sulfate grow short needles in larger quantities,distribution messy microcrystalline.Diamond crystals and crystallites were detected in the regular diffraction pattern,and the crystal conditions were further optimized.And the final optimized stubby slenderness of the two forms of crystal proteins,but the diffraction rate was extremely low and no valid crystal analysis data was obtained.The results showed that the aggregation status of PRRSV PL2 protein was concentration-dependent,protein concentration increased,and multimer increased significantly.The internal cysteine content of the protein is as high as 15%.It is speculated that disulfide bond mismatch may cause protein heterogeneity,resulting in polymorphism of protein crystals.(2)Detection of PL2 protein deubiquitination: In order to understand the PL2 deubiquitinase activity and characteristics,the ubiquitylated proteins RIG-I,Nsp1α,NEMO,which can be used for Lys48,Lys63,or linear ubiquitination,were selected.After the action of ubiquitinated molecules,prokaryotic expression of purified PL2 protein was added to study its deubiquitination characteristics.Construction of the PL2 karyotic expression vector and co-transfected with ubiquitin of various specific sites to detect the deubiquitination mode of PL2.The results showed that the PL2 protein mainly undergoes deubiquitylation of the ubiquitinated protein at K48;it also has a slight cleavage activity for linear ubiquitination K63 polyubiquitin chain.In summary,purified PL2 protein exhibits poly-characteristics,high disulfide bonds affect the formation of regular crystals and diffraction patterns;recombinant PL2 protein has de-ubiquitinating enzyme activity,and plays a important role in the deubiquitination of the K48,K63 and linear ubiquitination modifying protein.The study of the biochemical and structural characteristics of the PL2 deubiquitinase from PRRSV contributes to the understanding of the pathogenic molecular mechanism of PRRSV.