Analysis Of Difference In NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype ? And ?

Japanese encephalitis virus(JEV),a mosquito-borne flavivirus,has a single-stranded,positive sense RNA genome encoding three structural proteins and seven non-structural proteins.After translation,the polyprotein is proteolytically cleaved at junction sites between each viral protein by both host cell and/or viral NS2B-NS3 proteases,which is essential or viral polyprotein maturation and viral replication.JEV is phylogenetically classified into five genotypes(genotype I to V).The genotype I(GI)replicates more efficiently than genotype III(GIII)in animals,and this difference is considered to be one of the reasons for the JEV genotype shift.In this research,we compared the enzymatic activity of NS2B-NS3 proteases between GI and GIII to gain an insight into regulation of replication of GI and GIII viruses.In our first study,eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein.Artificial green fluorescent protein(GFP)substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle(ST)cells in the presence and absence of JEV infection or co-expressed in E.coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2 B.The cleavage of GFP substrates was examined by western blot.Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases,all sites were found to be cleaved by host cell proteases with different efficiencies.The sites at internal C,NS2A/NS2 B,NS2B/NS3 and NS3/NS4 A junctions,but not the sites at internal NS3,internal NS4 A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease in prokaryotic model.These data provide insight into the proteolytic processing of polyprotein,which is useful for understanding JEV replication and pathogenesis.In our second study,the recombinant NS2B(H)-NS3(pro)proteases were prepared in E.coli and used to compare the enzymatic activity between GI and GIII NS2B-NS3 proteases.The GI NS2B(H)-NS3(pro)was able to cleave the sites at internal C,NS2A/NS2 B,NS2B/NS3 and NS3/NS4 A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro).Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro)proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro)was significantly higher than that of GI NS2B(H)-NS3(pro).There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro),which may be responsible for the difference in enzymatic activities between GI and GIII proteases.Therefore,recombinant mutants were generated by exchanging NS2B(H)and NS3(pro)domains between GI and GIII NS2B(H)-NS3(pro)and subjected to protease activity analysis.Substitution of NS2B(H)significantly altered the protease activities,as compared to the parental NS2B(H)-NS3(pro),suggesting that NS2B(H)played an essential role in regulation of NS3(pro)protease activity.To further identify the amino acids responsible for the difference in protease activities,a seral of substitution mutants including the individual and combined mutations at the variant residue 55 and 65 of NS2B(H)were generated and subjected to protease activity analysis.Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro)protease,whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro)protease.Overall,these data demonstrated that NS2B-55 and NS2B-65 variations in hydrophilic domain of NS2 B cocontributed to the difference in NS2B(H)-NS3(pro)protease activities between GI and GIII.These observations gain an insight into the role of NS2 B in regulation of NS3 protease activities,which is useful for understanding the replication of GI and GIII viruses.In conclusion,the cleavage sites mediated by NS2B-NS3 protease in JEV polyprotein were identified using recombinant NS2B(H)-NS3(pro)protease in E.coli model.The sites at internal C,NS2A/NS2 B,NS2B/NS3 and NS3/NS4 A junctions,but not the sites at internal NS3,internal NS4 A and NS4B/NS5 junctions were identified to be cleaved by both NS2B-NS3 protease of GI and GIII viruses.Later,the enzymatic activity between GI and GIII NS2B-NS3 proteases were compared using the recombinant NS2B(H)-NS3(pro)proteases in a model of fluorogenic peptide substrate.The proteolytical processing activity of GIII NS2B(H)-NS3(pro)was significantly higher than that of GI NS2B(H)-NS3(pro).The NS2B-55 and NS2B-65 variations in hydrophilic domain of NS2 B co-contributed to the difference in NS2B(H)-NS3(pro)protease activities between GI and GIII.These data provide an insight ino the proteolytic processing of polyprotein as well as the role of NS2 B in regulation of NS3 protease activities.