Structural Determination And Functional Investigation On Classical Swine Fever Virus Nonstructural Protein 3

Classical swine fever(CSF)is a highly lethal disease of swine,as its pathogen,classical swine fever virus(CSFV)is a member of pestivirus,which belongs to Flaviviridae.The genome of CSFV is a single-stranded positive-sense RNA,with length of about 12.3 kb.The CSFV genome contains an open reading frame(ORF)which was flanked by two terminal nontranslated region(NTR).The ORF encodes a polyprotein,which was processed into 12 mature proteins(Npro,C,Erns,E1,E2,p7,NS2,NS3,NS4A,NS4B,NS5A,NS5B)by viral and host proteases.The nonstructural protein 3(NS3)is a multifunctional protein,and plays important roles in the viral genome replication and infectious virus production.In this study,we investigated on the structure and functiona of CSFV NS3 and the following results were obtained:We constructed a recombinant NS3 by fusing the NS4A protease cofactor segment(PCS)to its N terminus,hereafter NS4APCS-3,then expressed by prokaryotic expression system.An S163A mutation was introduced to NS3 to abolish the serine protease activities that may lead to autocleavage.Through large-scale expression of E.coli,affinity chromatography,ion exchange chromatography and gel filtration chromatography isolation,the NS4APCS-3S163a was purified to homogeneity.After crystallization screening and optimization,we finally obtained octahedral-shaped crystals with high quality.Through X-ray diffraction and data collection,molecular displacement and model optimization,we solved the crystal structure of NS4APCS-3S163A at a 2.35 A resolution.The solved structure displays a novel closed conformation,the five residues of NS3 C terminus inserted into the protease pocket,presented an NS3-NS4A cis-cleavage-related conformation,and the protease approaching the "back" of the helicase,mainly interacted with helicase D3,forming a 2243 A2 intramolecular interface.Further structural analysis found a continuous basic zone on the NS3 surface,which was thought to be involved in the binding of helicase to its substrate,especially the basic zone on the protease surface,which implies the molecular mechanism of NS3 protease stimulation on its helicase activity.Based on the structural analysis of CSFV full-length NS3,the main intramolecular interface interactions can be defined into three clusters.The first cluster involves the entire P-side of the protease active site and the C-terminal tail(residues 679 to 683)of helicase D3,the second cluster includes a triple salt bridge charge network and a"hydrophobic cap" at its edge,the third cluster involves the protease ?7b-?8 hairpin of the N-barrel,a loop in helicase D3(residues 559 to 563),and the D2-D3 linker.According to the three clusters of interface interactions,we designed three sets of mutations,each was introduced in the context of recombinant NS3 protein and full-length virus genome respectively.Then we assess the effect of the interface mutations to NS3 helicase and ATPase activities,also to virus production and plaque formation.our data showed that the interface mutations reduced NS3 helicase activity in different degree,while have no effect on the ATP hydrolysis.In vivo assays showed that,the interface mutations significantly reduced the infectious virus production,as well as the capacity of plaque formation.We further study the continuous basic region on NS3 protease surface through mutation analysis.The H24(located in NS4APCS)and R50,K74,K94(located in NS3)were mutated to alanine and aspartic acid respectively,the data of biochemical assays showed that ATPase activity was not be significantly affected,while the helicase activity generally declined.Mutation analysis based on CSFV infectious clones showed that the virus production was significantly reduced.Collectively,both the virological and the helicase unwinding data indicated that the basic residues on NS3 protease surface can regulate the replication of CSFV genome by binding to helicase substrate.Our findings are helpful to deepen the understanding of the relationship between the structure and function of CSFV NS3,the regulatory mechanism of NS3 in viral genome replication and infectious virus production,also provide new theoretical bases for elucidating the pathogenic mechanism of CSFV and developing new disease control techniques.