Study On Structure And Function Of Enterovirus 71 3D Protein

Enterovirus 71(EV71)is one of the major pathogens of hand foot mouth disease(HFMD),which belongs to species A in family Picornaviridae,genus Enterovirus.As a neurotropic enterovirus,the clinical symptoms of EV71 infection showed different severity,mild cases are only manifested as fever and herpes in hand,foot and mouth,however,some severe cases are manifested as central nervous system complications,such as aseptic meningitis,encephalomyelitis and neurogenic pulmonary edema,and even death.At present,severe HFMD is mainly caused by EV71,the pathogenic mechanism of EV71 has not elucidated yet,and further study is needed.Our previous study showed that there were difference in replication ability and virulence between EV71 strains SDLY107 and SDLY1,the replication ability and virulence of high virulent strain SDLY107 were higher than low virulent strain SDLY1.And as a RNA-dependent RNA polymerase,3D protein plays a key role in virus replication.Therefore,in this study,3D region of high virulent strain SDLY107 was replaced by that of low virulent strain SDLYland the recombinant virus was rescued by reverse genetics technology.To explore the role of 3D protein in viral replication and virulence in vitro.At the same time,the 3D protein crystals of SDLY1 and SDLY107 were screened by using structural biology techniques.And then,we will analyse the crystal structure by X-ray diffraction,which will provide the reference for the accurate localization of virulence sites on 3D protein.Objective1.To construct and rescue recombinant virus SDLY 107(1-3D),and to explore the effect of 3D protein on viral replication and virulence in vitro.2.To screen 3D protein crystals of SDLY1 and SDLY107.To analyse the crystal structure and the influence on protein structure of 3D protein mutation sites from the two strains,finding out the significant mutation sites and providing the reference of the accurate location of virulence sites on 3D protein.Methods1.3D region of high virulent strain SDLY 107 was replaced by that of low virulent strain SDLY1.The infectious RNA was obtained by in vitro transcription,then transfected into Vero cells and recombinant virus SDLY 107(1-3D)gained after three passages.2.The recombinant virus was identified by PCR,quantitative real-time polymerase chain reaction(qRT-PCR)and indirect immunofluorescence assay(IFA),and then verified by sequencing.The virus was harvested by Vero cells and the virus titer was determined by 50%terminal method.3.The RD cells were infected with SDLY1,SDLY107 and SDLY107(1-3D)(MOI=100)and cultured at 37? and 39.5?,respectively.The supernatant was collected every 12 hours.The virus growth curves were determined by qRT-PCR and the difference of replication ability of different strains was compared.4.The RD cells were infected with SDLY1,SDLY107 and SDLY107(1-3D)(MOI =10)and cultured at 37? and 39.5?;respectively.To detect cell injurys of different strains by actate dehydrogenase(LDH)test and CCK-8 test after 48 hours.5.The 3D fragments of SDLY1 and SDLY107 were obtained by PCR,and then inserted into prokaryotic expression vector PET32a(+)and pEGX-6P-1 respectively to construct recombinant plasmids.6.The recombinant plasmids were transformed into Escherichia coli(E.coli)DE3 and protein expression induced by IPTG The bacteria were collected for ultrasonication,and supernatant containing 3D protein were purified by affinity chromatography,ion exchange chromatography and molecular sieve chromatography.3D protein crystals were screened by sitting drop method.Results1.Cytopathic effect(CPE)was observed after infectious RNA transfected into Vero cells and cultivation to the third generation.PCR,qRT-PCR,IFA and sequencing results confirmed that the recombinant virus SDLY 107(1-3D)was successfully rescued.The CCID50 of SDLY1,SDLY107 and SDLY107(1-3D)were 10-6.0/ml,10-6.5/ml and 10-6.5/ml.2.The virus growth curves of different strains showed that the replication ability of the recombinant virus SDLY107(1-3D)was slightly weaker than SDLY107 and stronger than SDLY1 at 37?.At 39.5?,the replication ability of SDLY107 was sligltly decreased,and the replication ability of SDLY1 and SDLY107(1-3D)were decreased significantly,SDLY107(1-3D)was slightly stronger than SDLY1.3.The results of LDH test and CCK-8 test showed that the effect on cell injury of SDLY107(1-3D)was weaker than SDLY107 and stronger than SDLY1 at 37?,the difference was statistically significant(P<0.05).The effect on cell injury of SDLY1 and SDLY107(1-3D)were significantly decreased at 39.5?,SDLY107(1-3D)was stronger than SDLY1,and the difference was statistically significant(P<0.05).4.The 3D fragments were amplified by PCR and inserted into prokaryotic expression vector PET32a(+)and pEGX-6P-1,respectively.The recombinant plasmids PET32a-3D-1,PET32a-3D-107,pEGX-6P-3D-1' and pEGX-6P-3D-107'were constructed successfully.5.After protein expression and purification,high purity and high concentration 3D protein were obtained.The 3D protein expressed by PET32a-3D-1,PET32a-3D-107,pEGX-6P-3D-1' and pEGX-6P-3D-107' were 14.8mg/ml,12.5mg/ml,9.0mg/ml and 7.0mg/ml.6.The 3D protein expressed by PET32a-3D-1 and PET32a-3D-107 were not observed crystals.The 3D protein expressed by pEGX-6P-3D-1' and pEGX-6P-3D-107' were both observed crystal on condition of 0.4M potassium sodium tartrate tetrahydrate.Conclusions1.The recombinant virus SDLY107(1-3D)was constructed and rescued successfully.The replication ability and virulence of SDLY107(1-3D)were both decreased,and it was especially obvious at high temperature,which indicated that the 3D protein had the virulence site.2.The prokaryotic expression system of 3D protein was constructed,high purity and high concentration 3D protein were obtained,and the 3D protein crystals of SDLYI and SDLY107 were screened.The results above lay the foundation for analysing the 3D protein structure and the effect of the mutated sites on 3D protein structure to find out the significant mutation sites of the 3D protein.