Structural And Functional Insights Into Coxsackievirus A6 And Its Complex With A Neutralizing Antibody

Hand,foot and mouth disease(HFMD)is a global infectious disease caused by human enterovirus,which mainly affects infants and children and has become a severe public health problem.China is the main epidemic area of HFMD,the annual report cases are more than one million in recent years.HFMD is caused by a few serotypes of enteroviruses,most frequently coxsackievirus A16(CAV16)and enterovirus 71(EV71).Coxsackievirus A6(CVA6),a member of the human Enterovirus species A,has recently emerged as one of the major pathogens of HFMD in children worldwide,which underscores the critical need for developing an effective vaccine and therapy.However,the atomic structure,neutralizing epitope and antigenicity of CVA6 particles still remain unknown,which restricts the related research.In this study,with a combination of various methods of biochemistry,celluar biology and structural biology,the capsid structure and immune-dominant neutralizing epitope of CVA6 were performed.In addition,the characteristics and immunogenicity of CVA6 particles were tested to determine if the particles could serve as vaccine candidates.The atomic structures of the CVA6 particles,immune-complex and the neutralizing mechanism were revealed,which will provide the necessary support for studying the vaccine and therapeutic drugs.In this study,the biochemical analysis and immunogenicity of CVA6 particles were tested.According to the previous work,we established an efficient large-scale culture system for CVA6 virus,to can obtain higher titer of the culture virus.CVA6 was purified following routine protocols employing centrifugation,ultracentrifugation and biochemical analysis.Two types of particles were obtained and sedimented with the coefficient of 84S and 128S respectively.The 84S particles had a capsid protein composition consistent with the presence of VPO,VP1 and VP3 in SDS-PAGE gel electrophoresis,and showed an absence of packaged genome as verified by negative stain electron microscopy.The 128S particles composed of capsid proteins VP1,VP2,and VP3 but no VP4 and contained packaged genome as verified by negative staining.In addition,we demonstrates that 128S particles can infect cells efficiently.Hence,the 84S and 128S particles more like the procapsid and uncoating intermediates(or A-particles).It is well known that procapsids and mature virions are typically the dominant types of picornavirus particles,A-particles have been characterized as necessary entry intermediates during infection.However,unlike the elusive A-particles,CVA6 A-particles are biochemically and structurally stable both in vivo and in vitro.The purified particles were used to the evaluation of the immunogenicity and protective effects.The results showed that the aluminum hydroxide-absorbed inactivated CVA6 particles possessed good immunogenicity,and could confer good protective effect in CVA6 animal models.These results demonstrated that the two particles have good immunogenicity and potentiality for vaccine development.The structure of CVA6 particles were obtained by cryoEM with electron-counting technology.We determined the 3.3 A and 3.1 A cryoEM structure of the CVA6 84S and 128S particles respectively.Our atomic structures of both particles show that their capsids have essentially identical structures,which resemble uncoating intermediates of other enteroviruses.CVA6 A-particle contains characteristic structural features that are typically attributed solely to picornavirus A-particles,including large,open channels at the icosahedral 2-fold,quasi 3-fold axes,and a collapsed hydrophobic pocket.The density maps of the procapsid and A-particle reveal essentially identical,which explains why the two particles share similar antigenicity.The most significant differences among the CVA6,EV71 and CVA16 used for comparison occur at the four surface loops(BC,DE,EF and HI)of VP1,which often serve as the viral neutralizing epitopes and appear to play important role in receptor-binding.In addition,the cryoEM density map and atomic model of the A-particle together display unique interactions between its capsid and the packaged genome.Notably,the VP2 E37 and W38 residues interact with the RNA near the 2-fold axes;strong interactions between the N-termini of VP3(P3)and the genomic RNA were clearly apparent in the CVA6 A-particle density map.We used cryoEM and 3D image reconstruction methods to investigate the structure of the CVA6 A-particle in complex with the Fab portion of neutralizing antibody.According to the previous work,we demonstrated that the antibody 1D5 elicits a high neutralizing titer against CVA6,and also exhibits a high binding affinity for procapsid and A-particles.The epitope recognized by the 1D5 was also shown to be an immune-dominant antigenic site.We also determined a 3.8 A cryoEM structure of the CVA6 virion complexed with 1D5.This resolution is the highest in the current study of the structure of virus-antibody complexes.The A-particle-1D5 interaction interface includes surface loops on two VP1 proteins at 5-fold vertex from two adjacent protomers.Each Fab interacts with the BC,EF,and HI loops of the nearest VP1 subunit and the DE loop of the counter-clockwise-related VP 1.The atomic model of the CVA6 A-particle-1D5 immune-complex provides the key virus epitope,including BC loop(Leu98,Asp99),EF loop(Asp159,Gly160,Tyr164),HI loop(Ser236,Thr237).The escape mutants assay showed that mutant virus were resistant to 1D5,and further verified that L98P and G160D are important for the 1D5 neutralization.The interaction interface of Fab 1D5 involves five of the six total complementary determining regions.This Fab-VP 1 interaction includes an array of eight hydrogen bonds and van der Waals interactions.The H-bond also forms between the first residue of the light chain(ASPI)on one Fab and TYR66.This might explain why the CVA6 A-particle-1D5 is a rather stable immune-complex,which stabilize the capsid and thereby restrict conformational changes necessary to allow RNA release.The fluorescence-based thermal stability assays demonstrated that the 1D5 can stabilize the capsid.Furthermore,we examined 1D5 could inhibit virus-cellular binding.Altogether,1D5 may block CVA6 from attaching to cell surface and rigidify A-particles to prevent conformational changes required for subsequent RNA release.In a summary,we first revealed that the CVA6 contains two types of viral particles.The unique A-particle is the dominant types and is highly infectious,which represent a picornavirus anomaly.We demonstrated that both the CVA6 procapsid and A-particles have potential application.We first report the cryoEM structures of the CVA6 procapsid,A-particle,and A-particle-1D5 immune-complex,and reveal atomic resolution details about the neutralizing epitopes and molecular determinants in CVA6 related to virus neutralization.The results can provide an important basis for the development of C VA6 vaccine or antiviral treatment,as well as the mechanisms of viral assembly and infection.