| Hepatitis E virus(HEV)is a non-enveloped single-stranded,positive-sense RNA virus.It mainly divided into 4 genotypes with a single serotype.Hepatitis E caused by HEV is a worldwide public health problem,which results in high case-fatality rate among pregnant women and infants.Due to the lack of efficient HEV culture system,HEV infection and neutralization mechanism has not been elucidated.HEV ORF2 encodes a unique structural protein assembling into viral capsid.The capsid proteins are directly involved in virus adsorption,cell entry and induction of host immune responses since HEV is non-enveloped.Therefore,recombinant expressed HEV capsid proteins are the ideal materials for related studies.The studies of capsid protein structure and immunological epitopes can help to elucidate the structural basis and the infection mechanism of HEV,and thus provide important guidance for HEV prevention,diagnosis and treatment.HEV ORF2 encodes the only structural protein with 660 amino acids and aa459-606 encoded protein can form homodimer(E2s),which is the principal target of HEV neutralizing antibodies.Previously,we have expressed E2s in E.coli.The structure of E2s:8C11 complex was determined by crystallographic method and 8C11 recognition epitope have been elucidated.E2s can't self-assamble into particles,but the HEV ORF2 aal 12-606(p495)expressed in insect cells can self-assemble into T=1 icosahedral particle.E2s located in the 2-fold axis region as protruding spike.8C11 recognized the groove region of E2s.What is interestion is that if 8C11 binds the same epitope on p495,it will have steric hindrance between the bound 8C11 and the adjacent E2s on particle.In this study,we obtained p495 VLP and its cryo-EM structure.Then the VLP was used to study the interaction with 8C11.Firstly,p495 protein was expressed by baculovirus insect cell expression system and assembled into VLPs(Virus Like Particles).The VLPs with high purity(>90%)and homogeneous in size was obtained by precipitation and subsequently anion exchange chromatograph.We also explored the storage conditions and eventually verified the most suitable condition for the stability of p495 VLPs.Results of enzyme-linked immunoassay and mouse immunization showed that p495 VLPs had good immunoreactivity and immunogenicity that is equivalent to vaccine antigen p239 of Hecolin(?).Other experiments on cell level showed that p495 VLPs could successfully attach to HepG2 cells and neutralizing antibody 8C11 could block the attachment.And 3.4 A cryo-EM structure of p495 VLP was obtained.Then,the p495 VLPs were used to prepare immune complexes with neutralizing antibody 8C11.Compared with 3B6 complex,8C11 complex was extremely unstable.8C11 made the VLPs depolymerized after a certain time to form complex of p495 monomer,dimer or polymer.The structure p495:8C11 before depolymerization was obtained by using Cryo-EM single particle reconstruction.The resolutions were 4.0 A.Through the analysis of cryo-EM density maps,we found that E2s parts of p495:8C11 appeared to be distorted irregularity.The symmetry was destroyed which lead to the signal loss during the reconstruction.Several reconstruction methods had been tried to obtain an ideal structure but failed.This also showed that 8C11 will destroy the particle.As a control,the structure of p495:3B6(9.4 A)showed intact densities of both p495 and Fab.As the depolymerization caused by antibody was observed on VLPs,here we explored antibodies-mediated virus stability to verify if 8C11 can destroy native HEV.The result suggested that 8C11 has the potential for destroying native HEV.In this study,we expressed p495 VLPs in insect cells and elucidated the phenomena of 8C11 making the VLPs depolymerized based on the methods of biochemistry,molecular biology and structural biology.The depolymerization was caused by steric hindrance.The way to destroy a virus particle directly due to steric hindrance may be a new neutralization mechanism of antibody. |
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