Spatial configuration of hepatitis E virus antigenic domain

Abstract

Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis. When an HEV capsid protein containing a 52-amino-acid deletion at the C terminus and a 111-amino-acid deletion at the N terminus is expressed in insect cells, the recombinant HEV capsid protein can self-assemble into a T=1 virus-like particle (VLP) that retains the antigenicity of the native HEV virion. In this study, we used cryoelectron microscopy and image reconstruction to show that anti-HEV monoclonal antibodies bind to the protruding domain of the capsid protein at the lateral side of the spikes. Molecular docking of the HEV VLP crystal structure revealed that Fab224 covered three surface loops of the recombinant truncated second open reading frame (ORF2) protein (PORF2) at the top part of the spike. We also determined the structure of a chimeric HEV VLP and located the inserted B-cell tag, an epitope of 11 amino acids coupled to the C-terminal end of the recombinant ORF2 protein. The binding site of Fab224 appeared to be distinct from the location of the inserted B-cell tag, suggesting that the chimeric VLP could elicit immunity against both HEV and an inserted foreign epitope. Therefore, the T=1 HEV VLP is a novel delivery system for displaying foreign epitopes at the VLP surface in order to induce antibodies against both HEV and the inserted epitope.

FIG. 1.

Characterization of VLP-C-tag and VLP-Fab224. (A) Western blot assay of the C-terminally truncated ORF2 proteins with Fab224. M, molecular weight markers; W, peptides recovered from baculovirus-infected cells. (B) Diagram of the C-terminal markers. (C) Electron micrograph of frozen-hydrated VLP-C-tag. (D) Electron micrograph of frozen-hydrated VLP-Fab224. Black arrowheads indicate the Fab molecules attached to the VLP. Both particles showed an absence of density in the center. Note that the surface spikes in VLP-Fab224 appeared as longer thorn-like densities compared to those of VLP-C-tag.

FIG. 2.

The cryo-EM structure of HEV T=1 VLP in complex with anti-HEV antibodies. (A) Surface presentation of VLP-Fab224 (left) and VLP-Fab4 (right) viewed along one of the icosahedral 2-fold axes. One 5-fold axis and two adjacent 3-fold axes are marked with the corresponding number. In both reconstructions, 60 copies of Fab are attached to the lateral side of HEV VLP; however, the density of Fab4 molecules appears to be less than that of Fab224 molecules. (B) The viral surface is shown as a stereographic projection overlapped with a line drawing of an icosahedral asymmetric unit. The 5-fold and two adjacent 3-fold axes are marked with corresponding numbers, while the black triangle encloses the area of an icosahedral asymmetric unit. The surface residues are colored according to the distance from the center of the VLP, with red being the furthest away and blue representing the surface depressions. The Fab density is projected as white contour lines on the viral surface, and the outermost layer of density is drawn as thick white contour lines.

FIG. 3.

The binding site of Fab224 antibody. (A) The cryo-EM density map of VLP-Fab224 was fitted with the crystal structure of PORF2 and viewed along a bound Fab molecule. One PORF2 dimer is presented as a solid surface and colored light magenta for the S domain, blue violet for the M domain, and dark gray for the P domain. The neighboring dimers are drawn in ribbon mode and colored wheat. (B) Side view of a PORF2 dimer fitted into the cryo-EM density map. (C) A PORF2 dimer viewed along the 2-fold axis and overlapped with the cryo-EM density map. (D) Top view of a PORF2 dimer viewed along the 2-fold axis. The amino acids in PORF2 responsible for binding to Fab224 are labeled. The PORF2 dimer is presented as a solid surface and colored in gray, violet, and light magenta for the P domain, the M domain, and the S domain, respectively. The residues along the Fab binding interface are colored according to the element, with green for carbon, blue for nitrogen, and red for oxygen.

FIG. 4.

The structure of the chimeric HEV VLP carrying a B-cell tag. (A) Surface presentation of VLP-C-tag viewed along an icosahedral 2-fold axis. (B) The cryo-EM density map of VLP-C-tag (mesh) was fitted with the crystal structure of the PORF2 decamer (ribbon). (C) Ribbon representation of PORF2 dimer with one monomer colored gray and the other colored pink for the S1 domain, blue for the M domain, and lime for the P domain. The amino acids prior to the four internal insertion sites are marked in sphere mode with color coding representing the elements as described in the Fig. 3 legend. (D) The top view of the PORF2 dimer, showing the location of the non-VLP insertion sites.

FIG. 5.

Fitting of the PORF2 structure into the cryo-EM density map of HEV VLP-C-tag. The side view (A) and the top view (B) of the fitted PORF2 dimer (surface presentation) are overlapped with the cryo-EM density map of VLP-C-tag (mesh). The C-terminal residue A606 is located at the side of the protruding spike. One PORF2 dimer in the surface presentation is colored light magenta, blue violet, and gray for the S, M, and P domain, respectively. The ribbon representation shows the adjacent dimers. The amino acids in PORF2 responsible for binding to Fab224 are colored green for carbon, blue for nitrogen, and red for oxygen. Asterisks mark the location of the extra density that was not occupied with PORF2 coordinates.