Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure

Abstract

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-A resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.

Fig. 1.

Crystal structure of HEV-LP and comparison of capsid protein dimers of HEV-LP, rNV, SMSV, and CARMV. The S, M, and P domains of the HEV capsid protein are indicated by pink, green, and blue, respectively. (A) HEV-LP is composed of sixty capsid subunits forming icosahedral 2-, 3-, and 5-fold axes and indicating a T = 1 symmetry. (B) The ribbon diagram of a capsid subunit of HEV-LP (PDB accession code: 2ZTN) shows P, M, and S domains at the top, middle, and bottom, respectively. The disordered regions are shown with dashed lines. The S domain shows a jerry roll-like β-barrel structure conserved in some viruses. The conserved anti-parallel β-strands are indicated (B to I). (C) The ribbon diagrams of crystal structures of capsid protein dimers of HEV-LP and those of rNV (PDB accession code 1IHM), SMSV (PDB accession code 2GH8), and CARMV (PDB accession code 1OPO) are indicated. Each capsid protein monomer is colored in red and blue.

Fig. 2.

Interaction of capsid protein subunits of HEV-LP around the 5-fold axis. (A) The pentamer of the capsid protein of HEV-LP. The close-up surface diagram of the 5-fold axis showed from outside and inside of HEV-LP. Amino acid residues Asn-200 and Tyr-288 are shown in yellow and green, respectively. The close-up surface diagram of the 5-fold axis showed from outside of rNV, SMSV, and CARMV. The aromatic amino acids Phe-118 of rNV, Tyr-330 of SMSV, and Phe-145 of CARMV are indicated in green. The deduced interacting atoms are connected with dashed lines, and the distances are indicated. (B) Sucrose density fractionation assay using the wild-type or mutant capsid proteins (53 kDa) in which the amino acids composing the 5-fold axis were substituted. The capsid protein composing HEV-LP was found in the 5–9th fractions from the top, while that which failed to form particles was found in the top fractions. The molecular mass of approximately 64 kDa was a non-specific protein.

Fig. 3.

Characterization of monoclonal antibodies and mutant HEV-LPs. (A) Neutralization of binding (NOB) of HEV-LP to Huh7 cells by monoclonal antibodies to HEV-LP. After preincubation of HEV-LP (10 μg/mL) with each of the monoclonal antibodies (20 μg/mL) for 1 h at 37°C, the mixture was inoculated into Huh7 cells and incubated for 1 h at 4°C. HEV-LP (lined area) bound to cells was detected by flow cytometry. The filled area indicates mock-incubated cells. (B) Construction of HEV-LP mutants. Sixteen HEV-LP mutants, in which the surface amino acid residues of the P domain were substituted, were constructed. The protein bands of 100 ng each of the purified wild-type and mutant HEV-LPs were visualized by Coomassie brilliant blue staining after SDS/PAGE. (C) Reactivities of NOB antibodies with the mutant HEV-LPs. Immunoprecipitation analyses of a series of HEV-LPs by NOB (MAB1323 and MAB272) or non-NOB antibodies (MAB358 and MAB161). Immunoprecipitated HEV-LPs were detected by an anti-HEV capsid rabbit polyclonal antibody. (D) Binding capability of the mutant HEV-LPs to Huh7 cells. Wild-type or mutant HEV-LPs (10 μg/mL) were incubated with Huh7 cells for 1 h at 4°C, and then HEV-LP (lined area) bound to cells was detected by flow cytometry. The filled area indicates mock-incubated cells. The MFI is shown in each panel.

Fig. 4.

Amino acid residues involved in the recognition by NOB antibodies and in the binding to Huh7 cells. Surface diagrams of the capsid protein dimer from a lateral (Upper) or top (Lower) view. (A) Amino acids in HEV-LP involved in the complete loss (deep color) or reduction (light color) of reactivity to MAB1323 and MAB272 are shown in orange and green, respectively. (B) Amino acids in HEV-LP responsible for binding to Huh7 cells are shown in red. Domains S, M, and P are colored pink, blue and gray, respectively. The substitutions in the P domain of HEV-LP that exhibited no effect on the reactivity with NOB antibodies or the binding to Huh7 cells are shown in dark gray.