Hepatitis E virus (HEV) is a causative agent of acute hepatitis in humans and is primarily transmitted via the fecal-oral route. HEV is thus resistant to the low pH and digestive enzymes associated with the stomach and gastrointestinal tract. HEV regularly causes epidemics in many tropical and subtropical countries. In India, 101 outbreaks were confirmed by serological analysis in the state of Maharashtra in the last 5 years (6
), and the lifetime risk of HEV infection exceeds 60% (28
). Sporadic cases have also been reported in regions where HEV is endemic, as well as in areas where it is not endemic. Although some of these cases were associated with travel, many cases involved patients without a history of travel to regions where HEV is endemic. Accumulating evidence suggests that sporadic infection occurs through a zoonotic route and is not limited to developing countries. Seroprevalence suggests hepatitis E infection may also be prevalent in high-income countries (21
), such as the United States (17
), the United Kingdom (3
), and Japan (18
). The overall mortality rate of HEV infection during an outbreak generally ranges from 1 to 15%, and the highest mortality occurs in pregnant women, with fatality rates of up to 30% (19
The HEV virion is composed of a 7.2-kb single-stranded RNA molecule and a 32- to 34-nm icosahedral capsid. The HEV genome contains three open reading frames (ORFs). The capsid protein, encoded by the second open reading frame (ORF2), located at the 3′ terminus of the genome, comprises 660 amino acids and is responsible for most capsid-related functions, such as assembly, host interaction, and immunogenicity. Recombinant ORF2 proteins can induce antibodies that block HEV infection in nonhuman primates (12
). Four major antigenic domains were predicted to be located within the C-terminal 268 amino acids of the ORF2 protein; one domain was experimentally identified as a neutralization epitope in the Sar-55 ORF2 capsid protein (25
). However, the minimal peptide needed to induce anti-HEV neutralizing antibodies contains residues 459 to 607 of the ORF2 protein (33
), which is much longer than a linear antigenic epitope, suggesting that the neutralization epitope is conformational. Therefore, the detailed structure of the HEV capsid protein is required in order to understand the organization of HEV epitopes.
Currently, there are 1,600 HEV genomic sequences available through the International Nucleotide Sequence Database Collaboration. They are classified into four genotypes which vary by geographic distribution and host range (10
). In contrast, only a single serotype has been identified, suggesting that the immunodominant domain of HEV is highly conserved among genotypes. Antibodies from any one of the four genotypes cross-react with the capsid protein of genotype 1 (7
Like other hepatitis viruses, HEV does not propagate well in currently available cell culture systems. Hepatitis E preventive strategies so far rely on the use of ORF2-derived recombinant protein (16
). When expressed in insect cells, recombinant truncated ORF2 protein (PORF2), with 52 residues deleted from the C terminus and 111 residues deleted from the N terminus, self-assembles into virus-like particles (VLPs) (15
). Our previous structural analysis of recombinant HEV VLP by cryoelectron microscopy (cryo-EM) provided the first understanding of the quaternary arrangement of PORF2.
The essential assembly element of the PORF2 protein contained amino acids 125 to 600 (13
), and the reconstructed VLP displayed a T=1 icosahedral particle composed of 60 copies of truncated PORF2 (30
). Recently, crystal structures were reported for genotype 1 T=1 VLPs (31
), genotype 3 T=1 VLPs (32
), and genotype 4 T=1 VLPs (8
), revealing that PORF2 is composed of three domains, the S domain, M domain, and P domain. The T=1 icosahedral shell is composed of 60 copies of S domains, while the M domain binds tightly to the S domain and interacts with two 3-fold-related M domains to form a surface plateau at each of the 3-fold axes. Two P domains are tightly associated as a dimeric spike that protrudes from each of the icosahedral 2-fold axes. As a result, on a low-resolution cryo-EM density map, the HEV T=1 VLP appears as an icosahedral particle with 30 spikes (30
Although these VLPs are smaller (270 Å in diameter) than the native HEV virion (320 to 340 Å), oral administration of HEV VLPs to experimental animals can induce anti-HEV antibodies that bind to native HEV (14
). When a B-cell tag of 11 amino acids on glycoprotein D of herpes simplex virus was covalently coupled to the C-terminal end of PORF2 (after residue 608), the fusion protein retained the ability of PORF2 to assemble and form chimeric T=1 icosahedral VLPs that were capable of eliciting systemic and mucosal antibodies against both HEV capsid protein and the attached B-cell tag (20
). Therefore, the HEV T=1 VLP is a potential carrier for delivering not only HEV antigen but also foreign antigens or antiviral drugs to the host immune system. However, rational design of HEV-based delivery vectors requires detailed information on HEV VLP structure, as well as on HEV immunodominant domains.
Here, we identified antigenic structures using cryo-EM and three-dimensional reconstruction. Our results indicate that the binding footprint of a neutralizing antibody covers the lateral side of the P domain, while a B-cell tag at the C terminus does not alter the assembly of T=1 HEV VLP.