Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway*

Abstract

Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T‫1؍‬ capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T‫3؍‬ icosahedral lattice and contains RNA fragment in contrast to the RNA-free T‫1؍‬ capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids. Hepatitis E virus (HEV),3 the causative agent of acute hepatitis in humans, is primarily transmitted through contaminated water and generally results in epidemic outbreaks in many developing countries. Sporadic cases have also been reported between outbreaks in HEV-endemic regions as well as in nonendemic areas, and these cases are transmitted through zoonotic route. The overall mortality rates of HEV during outbreaks range from 1 to 15% in general, and the highest mortality occurs in pregnant women, with fatality rates of up to 30% (1).HEV consists of a non-enveloped icosahedral capsid and a single-stranded, positive-strand RNA genome of ϳ7.2 kb that encodes three open reading frames (ORFs) (2). The capsid protein, encoded by the ORF2, is composed of 660 amino acids and responsible for most capsid-related functions, such as virion assembly, host interaction, and immunogenicity. Like other hepatitis viruses, HEV is unable to propagate in currently available cell culture systems, and the research of HEV relies largely on the recombinant HEV capsid proteins (3-6). Virus-like particle (VLP) was obtained when the truncated HEV capsid protein was expressed in insect Tn5 cells with deletion of 52 residues from the C terminus and 111 residues from the N terminus (PORF2) (7). Our previous structural analysis of this HEV-VLP by cryo-electron microscopy (cryo-EM) provided a basic understanding of the quaternary arrangement of PORF2, where the reconstructed VLP displayed a Tϭ1 icosahedral particle composed of 60 copies of PORF2 (8). The essential element of PORF2 protein for Tϭ1 VLP assembly includes amino acids 125-600 (9). Recently, the structural information was further refined by the crystal structures of genotype-3 Tϭ1 VLP (10) and genotype-4 Tϭ1 VLP (11), which revealed the tertiary structure of PORF2 to the level of amino acids. However, the Tϭ1 VLPs used in these experiments were much smaller than that of the native virion, which has a diameter of 320 -340 Å, as determined by immuno-EM (12). There is still a need to investigate the assembly pathway of HEV capsid.HEV virion is hypothesized to be made of 180 copies of the capsid protein (8,11). To test this hypothesis, we screened for HEV genoty...

Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody

“…Meanwhile, this residue was also reported to be one of the several essential residues for host cell interaction [9]. To have a better spatial view of all the key epitope sites, i.e., the potential receptor binding sites, we mapped all the residues that have been shown to be involved in binding to the host cell ( Figure 7, Table 2 and Supplementary information, Figure S11) on the surface of T = 3 virion-sized particle [31] ( Figure 7A) and T = 1 HEV VLP [9,10,32] (Figure 7B). Interestingly all of these residues within E2 domain are located either in the dimerization region or in the groove region.…”

Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding

“…Deconvolution of contrast transfer function was done similarly as previously described (6,45), where these particles were centered by autocorrelation, bandpass-filtered, and subjected to multivariate statistical analysis, namely correspondence analysis and hierarchical clustering, and submitted for refinement using EMAN (46). The density maps were validated by the agreement between the calculated reprojections and the class averages of micrographic images at each angular step (Fig.…”

“…Samples of subtype C o-gp140ΔV2TV1 (hereinafter gp140) were biochemically characterized, purified, and imaged as previously described (6,12,45,46) (see also Figs. S3, S4, and S5).…”

“…1 A and D). Density maps were fitted to previously published X-ray coordinates of either unliganded SIV gp120 (7) or sCD4-bound HIV-1 gp120 (8) as previously described (6). To address the lack of unliganded gp41 coordinates, a cylinder with a radius of 17 Å and height of 48 Å, equivalent to the six-helix bundle, was placed at the threefold axis, thus conveying the maximum space that gp41 could ostensibly occupy.…”

A Comprehensive Study of Neutralizing Antigenic Sites on the Hepatitis E Virus (HEV) Capsid by Constructing, Clustering, and Characterizing a Tool Box

“…ORF2 exclusively encodes a structural protein that is 660 amino acids in length; with the N-terminal 112 residues responsible for the packaging of the viral RNA genome (14 -16). The generation of N-terminal truncated virus-like particles (aa 112-608) have identified 3 definitive domains: the S domain (aa 129 -319) forms the viral shell; the M domain (aa 320 -455) is associated with the S domain and involves the formation of the 2-, 3-, and 5-fold icosahedral symmetries of the HEV capsid; and the P domain (aa 456 -606, equivalent to the E2s domain) forms the protrusions that extend outward from the shell (17)(18)(19)(20)(21). Based on the high-resolution crystal structure, the E2s domain adopts a twisted anti-parallel ␤-barrelfold and maintains a tight dimeric structure (21,22).…”