Mutational Analysis of Essential Interactions Involved in the Assembly of Hepatitis E Virus Capsid

Abstract

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394 -606) and p239 (amino acids 368 -606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala 597 , Val 598 , Ala 599 , Leu 601 , and Ala 602 ; furthermore, the site involved in particle formation was located at amino acids 368 -394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.

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

“…Cloning, Expression, and Purification of p239 and Mutant Antigens-The gene encoding p239 was described previously (36). Genes for mutant antigens with the surface amino acids replaced by alanine (Ala) were prepared by site-directed mutagenesis using polymerase chain reaction (PCR).…”

“…Similar to the outer membrane protrusions on other viral surfaces (26 -30), the HEV E2s domain harbors the major neutralizing epitopes for protection (31)(32)(33)(34)(35). A series of recombinant proteins containing the E2s domain, which included the bacterially expressed truncated proteins pE2 (aa 394 -606) (22,23,36) and p239 (aa 368 -606) (37) and the baculovirus expression system expressed T ϭ 1 virus-like particle (21), protected non-human primates and humans efficiently against HEV infection and liver injury (32,34). Among the truncated proteins, p239 was successfully used in the only approved HEV vaccine (32).…”

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

“…The E2s, E2 and p239 genes of HEV were cloned as previously described [12,17]. All mutated constructs were generated with site-directed PCR reactions.…”

“…We have previously shown that a truncated form of this capsid protein, p239 (aa 368-606) can self-assemble into virus-like particles (VLPs) of ~23 nm in diameter and subsequently penetrate susceptible cells, similar to a live virus [12]. We later produced the recombinant vaccine, HEV239, and employed this in a randomized, double-blind, placebo-controlled, phase 3 clinical trial involving more than 100 000 volunteers [13].…”

“…The capsid protein is responsible for virion assembly, host interaction and immunogenicity and is formed by homodimeric subunits, which comprise a protrusion domain [9,10] (E2s: npg www.cell-research.com | Cell Research aa 455-602 [11] or its N-terminal extension version E2: aa 394-606 [12]). These dimers project from the surface of the virus and initiate infection by interacting with host cells.…”