戊型肝炎病毒衣壳蛋白同源二聚体的相互作用结构域

为了探讨戊型肝炎病毒衣壳蛋白同源二聚体形成的关键区域和相互作用结构域，以及二聚体形成与主要天然中和表位的形成之间的关系，通过末端缺失、定点突变技术研究戊型肝炎病毒（HEV）ORF2的aa394-aa606片段NE2的聚合现象，发现其C端的aa597-aa602（AVAVLA）疏水区是该片段同源聚合的核心区域，提高该区域氨基酸的亲水性将妨碍聚合现象的发生；半胱氨酸化学交联实验表明NE2形成同源二聚体时，aa597在空间位置上相接近，处于可生成化学键的距离，提示所处区域为疏水聚合的作用结构域；通过Blast程序估算核心区域的天然突变率，发现其疏水性高度保守；N端缺失实验表明，至少65个氨基酸既不影响同源聚合也不直接参与主要的天然中和表位的形成，但可协助中和表位构象的形成，而这种协助作用可被ORF2的末端肽段所代替。Aa597-aa602（AVAVLA）疏水区为戊肝病毒衣壳组装的第一步骤的核心区域，并与重要的天然中和表位的形成直接相关，从而为戊肝病毒疫苗的研究提供更详细的信息。

Interface Domain of Hepatitis E Virus Capsid Protein Homodimer

Hepatitis E is a main cause of acute viral hepatitis in developing countries where it occurs as sporadic cases and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. The approximately 7.5 kb positive-sense single-strand RNA genome includes three open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2) of 660 amino acid residues. We earlier showed that a bacterially expressed peptide, designated as NE2, located from amino acid residues 394 to 606 of ORF2, was found to aggregate into homodimer to at least hexamer. To understand the interface domains within this peptide vital for dimerization and formation of major neutralizing epitopes, NE2 protein underwent terminal-truncated and site-directed mutation. The hydrophobic region, ORF2 aa597-aa602 (AVAVLA), played a key role in oligomerization. Any amino acid residue of this region replaced with glutamic acid residue, the peptide can not refold as homodimer and/or oligomer. The immunoreactivities of these mutant peptides, blotted with anti-HEV neutralizing monoclonal antibody (8C11) and convalescent human sera, show associated to the formation of homodimer. The intermolecular contact region on homodimer was investigated by chemical cross-linking of two site-directed cysteines. When the alanine on aa597 site mutated with cysteine, two different homodimers were found in SDS-PAGE analysis. One (42kD) can be disassociated with 8mol/L urea, which is postulated to form by virtue of hydrophobic interaction, and the other (60kD) falls apart with the reductant DTT present. The exact conformation, generating the cross-linking reaction of cysteines, was further investigated by induced-oxidation on monomer and hydrophobic homodimer of A597C protein with GSH/GSSG. And the results revealed, it is the conformation of hydrophobic homodimer that induces the disulfide bond come into being, instead of the one of monomer. So the aa597 site was verified to be located on interface domain of hydrophobically interacting homodimeric complex. To evaluate the biological significance of hydrophobicity of interface domain, we searched natural variations as to the region on all available databases with NCBI blast program. All variations on these amino acid residues kept higher hydrophobicity, which suggests that the hydrophobic domain is critical for the assemblage and propagation of HEV. NE2 N-terminal deletions up to aa458 had no effect on dimerization and took no exact part in formation of major neutralizing epitopes, but the fragment may act as helper for the formation of major neutralizing epitopes on NE2. Interestingly, the C-terminus aa605-aa660 of ORF2 can also act as helper instead of the N-terminus of NE2. This study suggests an interface domain of NE2 might be vital for HEV capsomer assembly and formation of major neutralizing epitopes. These results may offer clues to the rational design of recombinant anti-HEV vaccine.