Different mutations at position 562 of the hepatitis E virus capsid proteins exhibit differential effects on viral neutralizing activity

Xu,Mingjie; Sun,Lizhi; Wang,Yan; Gao,Shuchun; Yang,Weihua; Li,Meng

doi:10.3892/etm.2020.9542

Different mutations at position 562 of the hepatitis E virus capsid proteins exhibit differential effects on viral neutralizing activity

Authors:
- Mingjie Xu
- Lizhi Sun
- Yan Wang
- Shuchun Gao
- Weihua Yang
- Meng Li
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Affiliations: Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong 250013, P.R. China, Department of Liver Disease, Jinan Infectious Disease Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
Published online on: November 27, 2020 https://doi.org/10.3892/etm.2020.9542
Article Number: 110
Copyright: © Xu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The hepatitis E virus (HEV) capsid protein pORF2 comprises three potential N‑linked glycosylation sites. One site, N562, is located at the cell attachment and neutralizing antigenic regions. The present study performed detailed analyses of the effects of specific amino acid substitutions at position 562 in the homodimerization, glycosylation, antigenicity, immunogenicity and neutralization activities of HEV pORF2. Recombinant HEV pORF2 glycoprotein E1 (amino acids 439‑617) and three mutant variants (N562L, N562C and N562K) were expressed in Pichia pastoris (P. pastoris) and SDS‑PAGE, Western blot analysis, tunicamycin assay, double‑antibody sandwich ELISA and in vitro PCR‑based neutralization assay were performed to characterize the different constructs. All proteins were indicated to be secreted by P. pastoris and formed homodimers. Tunicamycin assay revealed the glycosylated status of the wild‑type protein, but the mutants were indicated to be non‑glycosylated. All proteins were immunoreactive with a neutralizing monoclonal antibody but were not recognized by the antibody after denaturation into monomers. An in vitro PCR‑based neutralization assay using mouse antibodies indicated efficient neutralization against N562L, whereas antibodies against N562C and N562K were revealed to be non‑neutralizing. Collectively, the present study indicated that specific amino acid substitutions at position 562 serve crucial roles in the activity of the HEV neutralizing epitope.

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