Biological Effects Of N562 Glycosylation And Mutation Of Hepatitis E Virus ORF2-encoded Protein

Hepatitis E virus (HEV) is transmitted via the fecal-oral route, predominantly through contaminated water, and causes epidemic and sporadic cases of acute hepatitis E in developing countries of Asia, Africa and Latin America, where sanitary conditions are suboptimal. Hepatitis E is generally a mild disease but may be severe, especially in pregnant women, among whom the mortality rate reaches 20%. The mortality rate of chronic HEV infection is up to 70%, owing to the development of fulminant liver disease. Recently, chronic HEV infection has been reported in the immunosuppressed. Detection of HEV in farmed swine and feral animals and reports of HEV transmission to humans via consumption of raw or undercooked meat of deer, boars and pigs strongly suggest an additional zoonotic pathway for HEV transmission. Global distribution and frequent detection, especially in developing countries, indicate that HEV poses a public health threat across the globe.ORF2 encodes a single structural protein of 660 amino acid (aa), which forms viral capsid and is responsible for binding cellular receptors and eliciting neutralizing antibodies. pORF2 has been expressed in different truncated forms in Escherichia coli and insect cells and has been used as a target for development of a subunit vaccine against HEV. Our previous study revealed that recombinant protein p166(aa452-617) of HEV ORF2 could model HEV conformation neutralization epitopes, and different genotypes of HEV had the same neutralization epitopes. Rencently, the recombinant protein p179(aa439-617) of HEV ORF2 has undergone successful phase Ⅱ clinical testing in humans. The ORF2-encoded protein contains three potential N-glycosylation sites, N137, N310 and N562. in the S and P domains. Until recently, no efficient cell culture system existed for studying HEV genome replication and protein expression and processing. Hence, the role of glycosylation in virions assembly remains unclear. One of the potential glycosylation sites, N562. is highly conserved and located in the apical center of the spike, which comprises the cell-attachment region and neutralizing antigenic region. Location of the site suggests its important role in viral morphogenesis and immune responses. Here, using site-specific mutagenesis of truncated capsid protein p179 (439-617aa) expressed in Pichia pastoris, we explored the role of N562 in glycosylation and immune response. The major results of this research are as follows:1. The conservation of a facultative N-linked glycosylation(N-X-S/T) site at N562 of pORF2 in HEV strainsTo obtain the conservation of a facultative N-linked glycosylation(N-X-S/T) site at N562 of pORF2 in HEV strains,210 complete sequences of HEV ORF2 available in GenBank were collected and compared. A potential glycosylation motif 562NTT was found to be highly conserved, but six sequences with GenBank accession numbers AB189070, AB425831, AB593690, AB698071, AB740232 and JQ026407 were found to contain D562 which is incompatible with the N-linked glycosylation at this site. As new HEV strains are isolated, the N562 site is likely to see other variations.2. Immunologic properties of HEV capsid protein ORF2 and Site-directed mutagenesis expressed in Pichia pastoris strain SMD1168Here, we expressed a recombinant polypeptide (p179) comprising 439-617 aa of pORF2 in Pichia pastoris. A set of 4 mutant proteins containing substitutions of Q, D, P and Y in place of N at position 562 were also generated and expressed using same expression system. All proteins were successfully secreted from yeast. Using SDS-PAGE, Western Blot analysis, ELISA and tunicamycin treatment assay to explored the role of N562 in glycosylation and immune response, we obtained the following results:2.1 The mutants were designed by replacement of N562 with aa similar to N in molecular weight namely, Q, D P, Y, representing amide, acidic, cyclic and aromatic aa. Site-directed mutagenesis was conducted using the overlap-extension PCR approach.2.2 Secondary structure prediction showed that HEV p179N562, p179N562Q, p179N562D and p179N562P have the same secondary structure of including 61.45% random coils,35.20% (3-sheet and 3.35% a-helix. The secondary structure of p179N562Y was slightly different, with random coils in place of β-sheet at position 559 and 560aa.2.3 Observation of a greater molecular weight for the wt protein suggests that it was glycosylated while the mutants were either not glycosylated or glycosylated to a lower degree. To evaluate the glycosylation status of the wt protein and its mutants, experiment was conducted to block glycosylation using tunicamycin, which is an inhibitor of bacterial and eukaryotice N-acetylglucosamine transferases and prevents glycosylation of newly synthesized glycoproteins. Addition of tunicamycin resulted in increased electrophoretic mobility of the wt protein compared to wt protein from untreated cells. Susceptibility of p179N562 to tunicamycin treatment indicates glycosylation of the protein. However, electrophoretic mobility of the mutant proteins was not affected with tunicamycin treatment, suggesting that the mutants were not glycosylated.2.4 We showed that the wild-type (wt) protein, p179N562, and 2 mutant variants, pl79N562Q and pl79N562D, form homodimers but only the wt protein was shown to be glycosylated, suggesting a role of N562 in glycosylation but not dimerization. As homodimers, all 3 proteins were immunoreactive with the neutralizing monoclonal antibody (mAb) 5G5, monomers formed by denaturation, however, were not immunoreact. Two other mutant variants, p179N562P and p179N562Y, did not form homodimers but were immunoreactive with mAb 5G5. The wt protein was shown to be less immunoreactive than the other 4 variants in a double-antibody sandwich ELISA, suggesting a role of glycosylation of N562 in reduced antibody binding.3. Immunogenicity of the recombinant protein and in vitro neutralizing test of mouse seraThe recombinant proteins were expressed in Pichia pastoris strain SMD1168 and purified. To evaluate the effect of N562 glycosylation on the immunogenicity of p179, we immunized mice with equal amounts of the wt and the Q, D, P, and Y mutants. Sera were collected periodically and tested by ELISA for anti-HEV IgG. In addition, we evaluated the ability of the mice sera to neutralize human HEV with an in vitro PCR-based assay. The results are as follows:3.1 Mice were immunized with all 5 recombinant proteins as described in Materials and Methods. Mouse immune sera were pooled from all animals immunized with the same antigent every week post inoculation (wpi). All mice vaccinated with the recombinant proteins presented HEV-specific antibodies in sera at 3 wpi. As expected, antibody levels increased after boost inoculations, with peak titers at 8 wpi in mice immunized with p179N562, p179N562Q, p179N562D and p179N562P. The peak titer was observed in mice immunized with p179N562Y at 5 wpi. Overall, the kinetics of antibody responses were similar in all groups of mice immunized with all proteins, indicating that N562 substitutions and glycosylation status did not have a detectable effect on these kinetics.3.2 The ability of immune sera to neutralize HEV was determined using the in vitro PCR-based neutralization assay. The corresponding pools of pre-incubation sera were used as negative controls. The end-point neutralization titers of antibody against p179N562, p179N562Q, p179N562D, p179N562P and p179N562Y were 1:20,1:80,1:20.1:640 and 1:160, respectively.Conclusion:1. A potential glycosylation motif 562NTT was found to be highly conserved, but is also slightly variable. In some genotype 3 strains,562DTT replaced 562NTT, with loss of the glycosylation motif.2. The wild-type (wt) protein, p179N562 and the four mutants were all successfully secreted from yeast. The wt protein was glycosylated while the mutants were not.3. The wild-type (wt) protein, p179N562, and 2 mutant variants p179N562Q and p179N562D form homodimers, which dissociated into into monomers when heated. Two other mutant variants, p179N562P and p179N562Y, did not form homodimers4. As homodimers, p179N562, p179N562Q, p179N562D were immunoreactive with the neutralizing monoclonal antibody (mAb) 5G5; but were no longer reactive after denaturation into monomers. Heating can destroy the structure of the p179 dimer and the neutralizing epitope.5. p179N562, p179N562Q, p179N562D, p179N562P and p179N562Y showed good immunogenicity and antigenicity and elicited neutralizing antibodies. N562 is not involved in the formation of a neutralizing epitope on p179, but sugar chain at N562 probably masks the partly neutralizing epitopes, reducing the titer of neutralizing antibody. The monomeric p179N562P elicited the highest titer of neutralizing antibody, its potential as vaccine is worthy of further investigation.