Comparative analysis between HEV ORF3 protein and peptides with deduced amino acids borne by selected phages.

<p>The consensus sequence was mainly identified in the ORF3 C-terminus. Only the sequence from A93 to R114 of ORF3 is presented. “.” represents residues of deduced amino acids differing from the ORF3 C-terminus, “-”indicates no residues. The numbers at the top signify the position of the residue at the ORF3 C-terminus. The first “3” represents position 93, and the last “4” represents position 114.</p

Detection of BHK cells transfected with pcDNA3.1-ORF3 plasmid via indirect immunofluorescence assay using mAb-1.

<p>BHK cells transfected with pcDNA3.1 empty plasmid were used as control. Nuclei were stained with DAPI. Results obtained with mAb-2 were similar to those with mAb-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133282#pone.0133282.s003" target="_blank">S3 Fig</a>).</p

(a) Expression analysis of ORF3 protein at 5 h post-induction using SDS-PAGE.

<p><i>Lane M</i>, protein molecular weight marker. <i>Lane 1</i>, pET32a empty plasmid. The arrow signifies His tag protein. <i>Lane 2</i>, uninduced bacteria transformed with pET32a-ORF3. <i>Lane 3</i>, deposit of lysed bacteria transformed with pET32a-ORF3. The arrow signifies ORF3 protein fused to the His tag. <i>Lane 4</i>, supernatant of lysed bacteria transformed with pET32a-ORF3. The arrow represents His-tagged ORF3 protein. <b>(b) Analysis of purified His-tagged ORF3 proteins.</b><i>Lane 1</i>, purified ORF3 protein-tagged His. <i>Lane 2</i>, unpurified expression product.</p

Comparative analysis of ORF3 C-terminal amino acid sequences among the four HEV genotypes.

<p>Residues shaded in black represent those differing from genotype 1 HEV ORF3. The consensus continuous sequence "VVDLP" is presented in the red box. The number in brackets represents the GenBank accession no. of the selected virus.</p

Efficacy of biopanning for HEV ORF3 mAbs.

<p>Efficacy of biopanning for HEV ORF3 mAbs.</p

A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V¹⁰⁵DLP¹⁰⁸ Immunoactivity

<div><p>The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region.</p></div

ELISA binding analysis of mutant S⁹⁷APPLPPVVDLP¹⁰⁸ peptides to ORF3 mAb.

<p><b>A</b>ntigen names are shown on the <i>x</i> axis and OD₄₉₀ value of each peptide on the <i>y</i> axis. Schematic illustration showing that the P100A mutant maintains similar binding activity to mAb as wild-type SAPPLPPVVDLP. “**” represents significant differences (<i>P</i><0.01).</p

Deduced amino acid sequences of peptides borne by selected phages.

<p>① Isoleucine (I) has similar structure and characteristics as Leucine (L)</p><p>② Isoleucine (I) has similar structure and characteristics as Valine (V). I, L and V all belong to the branched chain amino acid group. The underline indicates the continuous consensus sequence within the C-terminal region of ORF3 protein.</p><p>N/A, not applicable.</p

ELISA analysis of binding of synthetic peptides to ORF3 mAb.

<p>The binding activity of each peptide is presented in individual histograms. The <i>x</i> axis represents peptide concentration and <i>y</i> axis shows the OD₄₉₀ value of each peptide at different concentrations. The color of each bar represents the ORF3 mAb serial concentration. Schematic illustration showing that only SAPPLPPVVDLPQLGL and SAPPLPPVVDLP react with the mAb,</p

Expression of three truncated ORF3 proteins and identification of the antigenic epitopes.

<p>(a) Bacteria containing three truncated ORF3 genes induced by 1 mM IPTG at 37°C for 5 h and identified via SDS-PAGE. <i>Lane M</i>, protein molecular weight marker. <i>Lanes 1</i>, <i>3</i>, <i>5</i>, uninduced bacteria containing ORF3-1, ORF3-2 and ORF3-3 truncated genes, respectively. <i>Lanes 2</i>, <i>4</i>, <i>6</i>, induced bacteria containing ORF3-1, ORF3-2 and ORF3-3 truncated genes, respectively. Arrows indicate the positions of the three recombinant proteins with a molecular weight of ~27 kDa. (b) Identification of the antigenic epitopes of ORF3 using mAb-1 via western blot. Only the ORF3-3 protein was recognized by this mAb, as indicated with the arrow. The lanes in (b) are one to one correspond to lanes in (a), but experiments were performed using two SDS gels. (c) Schematic representation of the truncated HEV ORF3 constructs and reactivity to mAbs. The names and lengths of the truncated constructs are indicated. Binding ability to ORF3 mAb-1 was determined based on western blot results. “+”, positive result and “-”, negative result. Results obtained with mAb-2 were similar to those with mAb-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133282#pone.0133282.s004" target="_blank">S4 Fig</a>), and ORF3 protein recognized by two mAbs via western blot was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133282#pone.0133282.s005" target="_blank">S5 Fig</a>.</p