Sucrose gradient analysis and electron micrographs of purified VLPs.

<p>Pellets derived from 293T cells transfected with prME or Ed395 were subjected to a 5 to 20% (wt/wt) sucrose gradient ultracentrifugation; each of the 10 fractions was subjected to Western blot analysis using a dengue-immune serum. (B) Gradient-purified particles from fractions 4 and 10 were subjected to immunogold labeling using anti-E mAb (1H10-6-7), stained with 2% PTA and photographed at the same magnification. White bar: 100 nm. (C) The size of particles (diameter) of fractions 4 and 10 derived from prME and fraction 10 derived from Ed395 were determined by measuring more than 130 particles from electron micrographs. The particles size (diameter) is shown in nm.</p

Expression of E protein and E protein ectodomain in the presence or absence of prM protein.

<p>(A) Schematic drawing of the DENV4 prME, prMEd395, E and Ed395 constructs. The C-terminus of E protein contains two α-helical domains (EH1 and EH2) in the stem region, followed by two transmembrane domains (ET1 and ET2) in the anchor region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Zhang1" target="_blank">[24]</a>. ss, signal sequence. (B, C) At 48 h, lysates of 293T cells transfected with each of the 4 constructs were subjected to Western blot analysis (B) using a dengue-immune serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Wang2" target="_blank">[39]</a> or immunoprecipitation (C) using two mouse anti-E mAbs (4G2 and 1H10-6-7) and a dengue-immune human serum (#13), followed by Western blot analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Hsieh1" target="_blank">[38]</a>. Anti-CD4 mAb was used a negative control. One representative experiment of two was shown. The size of molecular weight markers is shown in kDa.</p

Membrane association of E protein and E protein ectodomain in the presence or absence of prM protein.

<p>(A) Membrane flotation assay. At 48 h post-transfection, 293T cells were homogenized and centrifuged to clear nuclei and debris. Supernatants were mixed with 72% sucrose, overlaid with 55% and 10% sucrose and ultracentrifuged; each of the 9 fractions was subjected to Western blot analysis using a dengue-immune human serum, anti-calnexin or anti-β-actin mAbs. (B) Subcellular fractionation assay. At 48 h, 293T cells transfected with prME, prMEd395, E or Ed395 were resuspended in modified buffer B and disrupted by freeze-thaw <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Hsieh1" target="_blank">[38]</a>. After clearing the nuclei and debris, membrane fraction and pellets derived from soluble fraction by sucrose cushion ultracentrifugation were subjected to Western blot analysis using a dengue-immune human serum (upper panel) and then reprobing with anti-calnexin mAb (lower panel). (C) Membrane fraction and (D) pellets of the soluble fraction of each transfectant were treated with endo H (H) or PNGase F (F), and subjected to Western blot analysis using a dengue-immune human serum. Arrow heads indicate E or Ed395, and aster indicates deglycosylated E (dgE) or deglycosylated Ed395 (dgEd395). One representative experiment of two was shown. The size of molecular weight markers is shown in kDa.</p

The effects of N-linked glycan and treatment with ammonia chloride on the secretion of E protein ectodomain.

<p>(A) At 48 h, cell lysates and culture supernatants of 293T cells transfected with Ed395 or each N-linked glycosylation mutant were subjected to Western blot analysis using a dengue-immune human serum. (B) At 16 h, transfected 293T cells were replaced with fresh medium containing 20 mM NH₄Cl. At 48 h, cell lysates and culture supernatants were subjected to Western blot analysis. (C, D) The ratio of Ed395 in supernatants to Ed395 in cells without (C) or with (D) NH₄Cl treatment. The amounts of Ed395 in cells and supernatants were quantified by capture-ELISA.</p

Production, detergent sensitivity and electron micrographs of VLPs.

<p>(A) At 48 h, cell lysates and pellets derived from culture supernatants of 293T cells transfected with prME, prMEd395, E or Ed395 were subjected to Western blot analysis using a dengue-immune serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Wang2" target="_blank">[39]</a>. (B) The amounts of various forms of E protein in culture supernatants, including total E protein in supernatants (Total sup), E protein in pellets containing VLPs (pellet-VLPs) and soluble E protein in supernatants post-ultracentrifugation (post-pellet) were quantified by a capture-ELISA as described in Methods. Right graph shows the proportion of E protein in pellet-VLPs and post-pellet (% of total E protein). (C) Pellets derived from culture supernatants of each transfectant were treated with or without 1% Triton X-100, followed by 15 to 55% (wt/wt) sucrose gradient ultracentrifugation; each fraction was subjected to Western blot analysis using a dengue-immune serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Wang2" target="_blank">[39]</a>. (D) An aliquot of the above pellets was stained with 2% PTA and photographed at the same magnification. (E) An aliquot of above pellets were subjected to Western blot analysis using a dengue-immune serum (top), rabbit anti-M serum (middle) and anti-β-actin mAb (bottom). The size of molecular weight markers is shown in kDa. One representative experiment of three was shown.</p

Oligomeric status of E protein and E protein ectodomain in cells by sucrose gradient sedimentation analysis.

<p>(A) At 48 h, 293T cells transfected with prME, prMEd395, E or Ed395 were treated with Triton X-100; cell lysates and protein markers were subjected to 5 to 20% (wt/wt) sucrose gradient ultracentrifugation. Each of the 13 fractions was collected and subjected to Western blot analysis using a dengue-immune human serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Wang2" target="_blank">[39]</a>. (B, C) 5 to 20% sucrose gradient ultracentrifugation of lysates of 293T cells transfected with prMEd395 or Ed395. Each of the 14 fractions was collected and subjected to Western blot analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Wang2" target="_blank">[39]</a>. Aliquots of fractions 14 and 6 were digested with endo H or PNGase F and subjected to Western blot analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100641#pone.0100641-Wang2" target="_blank">[39]</a>. The size of molecular weight markers is shown in kDa. Arrow heads indicate Ed395 and the deglycosylated (dg) form.</p

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

<div><h3>Background</h3><p>The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.</p> <h3>Methodology/Principal Findings</h3><p>In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.</p> <h3>Conclusions/Significance</h3><p>A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.</p> </div

Determination of % anti-FL Abs in serum of a dengue patient by VLP-capture ELISA.

<p>(A) Serial dilutions of the serum were subjected to a capture ELISA using DENV1 WT and mutant VLPs containing mutations in the FL epitope (W101A+F108A). The bar graph displaying results from an anti-E ELISA shows that comparable amounts of WT and mutant VLPs were added based on recognition of E by pooled human dengue-immune sera. % anti-FL Abs = [1 – endpoint titer to mutant VLPs/endpoint titer to WT VLPs]×100%. (B) The same serum was subjected to a capture ELISA using DENV2 WT and mutant VLPs (W101A+F108A). Data are presented as in (A). Data are means with standard deviation of duplicates from one representative experiment of two. For endpoint titers, only means are shown.</p

Effect of WT and C-terminally truncated E proteins on the expression and stability of prM protein by pulse-chase experiment.

<p>(A) 293T cells transfected with WT prME or prME constructs with C-teriminal truncation were pulsed for 20 min with [³⁵S] methionine at 20 h post-transfection, and chased at 0 min and 90 min by immunoprecipitation with mixed human sera of confirmed dengue cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052600#pone.0052600-Lin1" target="_blank">[33]</a>, followed by 12% PAGE as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052600#s2" target="_blank">Methods</a>. One representative experiment of three is shown. The size of molecular weight markers is shown in kDa. Arrow heads indicate E and prM proteins. (B) Relative prM/E at 90 min was determined by the ratio of the intensity of prM band to truncated E band at 90 min divided by such ratio at 0 min as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052600#s2" target="_blank">Methods</a>. *<i>P</i> = 0.002, **<i>P</i> = 0.009, two-tailed Mann-Whitney test.</p

Schematic drawing of prM/E proteins after synthesis and summary of the effect of C-terminal E domains on the recognition of E protein by mAbs.

<p>(A) Schematic drawing of prM/E proteins on ER membrane after synthesis. The topology of the stem (MH, EH1, EH2) and anchor (MT1, MT2, ET1, ET2) regions on membrane were based on a cryo-EM study of DENV virions at high resolution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052600#pone.0052600-Zhang1" target="_blank">[18]</a>. The ectodomains of prM and E proteins were drawn disproportionately. SS: signal sequence. The numbers of E residues between domains were shown. (B) Summary of the effect of C-terminal E domains on the recognition of E protein by mAbs based on dot blot assay and capture ELISA. Epitope residues were determined by binding assays involving a panel of 67 alanine mutants of predicted surface-exposed E residues as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052600#pone.0052600-Lin1" target="_blank">[33]</a>. ↓ indicates reduced binding (R.I.<0.4 in dot blot assay or <i>P</i><0.05 in capture ELISA) to mutant E proteins (prMEd421, prMEd395, Ed421); →indicates binding was not reduced. ND, not done.</p