Figure 1.
Schematic drawing of DENV4 prME, prM and E constructs with serially C-terminal truncation and their expression.
(A) 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 [18]. The C-terminus of prM protein contains an α-helical domain (MH) and two transmembrane domains (MT1 and MT2). WT and a series of C-terminal truncation of prME, prM and E constructs were shown. (B) 293T cells were transfected with prM and its C-terminal truncation constructs in the presence or absence of E construct. (C) 293T cells were transfected with E and its C-terminal truncation constructs in the presence or absence of prM construct. (D) 293T cells were transfected with prME and its C-terminal truncation constructs. Cell lysates collected at 48 h post-transfection were subjected to Western blot analysis using human serum of a confirmed dengue case [31], [32]. One representative experiment of three is shown. The size of molecular weight markers is shown in kDa. Arrow heads indicate E and prM proteins.
Figure 2.
Effect of WT and mutant E proteins on the expression of prM protein.
293T cells were transfected with prM construct alone or prM construct and increasing amounts (0 to 2 µg) of WT E construct (A), E construct without the stem and anchor, Ed395 (B) or CD4D4SA construct containing the stem and anchor of E fused to the ectodomain of CD4 (C) [31]. Cell lysates collected at 48 h post-transfection were subjected to Western blot analysis using human serum of a confirmed dengue case [31], [32]. One representative experiment of three is shown. The size of molecular weight markers is shown in kDa. Arrow heads indicate prM, E, Ed395 or CD4D4SA protein.
Figure 3.
Effect of WT and C-terminally truncated E proteins on the expression and stability of prM protein by pulse-chase experiment.
(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 [33], followed by 12% PAGE as described in the Methods. 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 Methods. *P = 0.002, **P = 0.009, two-tailed Mann-Whitney test.
Figure 4.
Interaction between prM protein and WT/C-terminally truncated E proteins by radioimmunoprecipitation assay and sucrose gradient sedimentation analysis.
(A) 293T cells transfected with WT prME or prME constructs with C-terminal truncation were labeled with [35S] methionine at 20 h post-transfection, immunoprecipitated with an anti-E mAb FL0232, and subjected to 12% PAGE (left) as described in the Methods. One representative experiment of two is shown. The size of molecular weight markers is shown in kDa. Arrow heads indicate E and prM proteins. The prM/E index (right) was determined by the ratio of the intensity of prM band to truncated E band divided by such ratio of prM band to WT E band as described in the Methods. (B to F) Cell lysates derived from 293T cells transfected with WT prME or prME constructs with C-terminal truncation were subjected to 5 to 20% (wt/wt) sucrose gradient ultracentrifugation, and each of the 14 fractions was collected and subjected to Western blot analysis using serum from a confirmed dengue case [31], [32]. Long exposure of prM bands was shown for (E) and (F). The intensities of the E and PrM bands in each fraction were determined and presented as the percentage of total intensities of E and prM bands, respectively. One representative experiment of two is shown. The size of molecular weight markers is shown in kDa. Arrow heads indicate E and prM proteins.
Figure 5.
Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs.
(A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33]. Data are mean and standard errors from two experiments.
Figure 6.
Binding of C-terminally truncated E proteins in extracellular fluid to different human and mouse anti-E mAbs by capture ELISA.
(A) Human GR anti-E mAbs. (B) Human TS anti-E mAb. (C) Mouse CR anti-E mAb, DEN3-3, and TS anti-mAb, 1H10-6-7. Comparable amounts (0.6 ng each) of truncated E protein derived from prMEd421, prMEd395, Ed421 or Ed395 were added to 96 well plate pre-coated with mixed mouse mAbs (for testing human mAbs) or human dengue-immune serum (for testing mouse mAbs), followed by addition of each human or mouse mAb and secondary antibody as described in Methods. Data are means and standard errors of quadricates from one representative experiment of two. *P = 0.03, two-tailed Mann-Whitney test.
Figure 7.
Effect of E protein on the recognition of prM protein by human anti-prM mAbs.
(A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5. (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.
Figure 8.
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.
(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 [18]. 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 [33]. ↓ indicates reduced binding (R.I.<0.4 in dot blot assay or P<0.05 in capture ELISA) to mutant E proteins (prMEd421, prMEd395, Ed421); →indicates binding was not reduced. ND, not done.