Figure 1.
MR Binding to DV Is Mediated by the CRD4–7
(A) Binding of MR extracellular domain (MR-HA) and chimeric MR fusion protein constructs to mosquito cell–derived (C6/36) NGC strain DV2 and (B) soluble E protein (sE) produced in a human cell line, detected by ELISA. Closed bars, DV antigen; open bars, uncoated wells. The MR-HA protein was detected with an anti-murine MR antibody (MR5D3). Binding of the Fc fusion proteins to the DV/sE was detected with an anti-Fc antibody. (C) Inhibition of CRD4–7-Fc binding to mosquito cell–derived semi-purified DV2 and (D) sE, detected by ELISA as above. (E) Binding of CRD4–7-Fc to all four serotypes of DV, detected with an anti-Fc antibody. Strains examined were mosquito cell (C6/36)–derived virus of DV1 (Hawaii), DV2 (NGC), DV3 (H-87) and DV4 (H-241), and Vero cell–grown DV2 (16681). Differential binding to the serotypes may reflect coating levels, as indicated by (F) binding of rabbit anti-DV1–4 antibody. Normal rabbit immunoglobulin (RIg) was included as a control and both were detected with an anti-rabbit antibody. The low levels of CRD4–7-Fc binding to C6/36-grown DV2 in this panel reflect a shorter development time than in the other panels. Data are expressed as mean and SD of triplicate wells. Representative data from two to five independent experiments are shown. Recombinant fusion proteins contain the cysteine-rich (CR) domain, fibronectin type-II (FNII) domain, and various carbohydrate-recognition domains (CRD) of the MR.
Figure 2.
MR CRD4-7-Fc Binds Specifically to sE in Blot Overlay, and Binding Depends on N-Linked Sugars
sE and PNGaseF-treated sE were resolved by SDS-PAGE using 10% gels, transferred to nitrocellulose membranes, and (A) probed with MR CRD4–7-Fc. (B) Blots were subsequently stripped and reprobed with the anti-E protein antibody, 3H5.
Figure 3.
Production and Characterisation of Recombinant Soluble Dengue Virus E-Glycoprotein
(A) SDS-PAGE of sE protein preparation resolved on 10% gel and stained with Coomassie Brilliant Blue. Lane 1, sE preparation as eluted from NiNTA-agarose. Lane 2, PNGaseF-treated sE. Lane 3, PNGaseF alone.
(B) Western blot of sE before (Lane 1) and after treatment with PNGaseF (Lane 2) or EndoH (Lane 3), resolved under reducing conditions by 10% SDS-PAGE, and probed with the anti-DV E monoclonal antibody, 3H5.
(C) NP HPLC chromatograms of the complete pool of 2AB labelled glycans of sE glycoprotein, together with sequential digestions. a) Complete glycan pool, undigested, with structural representation of A2G2S2. b) Glycans digested with Arthrobacter ureafaciens sialidase (abs), which releases α2–6 and 3 linked sialic acids. Boxed sections show peaks containing α1–3 or 4 linked fucose residues, with structural representations of A2G2 and A2FG2. c) Glycans digested with abs and almond meal fucosidase (amf), which releases α1–3 and 4 linked fucose residues.
Key: A2, biantennary; G, galactose; F, fucose; S, sialic acid. Filled square, N-acetyl glucosamine; open circle, mannose; open diamond, galactose; filled star, sialic acid; open diamond with dot, fucose. The solid lines are β-linkage, dotted lines are α-linkage, and curved lines are unknown linkage.
Figure 4.
DV Binds to Cells Transfected with Human MR or DC-SIGN
The expression levels of (A) MR and (B) DC-SIGN on cells transfected with MR (3T3.hMR), DC-SIGN (3T3.DC-SIGN), or vector only (3T3) were assessed by flow cytometery. Receptor expression was detected with 15–2 (anti-hMR) or 120507 (anti-DC-SIGN) antibodies (pale and dark blue lines). Matched isotype control (purple and green lines) and secondary antibody only (brown and red lines) staining is included. Binding of NGC DV2 to cells transfected with (C) vector only (3T3), (D) MR (3T3.hMR), and (E) DC-SIGN (3T3.DC-SIGN) for 90 min on ice was detected by flow cytometry. The histograms show the binding of anti-DV2 antibody (3H5; brown and green lines) and isotype matched control antibody (blue and red lines). The relative fluorescence intensity was measured by FACSCalibur analysis and the data are normalised and presented as percent of maximum. Representative data from one of two independent experiments are shown.
Figure 5.
IL-4 and IL-13 Enhance Monocyte and MØ Susceptibility to DV Infection
Monocytes isolated from human peripheral blood were either treated with IL-4 or IL-13 or were left untreated for 2 d (day 2 MDMØ) prior to infection with dengue virus. Two days following infection, permeabilized cells were stained with antibody to dengue E protein (green) and nuclei stained with DAPI (blue).
(A) Low power image. (B) Single DV-infected cell confocal image showing DV protein distributed throughout the cytoplasm. Image represents a single x-y section through the middle of a cell. Fluorescence images are shown next to the corresponding transmission image. (C) The percentage of cells infected was counted by microscopy. Each line represents a single donor. (D) Monocytes were matured into MDMØ by 7 d incubation prior to treatment with IL-4 for 2 d (day 9 MDMØ). Cells were infected with DV and stained, and percent infected cells calculated.
Figure 6.
IL-4 Treatment Enhances Surface Expression of MR and DC-SIGN on Human MDMØ
The effects of 2 d IL-4 treatment (25ng/ml) of human monocytes on surface expression levels of (A) MR and (B) DC-SIGN were assessed by flow cytometery. Receptor expression was detected with 15–2 (anti-hMR) or 120507 (anti-DC-SIGN) antibodies (black lines) on untreated cells (open histogram) and IL-4-treated cells (filled histogram). Matched isotype control (grey lines) staining is included. The relative fluorescence intensity was measured by FACSCalibur analysis and the data are normalised and presented as percent of maximum. Representative data from one of eight donors are shown.
Figure 7.
Anti-MR and Anti-DC-SIGN Antibodies Inhibit the Ability of DV2 to Infect IL-4-Treated Monocytes
(A) IL-4-treated human monocytes were incubated in triplicate wells with medium alone (no block), 2mg/ml mannan, titrations of goat anti-human MR antiserum, or with normal goat serum (NGS). Treated cells were infected with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.5 in the presence of these inhibitors, incubated for 48 h and fixed.
(B) Monocytes were treated in triplicate wells, as above, including blocking with monoclonal antibodies specific for DC-SIGN (120507), DC-SIGNR (120604), and both DC-SIGN and DC-SIGNR (DC28 and 120612) or isotype controls (all at 5ug/ml) prior to infection with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.04 in the presence of these inhibitors. Following fixation, cells were immunolabelled with anti-DV E protein monoclonal antibody 3H5 and counted using a fluorescent microscope, and percent of cells infected calculated. Data are expressed as mean and SD of triplicate wells. Representative data from one of multiple donors are shown.
(C) The titre of infectious virus in the cell supernatant at 48 h post infection was determined by plaque assay. Due to variation between donors, the titre at 48 h in the absence of block was normalised to 100. Each data point is the average of three infected wells, each plaqued in triplicate, and the results from six donors are shown in this graph (mean represented by a bar). The p value was calculated by unpaired, two-tailed t test.