Fig 1.
Workflow including virus purification, lectin array and MALDI-TOF/TOF-MS analysis.
Fig 2.
Purification of DENV-2 particles from insect cells and infection of MDDC.
(A) DENV-2 virus was concentrated and purified via sucrose density gradient centrifugation. Fractions from the gradients were analyzed by SDS-PAGE, and dengue virus was mainly found in fractions4, 5 and 6 after centrifugation (upper panel). Double anti-DENV-2 antibody ELISA results were consistent with electrophoresis(lower panel).(B) Purified mature DENV-2 virions were negative stained and observed via transmission electron microscopy. Mature dengue virions were approximately 50 nm in diameter, surrounded by lipid bilayers, and had‘‘smooth” regions on their outer membranes. (C) Monocytes isolated from PBMCs were treated with 25 ng/ml IL-4 and 50 ng/ml GM-CSF for 7 days and infected with DENV-2 at an MOI = 0.1 for 48 hours. Two days after infection, the cells were permeabilized and analyzed for DENV-2 E protein expression using a 4G2 antibody. Nuclei were stained with DAPI (blue). (D) The expression of DENV-2 E was demonstrated by western blotting using anti-DENV-2 hyperimmune serum.
Fig 3.
N-Linked glycosylation status of purified DENV-2 was analyzed by SDS-PAGE and WB after PNGase F treatment.
(A) Purified virus was observed by SDS-PAGE. (B) Purified virus was digested with PNGaseF (+) or mock digested (-) and evaluated by SDS-PAGE. (C) Western blotting was performed to show the deglycosylation patterns of envelope proteins of DENV-2 virions using anti-DENV-2 hyperimmune serum.
Fig 4.
Glycan profiling of purified DENV-2 particles from insect cells by lectin microarrays.
(A) The layout of the lectin microarray. A total of 37 lectins were dissolved in manufacturer's recommended buffer to a concentration of 1 mg/mL and spotted onto homemade, epoxysilane-coated slides according to aprotocol from Stealth micro spotting pins (SMP-10B). Each lectin was spotted in triplicate per block, and triplicate blocks were placed on one slide. Cy3-labeled BSA was spotted as a location marker, and BSA was used as a negative control. (B) A profile of Cy3-labeled DENV-2 virions derived from insect cells bound to the lectin microarrays. Fluorescent images were scanned in a 70% photomultiplier tube at a 100% laser power setting with a Genepix 4000B confocal scanner. A representative portion of a slide with three replicates of the lectin array is shown. (C) The relative fluorescence intensities of cy3-labeled DENV-2 binding to 37 lectins. A total of 21 lectins out of 37 showed positive binding signals.
Fig 5.
Relative expression levels of DENV-2 glycan binders by lectin microarray.
The glycans binders were categorized into five types. (A) The GlcNAc binder DSA showed a stronger binding signal than the others. (B) (GlcNAc)n binders. (C) Bisecting and biantennary GlcNAc binders. (D) Mannose binders. (E) Gal binders. (F) Fucose binders. (G) Sialic acid binders.
Fig 6.
MALDI-TOF-MS spectra of N-glycans on purified mature DENV-2.
N-glycans from purified mature DENV-2 virions were separated and desalted as described in M&M. Lyophilized N-glycans were dissolved in MW, and an aliquot of a mixture with DHB solution was spotted onto an MTP Anchor Chip sample target and air-dried. MALDI-TOF-MS was performed in positive-ion mode. Experiments were performed in biological triplicate, and representative N-glycan spectra are shown. Peaks (signal-to-noise ratio >5) were selected for relative proportion analysis. Detailed structures were analyzed using Glyco Work bench software. Proposed structures are indicated by m/z values.
Fig 7.
Predicted structures and their molecular ions in MALDI Spectra of N-Glycans from purified mature DENV-2 virions.
Fig 8.
MALDI-TOF/TOF-MS/MS analysis of N-glycan precursor ions in MS spectra.
Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Structures of cleavage ions and m/z values are shown in tandem mass spectra. Three major N-glycan peaks are indicated: (A) m/z 1419.476, (B) m/z 1663.581, and (C)m/z 2028.714.
Fig 9.
Binding energies between DC-SIGN and selected glycans on the DENV-2 virus surface as predicted by mass spectra.
The 1–6 groups represent binding energies between DC-SIGN and a natural mannose glycan receptor (Hex3HexNAc2,NR), a high-mannose glycan (Hex9HexNAc2,HM), a hybridtype-N-glycan (Hex7HexNAc4,HY), a galactosylated glycan (Fuc1Hex6HexNAc5,GS) and asialylated complex type-Glycan (NeuAc1Fuc2Hex5H,SC), as well as a 6-glucose-hexose (6G).
Fig 10.
Docking analysis of DC-SIGN and glycan receptors.
A-D represent docking conformations of DC-SIGN and HM, HY, GS and SC. As is shown, the identified interactions mainly originated between glycans and the amino acid residues shown in blue.
Fig 11.
The proposed structural information of glycan on the surface of mature DENV-2 revealed by Mass Spectrometry and lectin microarray analysis.