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Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

Lei Y, Yu H, Dong Y, Yang J, Ye W, Wang Y, Chen W, Jia Z, Xu Z, Li Z, Zhang F - PLoS ONE (2015)

Bottom Line: By combining these methods, a high heterogeneity of DENV N-glycans was found.Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found.For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Preclinical Medicine, The Fourth Military Medical University, 169 Changle Xi Road, Xian, Shaanxi 710032 China.

ABSTRACT
DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0132122.g002: 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.

Mentions: Previous studies have shown that a heterogeneous population of virions that vary in maturation state are produced from DENV-infected cells[21]. It is well known that an incomplete cleavage of the envelope glycoprotein prM generates a mixture of mature (prM-less) and immature extracellular particles(prM-containing) during the dengue virus life cycle[22]. In vitro infections with immature and mature dengue viruses have shown that fully immature particles are non-infectious because they block virus-receptor interactions and membrane fusion activity via the prM protein. To characterize the glycans that decorate the surfaces of mature dengue virions, we optimized culture conditions with RPMI-1640 media and created a sucrose density gradient centrifugation method to harvest mature dengue virus. As indicated in Fig 2A, SDS-PAGE shows that dengue virus was mainly in fractions 4, 5 and 6 after centrifugation (upper panel). Double anti-DENV-2 antibody ELISA results were consistent with the results from electrophoresis(lower panel). The fraction that contained the purified mature virions was harvested. It is known that immature DENV particles have mosaic structures with ‘‘spiky” regions and that mature virions have‘‘smooth” regions, as demonstrated by structural studies. Furthermore, mature DENV-2 virions were negative-stained and observed using transmission electron microscopy. Fig 2B shows that the mature dengue virions were approximately 50 nm in diameter and surrounded by a lipid bilayer and had‘‘smooth” regions on their outer membranes.


Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

Lei Y, Yu H, Dong Y, Yang J, Ye W, Wang Y, Chen W, Jia Z, Xu Z, Li Z, Zhang F - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4514477&req=5

pone.0132122.g002: 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.
Mentions: Previous studies have shown that a heterogeneous population of virions that vary in maturation state are produced from DENV-infected cells[21]. It is well known that an incomplete cleavage of the envelope glycoprotein prM generates a mixture of mature (prM-less) and immature extracellular particles(prM-containing) during the dengue virus life cycle[22]. In vitro infections with immature and mature dengue viruses have shown that fully immature particles are non-infectious because they block virus-receptor interactions and membrane fusion activity via the prM protein. To characterize the glycans that decorate the surfaces of mature dengue virions, we optimized culture conditions with RPMI-1640 media and created a sucrose density gradient centrifugation method to harvest mature dengue virus. As indicated in Fig 2A, SDS-PAGE shows that dengue virus was mainly in fractions 4, 5 and 6 after centrifugation (upper panel). Double anti-DENV-2 antibody ELISA results were consistent with the results from electrophoresis(lower panel). The fraction that contained the purified mature virions was harvested. It is known that immature DENV particles have mosaic structures with ‘‘spiky” regions and that mature virions have‘‘smooth” regions, as demonstrated by structural studies. Furthermore, mature DENV-2 virions were negative-stained and observed using transmission electron microscopy. Fig 2B shows that the mature dengue virions were approximately 50 nm in diameter and surrounded by a lipid bilayer and had‘‘smooth” regions on their outer membranes.

Bottom Line: By combining these methods, a high heterogeneity of DENV N-glycans was found.Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found.For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Preclinical Medicine, The Fourth Military Medical University, 169 Changle Xi Road, Xian, Shaanxi 710032 China.

ABSTRACT
DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

No MeSH data available.


Related in: MedlinePlus