Investigation of the role of glycans for the biological activity of Semliki Forest virus grown in Aedes albopictus cells using inhibitors of asparagine-linked oligosaccharides trimming

Naim, Hussein Y.; Koblet, H.

doi:10.1007/bf01315564

Cited by 15 publications

(13 citation statements)

References 54 publications

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“…The deletion of the glycosylation sites in E1 did not affect heparin binding but decreased replication in BHK cells and virulence for mice, particularly for the E1-245 mutant, for which replication was impaired in C7/10 cells as well. Previous observations that altered alphavirus glycosylation does not affect the processing of pE2 (13,36,38) were confirmed. Interaction with the cellular glycosaminoglycan HS is an initial electrostatic binding event for the E2 glycoproteins of many strains of SINV, including TE12, the parent strain for these studies (6,7,26).…”

Section: Discussionsupporting

confidence: 66%

Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

Knight

Schultz

Kent

et al. 2009

J Virol

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). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.Sindbis virus (SINV), the prototype alphavirus in the family Togaviridae, is an arthropod-borne virus that cycles between mosquitoes and wild birds and causes summertime outbreaks of rashes and arthritis in humans (17, 34). In mice, SINV causes encephalomyelitis and serves as a useful model for the study of alphavirus infections of the central nervous system (CNS). The virion is enveloped and has icosahedral symmetry, with a single linear strand of positive-sense RNA surrounded by a capsid and two transmembrane glycoproteins, E1 and E2, that heterodimerize and then form trimeric spikes on the surface. Each of the viral glycoproteins has two N-linked glycosylation sites at E1 positions 139 and 245 (E1-139 and E1-245) and at E2 positions 196 and 318 (E2-196 and E2-318) that are used (4, 42). Some glycosylation is required for proper folding and transport of the glycoproteins, as total ablation of glycosylation by treatment with tunicamycin results in failure of E1 and E2 to be transported through the Golgi apparatus to the cell surface (31, 32).The type of carbohydrate, high-mannose carbohydrate or complex oligosaccharide, at a glycosylation site varies with the type and growth state of the cell infected (3,18,23,28,29,35,51). In insect cells, all sites have high-mannose sugars (22) due to the absence of N-acetylglucosaminyl-, galactosyl-, and sialyltransferases (5). In vertebrate cells, the distribution of complex and high-mannose carbohydrates is largely determined by the accessibility of the site to processing enzymes in the Golgi apparatus (23).E1 has an extracellular domain that contains the internal fusion peptide. The carbohydrate at E1-139 consists of complex oligosaccharides in vertebrate cell...

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Section: Discussionsupporting

confidence: 66%

Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

Knight

Schultz

Kent

et al. 2009

J Virol

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“…This strongly suggests that heterogeneity in the oligosaccharide chains occurs in a compartment beyond the ~-mannosidase I site, likely in the Golgi apparatus. This is supported by the finding that in the presence of swainsonine, which inhibits a-mannosidase II activity [-5, 12], heterogeneity is apparent and several oligosaccharide structures are revealed [22]. The data demonstrate that the inhibitors of oligosaccharide processing prevent the formation of low mannose or hybrid types of sugar chains.…”

Section: Oligosaccharides Of Semliki Forest Virus 57supporting

confidence: 69%

“…E 1 was resistant to Endo-H (lane 2). Endo-F/GF treatment, which de-N-glycosylates the viral proteins [22], caused faster migration of E~ and E2 ( Fig. 1 a, lane 4).…”

Section: Partial Characterization Of N-linked Oligosaccharides On Thementioning

confidence: 89%

“…Yet, the virions were formed at normal rates and in normal amounts. Furthermore, the particle/PFU ratios indicated that glycan modifications did not diminish the infectious capability [22]. However, addition of the glycan chains per se was required for virus maturation [22].…”

Section: Oligosaccharides Of Semliki Forest Virus 57mentioning

confidence: 99%

“…These glycoproteins contained both Endo-H resistant and Endo-H sensitive forms [21,22]. About 80% of the Endo-H resistant side chains had the structure of Man3GlcNAc2 that are Endo-D sensitive The Endo-H sensitive oligosaccharides are mainly of the high mannose types, and have the structures Mans_sGlcNAc:.…”

Section: Oligosaccharides Of Semliki Forest Virus 57mentioning

confidence: 99%

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Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells

Naim

Koblet

1992

Archives of Virology

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The structure of the N-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo-beta-N-acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography. The results demonstrated that the glycoproteins decorating the virus shed from infected cells have N-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells. However, the E1 glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminal N-acetylglucosamine. The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at different N-glycosylation sites as in the case of E2, or at the same N-glycosylation site as in the case of E1. In the presence of 1-deoxymannojirimycin, no structural heterogeneities in the glycan chains were found. This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes.

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Genetic and Biochemical Studies on the Assembly of an Enveloped Virus

Tellinghuisen

Perera

Kühn

Genetic Engineering: Principles and Methods

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Investigation of the role of glycans for the biological activity of Semliki Forest virus grown in Aedes albopictus cells using inhibitors of asparagine-linked oligosaccharides trimming

Cited by 15 publications

References 54 publications

Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells

Genetic and Biochemical Studies on the Assembly of an Enveloped Virus

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