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J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1368372


Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

Coronavirus/physiology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Liquid , Computational Biology/methods , Coronavirus/drug effects , Coronavirus Infections/veterinary , Gene Expression Regulation, Viral , Glycosylation/drug effects , Infectious bronchitis virus/physiology , Models, Molecular , Molecular Conformation , Molecular Weight , Neutralization Tests , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Poultry Diseases/virology , Protein Transport , Spectrometry, Mass, Electrospray Ionization , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Virus Replication/drug effects
Int J Biol Macromol ; 163: 1649-1658, 2020 Nov 15.
Article in English | MEDLINE | ID: covidwho-792418


The SARS-CoV-2 spike glycoproteins (SGPs) and human angiotensin converting enzyme 2 (ACE2) are the two key targets for the prevention and treatment of COVID-19. Host cell surface heparan sulfate (HS) is believed to interact with SARS-CoV-2 SGPs to facilitate host cell entry. In the current study, a series of polysaccharides from Saccharina japonica were prepared to investigate the structure-activity relationship on the binding abilities of polysaccharides (oligosaccharides) to pseudotype particles, including SARS-CoV-2 SGPs, and ACE2 using surface plasmon resonance. Sulfated galactofucan (SJ-D-S-H) and glucuronomannan (Gn) displayed strongly inhibited interaction between SARS-CoV-2 SGPs and heparin while showing negligible inhibition of the interaction between SARS-CoV-2 SGPs and ACE2. The IC50 values of SJ-D-S-H and Gn in blocking heparin SGP binding were 27 and 231 nM, respectively. NMR analysis showed that the structure of SJ-D-S-H featured with a backbone of 1, 3-linked α-L-Fucp residues sulfated at C4 and C2/C4 and 1, 3-linked α-L-Fucp residues sulfated at C4 and branched with 1, 6-linked ß-D-galacto-biose; Gn had a backbone of alternating 1, 4-linked ß-D-GlcAp residues and 1, 2-linked α-D-Manp residues. The sulfated galactofucan and glucuronomannan showed strong binding ability to SARS-CoV-2 SGPs, suggesting that these polysaccharides might be good candidates for preventing and/or treating SARS-CoV-2.

Coronavirus Infections/virology , Glucuronates/metabolism , Mannose/analogs & derivatives , Pneumonia, Viral/virology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Betacoronavirus/chemistry , Betacoronavirus/metabolism , Binding Sites , COVID-19 , Glucuronates/chemistry , Heparin/chemistry , Heparin/metabolism , Humans , Mannose/chemistry , Mannose/metabolism , Oligosaccharides/chemistry , Pandemics , Peptidyl-Dipeptidase A/metabolism , Phaeophyta/chemistry , Polysaccharides/chemistry , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Structure-Activity Relationship
Science ; 369(6501): 330-333, 2020 07 17.
Article in English | MEDLINE | ID: covidwho-187772


The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

Betacoronavirus/chemistry , Polysaccharides/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Binding Sites , COVID-19 , Coronavirus Infections , Glycopeptides/chemistry , Glycopeptides/immunology , Glycosylation , Humans , Mass Spectrometry , Models, Molecular , Oligosaccharides/chemistry , Pandemics , Pneumonia, Viral , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology