Dataset Information

Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV.

ABSTRACT: The spike (S) glycoprotein of coronaviruses is known to be essential in the binding of the virus to the host cell at the advent of the infection process. To study the maturation pathway of the S glycoprotein of the severe acute respiratory syndrome (SARS)-coronavirus (CoV) within the host cell, a T7/vaccinia virus-based expression system coupled to immunoprecipitation with anti-S antibodies was used to test and analyze different forms of the S glycoprotein. The state of maturity of the S glycoprotein can be deduced from its sensitivity to hydrolysis by endoglycosidase H (EndoH) or N-glycosidase F (N-Gly F). A fully matured S glycoprotein will be modified with complex oligosaccharides which makes it resistant to cleavage by EndoH but not by N-Gly F. By exploiting this characteristic, it is then possible to determine which forms of the immunoprecipitated S protein are properly processed by the host cell. With this system, many different constructs of the S glycoprotein can be analyzed in parallel thus providing another method by which to study the functional domains of S involved in membrane fusion event that occurs during viral infection.

SUBMITTER: Shen S

PROVIDER: S-EPMC7120769 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV.

Shen Shuo S Tan Timothy H P TH Tan Yee-Joo YJ

Methods in molecular biology (Clifton, N.J.) 20070101

The spike (S) glycoprotein of coronaviruses is known to be essential in the binding of the virus to the host cell at the advent of the infection process. To study the maturation pathway of the S glycoprotein of the severe acute respiratory syndrome (SARS)-coronavirus (CoV) within the host cell, a T7/vaccinia virus-based expression system coupled to immunoprecipitation with anti-S antibodies was used to test and analyze different forms of the S glycoprotein. The state of maturity of the S glycopr ...[more]

PMID: 17502675

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Project description:The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E, and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

Dataset Information

Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV.

Publications

Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV.

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