Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein

J Virol. 2022 Feb 9;96(3):e0162621. doi: 10.1128/JVI.01626-21. Epub 2021 Nov 24.

Abstract

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.

Keywords: COVID-19; Golgi; SARS-CoV-2; coronavirus; disulfide; glycosylation; membrane protein; spike glycoprotein; viral protein; virus entry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Neutralizing / immunology
  • COVID-19 / virology*
  • Disulfides
  • Gene Expression Regulation, Viral
  • Glycosylation
  • Humans
  • Models, Molecular
  • Neutralization Tests
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Protein Transport
  • Recombinant Proteins
  • SARS-CoV-2 / physiology*
  • Spike Glycoprotein, Coronavirus / chemistry
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / isolation & purification
  • Spike Glycoprotein, Coronavirus / metabolism*
  • Structure-Activity Relationship

Substances

  • Antibodies, Neutralizing
  • Disulfides
  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • spike glycoprotein, SARS-CoV