Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry

Mol Ther. 2021 Jun 2;29(6):1984-2000. doi: 10.1016/j.ymthe.2021.02.007. Epub 2021 Feb 10.

Abstract

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Keywords: 2019-nCoV; COVID-19; SARS-CoV-2; bioluminescence; bioreporter; coronavirus; high-throughput screening; viral entry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2 / antagonists & inhibitors
  • Angiotensin-Converting Enzyme 2 / chemistry*
  • Angiotensin-Converting Enzyme 2 / genetics
  • Angiotensin-Converting Enzyme 2 / immunology
  • Antibodies, Neutralizing / pharmacology*
  • Asparagine / chemistry
  • Asparagine / metabolism
  • Binding Sites
  • Biological Assay*
  • COVID-19 / diagnosis
  • COVID-19 / drug therapy
  • COVID-19 / immunology
  • COVID-19 / virology
  • Genes, Reporter
  • Glycosylation / drug effects
  • HEK293 Cells
  • Host-Pathogen Interactions / drug effects
  • Host-Pathogen Interactions / genetics
  • Humans
  • Lectins / pharmacology*
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Measurements
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Secondary
  • Receptors, Virus / antagonists & inhibitors
  • Receptors, Virus / chemistry*
  • Receptors, Virus / genetics
  • Receptors, Virus / immunology
  • SARS-CoV-2 / drug effects
  • SARS-CoV-2 / growth & development
  • SARS-CoV-2 / immunology
  • Spike Glycoprotein, Coronavirus / antagonists & inhibitors
  • Spike Glycoprotein, Coronavirus / chemistry*
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology
  • Virus Internalization / drug effects

Substances

  • Antibodies, Neutralizing
  • Lectins
  • Receptors, Virus
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • Asparagine
  • Luciferases
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2