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

Taha Azad; Ragunath Singaravelu; Zaid Taha; Taylor R Jamieson; Stephen Boulton; Mathieu J F Crupi; Nikolas T Martin; Emily E F Fekete; Joanna Poutou; Mina Ghahremani; Adrian Pelin; Kazem Nouri; Reza Rezaei; Christopher Boyd Marshall; Masahiro Enomoto; Rozanne Arulanandam; Nouf Alluqmani; Reuben Samson; Anne-Claude Gingras; D William Cameron; Peter A Greer; Carolina S Ilkow; Jean-Simon Diallo; John C Bell

doi:10.1016/j.ymthe.2021.02.007

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.

Authors

Taha Azad¹, Ragunath Singaravelu¹, Zaid Taha¹, Taylor R Jamieson¹, Stephen Boulton¹, Mathieu J F Crupi¹, Nikolas T Martin¹, Emily E F Fekete¹, Joanna Poutou¹, Mina Ghahremani², Adrian Pelin¹, Kazem Nouri³, Reza Rezaei¹, Christopher Boyd Marshall³, Masahiro Enomoto³, Rozanne Arulanandam⁴, Nouf Alluqmani¹, Reuben Samson⁵, Anne-Claude Gingras⁵, D William Cameron¹, Peter A Greer⁶, Carolina S Ilkow¹, Jean-Simon Diallo¹, John C Bell⁷

Affiliations

¹ Center for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
² Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
³ Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada.
⁴ Center for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.
⁵ Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
⁶ Department of Pathology and Molecular Medicine, Queens University, Kingston, ON K7L 3N6, Canada.
⁷ Center for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada. Electronic address: [email protected].

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 / immunology
COVID-19 / virology
COVID-19 Drug Treatment
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