Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

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Abstract

The Spike protein of SARS-CoV-2, its receptor binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

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All data generated or analysed during this study are included in the manuscript and supporting files. All plasmids generated by the authors will be deposited at Addgene.

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Qi Yang

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
Qi Yang, Co-author of a provisional patent application.(63/079,667).
Thomas A Hughes

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
Thomas A Hughes, Co-author of a provisional patent application.(63/079,667).

"This ORCID iD identifies the author of this article:" 0000-0002-7887-6876
Anju Kelkar

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
Anju Kelkar, Co-author of a provisional patent application.(63/079,667).
Xinheng Yu

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
No competing interests declared.
Kai Cheng

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
No competing interests declared.
Sheldon Park

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
No competing interests declared.
Wei-Chiao Huang

Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
No competing interests declared.
Jonathan F Lovell

Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-9052-884X
Sriram Neelamegham

Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States

For correspondence
neel@buffalo.edu

Competing interests
Sriram Neelamegham, Co-author of a provisional patent application.(63/079,667).

"This ORCID iD identifies the author of this article:" 0000-0002-1371-8500

Funding

National Institutes of Health (HL103411)

Sriram Neelamegham

National Institutes of Health (GM133195)

Sriram Neelamegham

National Institutes of Health (GM126537)

Sriram Neelamegham

National Institutes of Health (GM139160)

Sheldon Park
Sriram Neelamegham

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.