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.

Data availability

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.

Article and author information

Author details

  1. 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).
  2. 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).
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7887-6876
  3. 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).
  4. Xinheng Yu

    Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States
    Competing interests
    No competing interests declared.
  5. Kai Cheng

    Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States
    Competing interests
    No competing interests declared.
  6. Sheldon Park

    Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, United States
    Competing interests
    No competing interests declared.
  7. Wei-Chiao Huang

    Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, United States
    Competing interests
    No competing interests declared.
  8. Jonathan F Lovell

    Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9052-884X
  9. 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).
    ORCID icon "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.

Reviewing Editor

  1. Malcolm J McConville, The University of Melbourne, Australia

Version history

  1. Received: July 29, 2020
  2. Accepted: October 24, 2020
  3. Accepted Manuscript published: October 26, 2020 (version 1)
  4. Version of Record published: November 24, 2020 (version 2)

Copyright

© 2020, Yang et al.

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.

Metrics

  • 8,510
    Page views
  • 1,298
    Downloads
  • 123
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Qi Yang
  2. Thomas A Hughes
  3. Anju Kelkar
  4. Xinheng Yu
  5. Kai Cheng
  6. Sheldon Park
  7. Wei-Chiao Huang
  8. Jonathan F Lovell
  9. Sriram Neelamegham
(2020)
Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration
eLife 9:e61552.
https://doi.org/10.7554/eLife.61552

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Rui-Qiu Yang, Yong-Hong Chen ... Cheng-Gang Zou
    Research Article Updated

    An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Daniel Muñoz-Reyes, Levi J McClelland ... Maria Jose Sanchez-Barrena
    Research Article

    The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.