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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SciScore for 10.1101/2020.10.15.339838: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: All cells were mycoplasma negative. Table 2: Resources
Antibodies Sentences Resources Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R&D Systems (Minneapolis, MA) Goat anti-human ACE2 polyclonal antibodysuggested: Noneanti-human ACE2suggested: NoneThese studies used Alexa-647 conjugated anti-ACE2 (MAB9332, R&D systems) and anti-RBD (Sino Biologicals) antibodies. anti-ACE2suggested: Noneanti-RBDsuggested: NoneExperimental Models: Cell Lines Sentence… SciScore for 10.1101/2020.10.15.339838: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: All cells were mycoplasma negative. Table 2: Resources
Antibodies Sentences Resources Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R&D Systems (Minneapolis, MA) Goat anti-human ACE2 polyclonal antibodysuggested: Noneanti-human ACE2suggested: NoneThese studies used Alexa-647 conjugated anti-ACE2 (MAB9332, R&D systems) and anti-RBD (Sino Biologicals) antibodies. anti-ACE2suggested: Noneanti-RBDsuggested: NoneExperimental Models: Cell Lines Sentences Resources Stable HEK293T/ACE2 cells were kindly provided by Michael Farzan (Scripps Research, Jupiter, FL). HEK293T/ACE2suggested: NoneCRISPR-Cas9 Knockout cell lines: HEK293T CRISPR-Cas9 cells were generated by transfecting wild-type 293T cells with pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene, Plasmid# 42230) carrying single-guide RNA (sgRNA) targeting either the Core-1 β3Gal-T (C1GalT1, target site: GCAGATTCTAGCCAACATAA) or β1,2 GlcNAc-transferase (MGAT1, GTGGGGCGCTATCCTCTTTGTGG). 293Tsuggested: NoneSpike-mutant that were produced in either: a. wild-type HEK293T, b. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Experimental Models: Organisms/Strains Sentences Resources Molecular biology: A plasmid containing the Spike protein (2019-nCov_pcDNA3.1(+)-P2A-eGFP [v1]) was kindly provided by Dr. Haisheng Yu (Institute of Laboratory Animal Science, Peking Union Medical College). +)-P2A-eGFP [ v1]suggested: NoneSoftware and Algorithms Sentences Resources Unless mentioned otherwise, all other reagents were from either Thermo-Fisher or Sigma Chemicals. Thermo-Fisher or Sigmasuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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