Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

  1. Liping Zhang
  2. Matthew Mann
  3. Zulfeqhar Syed
  4. Hayley M. Reynolds
  5. E Tian
  6. Nadine L. Samara
  7. Darryl C. Zeldin
  8. Lawrence A. Tabak
  9. Kelly G. Ten Hagen

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Abstract

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a unique furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation. Here, we show that O-glycosylation near the furin cleavage site is mediated by specific members of the GALNT enzyme family and is dependent on the novel proline at position 681 (P681). We further demonstrate that O-glycosylation of S decreases furin cleavage. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the potential role of P681 mutations in the recently identified, highly transmissible B.1.1.7 variant.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.05.429982: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membranes were blocked with Odyssey Blocking Buffer (PBS-based) (Li-COR), then incubated with anti-V5 antibody (dilution 1:1000, Invitrogen) or anti-S1 antibody (dilution 1:1000, GeneTex) overnight at 4 °C.
    anti-V5
    suggested: None
    anti-S1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expression of Spike and GALNTs in Drosophila S2R+ cells/Vero E6 cells and western blotting: The plasmids pIB-spike-V5 with or without pIB-GALNTs-FLAG were transfected to S2R+ cells (DGRC) using Effectene transfection reagent (Qiagen) according to the instructions.
    E6
    suggested: None
    For expression of Spike in Vero E6 cells (ATCC), the cells were transfected with pcDNA3.1-spike-V5 with or without pcDNA3.1-GALNTs-FLAG (Genscript) using Lipofectamine 3000 reagent (Invitrogen) according to the instructions.
    Vero E6
    suggested: None
    Enzyme assays: Expression of recombinant GALNTs was performed either using Pichia pastoris or COS7 cells as described previously and referenced in the main text.
    COS7
    suggested: CLS Cat# 605470/p532_COS-7, RRID:CVCL_0224)
    Software and Algorithms
    SentencesResources
    S), human ACE2 and GALNTs: The codon optimized cDNA of full-length spike (Genscript) was amplified by PCR and digested by HindIII and NotI, then cloned into pIB/V5-His vector (Invitrogen), fused with V5 epitope at the C-terminus.
    GALNTs
    suggested: None
    Images were processed using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    We subset the data for the Epithelial cell compartment from the upper and lower airways atlas and the expression of GALNTs and ACE2 is illustrated as dot plots using the R ggplot2 (v.3.3.2) library.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Individual dot plots for upper and lower airways were assembled in Adobe Illustrator (v.24.3) to generate a final figure.
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)

    Results 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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.02.05.429982 on bioRxiv