Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin

  1. Tiffany Tang
  2. Javier A. Jaimes
  3. Miya K. Bidon
  4. Marco R. Straus
  5. Susan Daniel
  6. Gary R. Whittaker

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2’ site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.10.04.325522: (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
    SARS-CoV-2 and SARS-CoV S was detected using a rabbit polyclonal antibody that recognizes the S2 domain (Cat: 40590-T62, Sinobiological) and an AlexaFluor 488 goat anti-rabbit secondary anti-body.
    SARS-CoV-2
    suggested: None
    anti-rabbit
    suggested: None
    MLV protein was detected using a mouse monoclonal antibody that recognizes the MLV p30 protein (Cat: ab130757, abcam) and an AlexaFluor 488 goat antimouse secondary antibody.
    antimouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, plasmids, and reagents: HEK293T (accession no: CRL-11268, ATCC), Vero E6 (accession no: CRL-1586, ATCC), and Calu-3 (accession no: HTB-55) cells were maintained at 37°C and 5% CO2 incubator and cultivated in Dulbecco’s Modified Eagle Medium (Cellgro) supplemented with 10% Hyclone FetalClone II (GE) and 10 mM HEPES (Cellgro) (DMEMc).
    Vero E6
    suggested: None
    Briefly, HEK293T cells were seeded to 30% confluency.
    HEK293T
    suggested: None

    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 10.1101/2020.10.04.325522 on bioRxiv