The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2

  1. Helena Winstone
  2. Maria Jose Lista
  3. Alisha C. Reid
  4. Clement Bouton
  5. Suzanne Pickering
  6. Rui Pedro Galao
  7. Claire Kerridge
  8. Katie J. Doores
  9. Chad M. Swanson
  10. Stuart J. D. Neil

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Abstract

The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell.

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  1. Version published to 10.1128/jvi.02422-20
  2. ScreenIT

    SciScore for 10.1101/2020.12.19.423592: (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
    Following incubation, cells were washed with 1X PBS and incubated with secondary antibody, goat anti-human IgG (Fc) peroxidase conjugate (Sigma A0170) for 45 min.
    anti-human IgG
    suggested: (Sigma-Aldrich Cat# A0170, RRID:AB_257868)
    Membranes were blocked in milk prior to detection with specific antibodies: 1:1000 ACE2 rabbit (Abcam, Ab108209), 1:1000 TMPRSS2 rabbit (Abcam, Ab92323), 1:2000 Actin mouse (Abcam, Ab6276), 1:5000 GAPDH rabbit (Abcam, Ab9485), 1:5000 HSP90 mouse (Genetex, Gt×109753), 1:50 HIV-1 p24Gag mouse (Chesebro, Wehrly, Nishio, & Perryman, 1992), 1:1000 Spike mouse (Genetex, Gtx632604), 1:3000 IFITM1 mouse (Proteintech, 60074-1-Ig), 1:3000 IFITM2 rabbit (Proteintech, 12769-1AP), 1:3000 IFITM3 rabbit (Proteintech, 11714-1-AP).
    ACE2
    suggested: None
    Ab108209
    suggested: (Abcam Cat# ab108209, RRID:AB_10862654)
    Actin
    suggested: (Abcam Cat# ab6276, RRID:AB_2223210)
    Ab6276
    suggested: (Abcam Cat# ab6276, RRID:AB_2223210)
    GAPDH
    suggested: (Abcam Cat# ab9485, RRID:AB_307275)
    Ab9485
    suggested: (Abcam Cat# ab9485, RRID:AB_307275)
    HIV-1 p24Gag mouse (Chesebro, Wehrly, Nishio,
    suggested: None
    Perryman, 1992
    suggested: None
    IFITM2
    suggested: (Proteintech Cat# 12769-1-AP, RRID:AB_2122089)
    IFITM3
    suggested: (Proteintech Cat# 11714-1-AP, RRID:AB_2295684)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and plasmids: 293T-17 (ATCC), A549-ACE2, A549-ACE2-TMPRSS2, Calu3 (ATCC), and A549-ACE2 expressing the individual IFITM proteins were cultured in DMEM (Gibco) with 10% FBS (Invitrogen) and 200ug/ml Gentamicin (Sigma).
    Calu3
    suggested: RRID:CVCL_EQ19)
    Mutants of spikes or IFITMs were generated with Q5® Site-Directed Mutagenesis Kit (E0554) following the manufacturers instructions: SARS-CoV-2 spike ΔPRRA (AGAAGCGTGGCCAGCCAG, GCTATTGGTCTGGGTCTGGTAG), SARS-CoV-1 spike PRRA (AGAGCCCGGAGCACCAGCCAGAAA, TCTAGGCAGCAGAGACACGGTGTG), IFITM2 Y19A (GCCTCCCAACgctGAGATGCTCAAGGAGGAG, TGGCCGCTGTTGACAGGA), IFITM2 Y19F (GCCTCCCAACtttGAGATGCTCAAGGAG, TGGCCGCTGTTGACAGGA) A549 stable cell lines expressing ACE2 (pMIGR1-puro), TMPRSS2 (IRES-neo.
    A549
    suggested: None
    Passage of SARS-CoV-2: PHE England strain 2 was propagated in Vero-E6 cells and titred by standard method.
    Vero-E6
    suggested: None
    Infection with replication competent SARS-CoV-2: 1.5×105 A549-ACE2 cells were infected for 1h at 37°C with SARS-CoV-2 replication competent with an MOI of 0.005.
    A549-ACE2
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

  3. Version published to 10.1101/2020.12.19.423592 on bioRxiv