Effect of mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell-cell fusion function

  1. Chelsea T. Barrett
  2. Hadley E. Neal
  3. Kearstin Edmonds
  4. Carole L. Moncman
  5. Rachel Thompson
  6. Jean M. Branttie
  7. Kerri Beth Boggs
  8. Cheng-Yu Wu
  9. Daisy W. Leung
  10. Rebecca E. Dutch

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Abstract

The SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S cell-cell fusion. Additionally, we examine S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this highly sought-after therapeutic target.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.24.428007: (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
    A549 and human colon carcinoma LoVo cells (both purchased from ATCC) were cultured in F12 Kaighns Modification media (GE Healthcare) with 10% FBS and 1% penicillin/streptomycin. Plasmids, Antibodies, and Mutagenesis: pCAGGS-SARS-CoV-2 spike was obtained from BEI Resources.
    A549
    suggested: None
    Antibodies anti-SARS spike glycoprotein (ab252690) and anti-hACE2 (ab15348) were purchased from Abcam, and anti-TMPRSS2 (H-4) was purchased from Santa Cruz Biotechnology, Inc. Gel electrophoresis and western blotting: Proteins were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
    anti-SARS spike glycoprotein
    suggested: None
    anti-hACE2
    suggested: None
    After blocking with 5% milk in tris-buffered saline + Tween-20 (tTBS) for 1 hour, membranes were incubated with respective antibodies (anti-SARS S 1:5000 dilution, anti-TMPRSS2 1:1000 dilution, anti-hACE2 1:1000 dilution) at 4°C overnight.
    anti-TMPRSS2
    suggested: None
    Cell lysates were centrifuged at 55,000 rpm for 10 minutes, and the supernatant was incubated with anti-SARS S polyclonal antibody at 4°C for three hours.
    anti-SARS
    suggested: None
    Cells were labeled with anti-SARS S antibody (1:2000 dilution) in blocking buffer overnight at 4°C or for three to five hours at room temperature.
    anti-SARS S
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549 and human colon carcinoma LoVo cells (both purchased from ATCC) were cultured in F12 Kaighns Modification media (GE Healthcare) with 10% FBS and 1% penicillin/streptomycin. Plasmids, Antibodies, and Mutagenesis: pCAGGS-SARS-CoV-2 spike was obtained from BEI Resources.
    LoVo
    suggested: None
    Effector cells (Vero or A549s) were plated in 12-well plates at 70-90% confluency and transfected with 1μg of total DNA (0.4μg of a T7 promoted luciferase plasmid, 0.6μg of wild-type (wt) or mutant S protein or S protein with additional proteases).
    Vero
    suggested: None
    Time Course Immunoprecipitation: 2μg of wt or mutant S was transfected into Vero or A549 cells using the Lipofectamine 3000 system (Invitrogen; ratios described above).
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    A549 and human colon carcinoma LoVo cells (both purchased from ATCC) were cultured in F12 Kaighns Modification media (GE Healthcare) with 10% FBS and 1% penicillin/streptomycin. Plasmids, Antibodies, and Mutagenesis: pCAGGS-SARS-CoV-2 spike was obtained from BEI Resources.
    Mutagenesis
    suggested: None
    Bands were quantified using band densitometry using the ImageQuant software (GE Healthcare).
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    Slides were imaged on an Axiovert 200M (Zeiss) at 63x magnification using Metamorph to collect Z-stacks and processed using Nikon NIS Elements.
    Metamorph
    suggested: None
    Statistical analysis: Statistical analysis was performed using Prism 7 for Windows (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

  2. Version published to 10.1101/2021.01.24.428007 on bioRxiv