Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein

  1. Shijian Zhang
  2. Eden P. Go
  3. Haitao Ding
  4. Saumya Anang
  5. John C. Kappes
  6. Heather Desaire
  7. Joseph G. Sodroski

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Abstract

The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines.

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  1. Version published to 10.1128/jvi.01626-21
  2. ScreenIT

    SciScore for 10.1101/2021.04.01.438120: (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 variableCell lines: The wild-type SARS-CoV-2 S glycoprotein, with Asp 614, was inducibly expressed in Lenti-x-293T human female kidney cells from Takara Bio (Catalog #: 632180).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Samples were Western blotted with 1:2,000 dilutions of rabbit anti-SARS-Spike S1, mouse anti-SARS-Spike S1, rabbit anti-SARS-Spike S2 or a 1:5,000 dilution of mouse anti-β-actin as the primary antibodies.
    anti-SARS-Spike
    suggested: None
    anti-SARS-Spike S2
    suggested: None
    anti-β-actin
    suggested: None
    HRP-conjugated anti-rabbit or anti-mouse antibodies at a dilution of 1:5,000 were used as secondary antibodies in the Western blots.
    HRP-conjugated
    suggested: None
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    Beads were washed three times and samples were Western blotted with a mouse anti-S1 antibody.
    anti-S1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lenti-x-293T cells were grown in DMEM with 10% FBS supplemented with L-glutamine and Pen-Strep.
    Lenti-x-293T
    suggested: None
    On the day prior to transfection, 293T cells were seeded in 6-well plates at a density of 1 x 106/well.
    293T
    suggested: None
    Purification of the S glycoproteins: To express the SARS-CoV-2 S glycoprotein for purification, 293T-S cells were induced with 1 µg/ml doxycycline for two days.
    293T-S
    suggested: RRID:CVCL_LC70)
    Approximately 4 x 104 293T-ACE2 cells were then added to each well, and the cultures were maintained for an additional 24 h at 37°C before luciferase activity was measured.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Software and Algorithms
    SentencesResources
    The adjusted integrated volumes of S, S1 and S2 bands from unsaturated Western blots were calculated using Fiji ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Neutralization activity was calculated from the reduction in luciferase activity compared to controls, using GraphPad Prism 8 (GraphPad Software Inc.).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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 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.

  3. Version published to 10.1101/2021.04.01.438120v1 on bioRxiv