Functional properties of the spike glycoprotein of the emerging SARS-CoV-2 variant B.1.1.529

  1. Qian Wang
  2. Saumya Anang
  3. Sho Iketani
  4. Yicheng Guo
  5. Lihong Liu
  6. Phinikoula S. Katsamba
  7. Lawrence Shapiro
  8. David D. Ho
  9. Joseph G. Sodroski

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Abstract

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  1. Version published to 10.1016/j.celrep.2022.110924
  2. ScreenIT

    SciScore for 10.1101/2021.12.27.474288: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The study protocol was approved by the CUIMC Institutional Review Board (IRB) and all participants provided written informed consent.
    Consent: The study protocol was approved by the CUIMC Institutional Review Board (IRB) and all participants provided written informed consent.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cell lysates and virions were analyzed by Western blotting with the following primary antibodies: rabbit anti-SARS-Spike S1 (Sino Biological, Cat# 40591-T62), rabbit anti-SARS-Spike S2 (Sino Biological, Cat# 40590-T62), rabbit anti-p55/p24/p17 (Abcam, Cambridge, MA (Cat# ab63917)), mouse anti-VSV NP (Millipore, Burlington, MA (Cat# MABF2348)) or mouse anti-GAPDH (Millipore, Cat# CB1001).
    anti-SARS-Spike
    suggested: None
    anti-SARS-Spike S2
    suggested: None
    anti-p55/p24/p17
    suggested: None
    anti-VSV NP
    suggested: None
    anti-GAPDH
    suggested: None
    The Western blots were developed with the following secondary antibodies: HRP-conjugated anti-rabbit antibody (Cytiva, Marlborough, MA (NA934-1ML)) or HRP-conjugated goat antimouse antibody (Jackson ImmunoResearch, West Grove, PA (Cat# 115-035-008)).
    anti-rabbit
    suggested: (GE Healthcare Cat# NA934, RRID:AB_772206)
    antimouse
    suggested: (C. Birchmeier - Max Delbruck Center for Molecular Medicine, Berlin, Germany Cat# Guinea pig anti-mouse Lbx1 polyclonal antibody, RRID:AB_2532144)
    S1, S2 and NP were detected as described above; huACE2-Fc bound to the pseudovirus particles was detected with Peroxidase AffiniPure goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch).
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: HEK293T, 293T-ACE2 (BEI), Vero-E6, and COS-1 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml penicillin-streptomycin (Thermo Fisher Scientific, Cambridge, MA).
    COS-1
    suggested: None
    Expi293 cells were maintained in suspension culture directly in Expi293TM Expression Medium, supplemented with penicillin and streptomycin, and were incubated at 37°C in a humidified atmosphere of 8% CO2 in air and on a shaker platform rotating at 125 rpm.
    Expi293
    suggested: RRID:CVCL_D615)
    Vero-E6 and COS-1 are from African green monkey kidneys.
    Vero-E6
    suggested: None
    S glycoprotein expression, processing and incorporation into pseudovirus particles: HEK293T cells were transfected to produce VSV- and HIV-based particles pseudotyped with SARS-CoV-2 S glycoprotein variants, as described above.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    To measure the infectivity of pseudovirus variants on target cells expressing different levels of human ACE2, serial dilutions (from 2 μg to 24.7 ng) of the human ACE2 expressor plasmid (Addgene, Watertown, MA (Cat# 1786)) were transfected into 293T cells in 12-well plates using 1 mg/ml PEI.
    293T
    suggested: None
    Approximately 3 x 104 target cells (Vero-E6 or 293T-ACE2 cells) per well were then added.
    293T-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    METHOD DETAILS: Plasmid constructs: The codon-optimized SARS-CoV-2 spike (S) gene (Sino Biological, Wayne, PA) encoding the S glycoprotein lacking 18 amino acids at the carboxyl terminus was cloned into the pCMV3 vector.
    pCMV3
    suggested: RRID:Addgene_161029)
    Then, 7.5 μg of SARS-CoV-2 S glycoprotein expressor plasmid and 7.5 μg pHIV-1NL4-3ΔEnv-NanoLuc reporter construct were cotransfected into the HEK293T cells using Polyethylenimine (Polysciences).
    pHIV-1NL4-3ΔEnv-NanoLuc
    suggested: None
    Software and Algorithms
    SentencesResources
    S, S1, and S2 band intensities from unsaturated Western blots were calculated using ImageJ Software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The concentrations of huACE2-Fc that achieved an OD450 of 0.8 and and the serum endopoint dilution that achieved an OD450 value > 3-fold over background were calculated by fitting the data in five-parameter dose-response curves in GraphPad Prism 9 (GraphPad Software Inc.
    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.
    • Thank you for including a protocol registration statement.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.12.27.474288v1 on bioRxiv