The D614G mutation redirects SARS-CoV-2 spike to lysosomes and suppresses deleterious traits of the furin cleavage site insertion mutation

  1. Chenxu Guo
  2. Shang-Jui Tsai
  3. Yiwei Ai
  4. Maggie Li
  5. Eduardo Anaya
  6. Andrew Pekosz
  7. Andrea Cox
  8. Stephen J. Gould

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress occurs by lysosomal exocytosis. We show that the Spike D614G mutation enhances Spike trafficking to lysosomes, drives Spike-mediated reprogramming of lysosomes, and reduces cell surface Spike expression by ~3-fold. D614G is not a human-specific adaptation. Rather, it is an adaptation to the earlier furin cleavage site insertion (FCSI) mutation that occurred at the genesis of SARS-CoV-2. While advantageous to the virus, furin cleavage of spike has deleterious effects on spike structure and function, inhibiting its trafficking to lysosomes and impairing its infectivity by the transmembrane serine protease 2(TMPRSS2)-independent, endolysosomal pathway. D614G restores spike trafficking to lysosomes and enhances the earliest events in SARS-CoV-2 infectivity, while spike mutations that restore SARS-CoV-2’s TMPRSS2-independent infectivity restore spike’s trafficking to lysosomes. Together, these and other results show that D614G is an intragenic suppressor of deleterious traits linked to the FCSI and lend additional support to the endolysosomal model of SARS-CoV-2 egress and entry.

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  1. Version published to 10.1126/sciadv.ade5085
  2. ScreenIT

    SciScore for 10.1101/2020.12.08.417022: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Following Johns Hopkins Medicine Institutional Review Board (IRB) approval, plasma samples were obtained under informed consent from healthy donors prior to the COVID-19 pandemic (JHM IRB NA_0004638) as described (Cox et al., 2005).
    Consent: Following Johns Hopkins Medicine Institutional Review Board (IRB) approval, plasma samples were obtained under informed consent from healthy donors prior to the COVID-19 pandemic (JHM IRB NA_0004638) as described (Cox et al., 2005).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ThermoFisher was the source for rabbit antibodies directed against LAMTOR1 (#8975S), ERGIC3 (#16029-1-AP), ERGIC53 (#13364-1-AP), and calnexin (#PA5-34665) and for mouse monoclonal antibodies to EEA1 (#48453) and Lamp2 (MA1-205).
    LAMTOR1
    suggested: None
    ERGIC3
    suggested: (Proteintech Cat# 16029-1-AP, RRID:AB_2098446)
    #16029-1-AP
    suggested: (Proteintech Cat# 16029-1-AP, RRID:AB_2098446)
    ERGIC53
    suggested: None
    #13364-1-AP
    suggested: (Proteintech Cat# 13364-1-AP, RRID:AB_2135994)
    calnexin ( #PA5-34665 )
    suggested: None
    EEA1
    suggested: None
    Lamp2
    suggested: (Thermo Fisher Scientific Cat# MA1-205, RRID:AB_2662613)
    Rabbit antibodies to mTOR were obtained from Cell Signaling (#2972S)
    mTOR
    suggested: (Cell Signaling Technology Cat# 2972, RRID:AB_330978)
    Mouse monoclonal antibodies to GM130 (#610822) and CD81 (#555675) were obtained from BD.
    GM130
    suggested: (BD Biosciences Cat# 610822, RRID:AB_398141)
    CD81
    suggested: (BD Biosciences Cat# 555675, RRID:AB_396028)
    Mouse monoclonal antibody directed against CD9 (#312102) was obtained from BioLegend.
    CD9
    suggested: (BioLegend Cat# 312102, RRID:AB_314907)
    Fluorescently labeled secondary antibodies specific for human Igs (IgG, IgM, and IgA; pan Ig), human IgG, rabbit IgG, or mouse IgG were obtained from Jackson ImmunoResearch
    human Igs ( IgG
    suggested: None
    human IgG
    suggested: None
    rabbit IgG
    suggested: None
    mouse IgG
    suggested: None
    Nihal Altan-Bonnet for generous gift of the anti-BiP/GRP78 antibodies, the Lamp1 monoclonal, and polyclonal antibody to the KDEL receptor.
    anti-BiP/GRP78
    suggested: None
    Lamp1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines, cell culture, transfections: HEK293 cells (ATCC) were cultured in complete medium (DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin solution (10,000 units/ml)).
    HEK293
    suggested: None
    The clinical isolates SARS-CoV-2/USA/MD-HP00076/2020 (Spike D614; GenBank: MT509475.1) and SARS-Cov-2/USA/DC-HP00007/2020 (Spike G614; GenBank: MT509464.1) were isolated using published procedures (Gniazdowski et al., 2020) and virus stocks were grown on VeroE6TMPRSS2 cells.
    VeroE6TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    Images were assembled into figures using Adobe Illustrator.
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    Quantitative analysis of image files was performed using ImageJ and proprietary software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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 10.1101/2020.12.08.417022v1 on bioRxiv