SARS-CoV-2 infects cells following viral entry via clathrin-mediated endocytosis

  1. Armin Bayati
  2. Rahul Kumar
  3. Vincent Francis
  4. Peter S. McPherson

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Abstract

With more than 51 million cases and 1.3 million deaths, and with the resulting social upheaval, the COVID-19 pandemic presents one of the greatest challenges ever to human society. It is thus vital to fully understand the biology of SARS-CoV-2, the causative agent of COVID-19. SARS-CoV-2 uses its spike glycoprotein to interact with the cell surface as a first step in the infection process. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, we now demonstrate that following engagement with the plasma membrane, SARS-CoV-2 undergoes rapid clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system, and importantly clathrin-heavy chain knockdown, which blocks clathrin-mediated endocytosis, reduces viral infectivity. This discovery reveals important new information about the basic biology of SARS-CoV-2 infectivity.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.13.201509: (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 AuthenticationContamination: Cell lines were monthly checked for mycoplasma contamination using the mycoplasma detection kit (biotool cat# B39038).

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: SARS-Cov-2 spike protein antibody is from GeneTex (GTX632604).
    Antibodies: SARS-Cov-2 spike protein antibody
    suggested: None
    Antibodies: SARS-Cov-2 spike protein
    suggested: None
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    ACE2 antibody is from GeneTex (GTX01160).
    ACE2
    suggested: None
    GTX01160
    suggested: None
    GAPDH antibody is from OriGene (TA802519).
    GAPDH
    suggested: (OriGene Cat# TA802519, RRID:AB_2626378)
    TA802519
    suggested: (OriGene Cat# TA802519, RRID:AB_2626378)
    Conjugated Transferrin antibody is from ThermoFisher Scientific (T23366)
    T23366
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: HEK-293T and A549 cell lines were obtained from ATCC (CRL-1573,
    HEK-293T
    suggested: None
    A549
    suggested: None
    HEK-293T-ACE2 stable cell line was obtained from Dr. Jesse D. Bloom (Crawford et. al., 2020).
    HEK-293T-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Quantification: Internalization of purified spike: Image J was used to measure the fluorescence intensity.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Statistics: Graphs were prepared using GraphPad Prism 6 software.
    GraphPad Prism
    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 35. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2020.07.13.201509 on bioRxiv