Ultrastructural insight into SARS-CoV-2 attachment, entry and budding in human airway epithelium

  1. Andreia L Pinto
  2. Ranjit K Rai
  3. Jonathan C Brown
  4. Paul Griffin
  5. James R Edgar
  6. Anand Shah
  7. Aran Singanayagam
  8. Claire Hogg
  9. Wendy S Barclay
  10. Clare E Futter
  11. Thomas Burgoyne

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Abstract

Ultrastructural studies of SARS-CoV-2 infected cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Many studies are limited by the lack of access to appropriate cellular models. As the airway epithelium is the primary site of infection it is essential to study SARS-CoV-2 infection of these cells. Here, we examined human airway epithelium, grown as highly differentiated air-liquid interface cultures and infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant (Variant of Concern 202012/01) by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Two key SARS-CoV-2 entry molecules, ACE2 and TMPRSS2, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistent with these observations, extracellular virions were frequently seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion at the apical plasma membrane demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein. Profiles strongly suggesting viral budding from the membrane was observed in these compartments and this may explain how virions gain their S glycoprotein containing envelope.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.10.439279: (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
    Antibodies against ACE2 (Abcam and Sigma-Aldrich), TMPRSS2 (Novus Biologicals), HA (Biolegend) and Myc (Santa Cruz), Ezrin (Santa Cruz) were used.
    ACE2
    suggested: (Abcam Cat# 3149-1, RRID:AB_2242331)
    TMPRSS2
    suggested: None
    HA
    suggested: (BioLegend Cat# 660001, RRID:AB_2563417)
    Myc (Santa Cruz), Ezrin (Santa Cruz)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Isolates were passaged twice in Vero cells before being used to infect HAE cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    HeLa cells and sections were permeabilised using 0.2% saponin in PBS for 20 mins at room temperature before incubating in blocking solution containing 0.02%, 1% BSA in PBS for 30 mins at room temperature.
    HeLa
    suggested: None
    Software and Algorithms
    SentencesResources
    Antibodies against ACE2 (Abcam and Sigma-Aldrich), TMPRSS2 (Novus Biologicals), HA (Biolegend) and Myc (Santa Cruz), Ezrin (Santa Cruz) were used.
    Novus Biologicals
    suggested: (Novus Biologicals, RRID:SCR_004286)
    Images were acquired using a Leica SP8 confocal and analysed using ImageJ.
    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 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 22. 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 10.1101/2021.04.10.439279v1 on bioRxiv