β-Coronaviruses use lysosomal organelles for cellular egress

  1. S Ghosh
  2. TA Dellibovi-Ragheb
  3. E Pak
  4. Q Qiu
  5. M Fisher
  6. PM Takvorian
  7. C Bleck
  8. V Hsu
  9. AR Fehr
  10. S Perlman
  11. SR Achar
  12. MR Straus
  13. GR Whittaker
  14. CAM de Haan
  15. G Altan-Bonnet
  16. N Altan-Bonnet

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Abstract

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that β-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.25.192310: (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 TGN46, Golgin 97
    TGN46
    suggested: None
    , Mannose 6-Phosphate Receptor, cathepsin (Abcam), SARS-CoV2 M (antibodies-online.com) and luminal epitope of LAMP1 (RND Systems) were purchased.
    cathepsin ( Abcam)
    suggested: (Abcam Cat# ab105369, RRID:AB_10865688)
    antibodies-online.com
    suggested: None
    LAMP1
    suggested: None
    Immunogold labelling was carried out on thawed sections with anti-GFP (2.5 mg/ml, rabbit, Rockland, 600-401-215) and anti-M (1:50, mouse) antibodies.
    anti-GFP
    suggested: (Biomol Cat# 600-401-215S, RRID:AB_2612814)
    anti-M
    suggested: None
    mouse
    suggested: None
    Mouse primary antibodies were detected with polyclonal rabbit anti-mouse immunoglobulin Gs (0.5 mg/ml).
    anti-mouse immunoglobulin
    suggested: None
    Cells were then immediately resuspended with ice-cold 2% PFA for 15 min, permeabilized with ice-cold 90% Methanol for 15 min, washed with FACS buffer and stained for phospho-ERK (E10 clone, Cell Signaling Technology) and anti-mouse secondary antibody (Jackson Immunochemicals).
    phospho-ERK
    suggested: (DB Biotech Cat# DB 013, RRID:AB_2315651)
    E10
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 cells were grown in Millicell EZ 8-well glass slides (Millipore) in infection media (EMEM, 4% FBS (Corning)) to a confluency of 90 – 100%.
    Vero E6
    suggested: None
    Upregulation of HLA-F open conformers upon MHV infection: HeLa-mCC1a cells were plated and infected (or not) with MHV for 24 hr.
    HeLa-mCC1a
    suggested: None
    . 5.104 HeLa cells were washed with Jurkat cell culture medium then resuspended with 5.104 Jurkat cells, spun at 100g for 15s and incubated at 37°C for 15min (some wells received only Jurkat cells or Jurkat cells and 1µMol of phorbol 12-myristate 13-acetate for negative and positive controls, respectively).
    HeLa
    suggested: None
    The levels of open HLA-F conformers were quantified by monitoring Jurkat cell activation (itself estimated geometric mean of phosphor-ERK staining in FSCint Jurkat cells).
    Jurkat
    suggested: TKG Cat# TKG 0209, RRID:CVCL_0065)
    Experimental Models: Organisms/Strains
    SentencesResources
    C57Bl6 Rag1-/- OT-1 TCR Transgenic mouse splenocytes were then harvested, cleared of their red blood cells by ACK lysis, added and spun onto macrophages (102 cells per well), and incubated for 6 hr.
    C57Bl6
    suggested: None
    Software and Algorithms
    SentencesResources
    Zen software (Carl Zeiss USA) and Image J (NIH) were used for all image analysis.
    Zen
    suggested: None
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Images were analyzed using Zen Blue software.
    Zen Blue
    suggested: (ZEN Digital Imaging for Light Microscopy, RRID:SCR_013672)
    Data were compensated and processed using FlowJo (TreeStar) and a custom-written Python pipeline (python.org).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Python
    suggested: (IPython, RRID:SCR_001658)
    Statistical Analysis: All graphs were plotted and unpaired two-tailed Student-t Test was performed using the GraphPad Prism 8 software or the SciPy Statistics library in Python. p values were considered significant for p< 0.05 unless otherwise indicated and denoted as */** and the corresponding values are mentioned in the figures.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    SciPy
    suggested: (SciPy, RRID:SCR_008058)

    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 32. 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 10.1101/2020.07.25.192310v1 on bioRxiv