Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

  1. Yuan-Lin Kang
  2. Yi-ying Chou
  3. Paul W. Rothlauf
  4. Zhuoming Liu
  5. Timothy K. Soh
  6. David Cureton
  7. James Brett Case
  8. Rita E. Chen
  9. Michael S. Diamond
  10. Sean P. J. Whelan
  11. Tom Kirchhausen

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Abstract

The membrane fusion proteins of viral pathogens as diverse in their replication strategies as coronaviruses and filoviruses depend, for their functional activity, on proteolytic processing during cell entry. Endosomal cathepsins carry out the cleavages. We have constructed chimeric forms of vesicular stomatitis virus (VSV) bearing the fusion proteins of Zaire ebolavirus (ZEBOV) or SARS coronavirus 2 (SARS-CoV-2) and shown that two small-molecule inhibitors of an endosomal lipid kinase (PIKfyve) inhibit viral infection by preventing release of the viral contents from endosomes. Both inhibitory compounds cause distension of Rab5 and Rab7 subcompartments into small vacuoles. One of them (Apilimod) also inhibits infection of cells by authentic SARS-CoV-2. The results point to possibilities for host targets of antiviral drugs.

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  1. Version published to 10.1073/pnas.2007837117
  2. Version published to 10.1101/2020.04.21.053058v3 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.04.21.053058: (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
    Plates were washed and sequentially incubated with 1 µg/mL of CR3022 anti-spike antibody (51) and HRP-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA.
    anti-spike
    suggested: None
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero C1008 [Vero 76, clone E6, Vero E6] (ATCC CRL-1586) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, and penicillin and streptomycin.
    Vero C1008
    suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
    Virus was passaged once in Vero CCL81 cells (ATCC) and titrated by focus-forming assay also on Vero E6 cells.
    Vero CCL81
    suggested: None
    Vero E6 cell monolayers were pretreated for 1 h at 37°C with serial dilutions of Apilimod at the indicated concentrations.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Viruses: Recombinant VSV (Indiana serotype) expressing MeGFP alone which initiates fusion at pH<6.2 (VSV-MeGFP) (45) (or in combination with V269H GP, VSV-MeGFP-V269H), RABV GP (VSV-MeGFP-RABV) (46), LASV GP (VSV-MeGFP-LASV) (7), LCMV GP (VSV-MeGFP-LCMV), Zaire EBOV GP (VSV-MeGFP-ZEBOV) (47) or SARS-CoV-2 S Wuhan-Hu-1 strain (VSV-eGFP-SARS-CoV-2 – description to be published elsewhere) were used for infection, entry and live cell imaging assays.
    SARS-CoV-2 S Wuhan-Hu-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Fluorescent intensity from 20,000 single cells from a single round of infection was determined by flow cytometry using a BD FACSCanto™ II equipped with DIVA software package.
    DIVA
    suggested: None
    The percentage of GFP cells was determined using FlowJo software (Tree Star Industries, Ashland, OR).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Data were processed using Prism software (GraphPad Prism 8.0) and viral titers are reported as percent inhibition relative to mock-treated SARS-CoV-2 infected cells.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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.
    • No protocol registration statement was detected.

    About SciScore

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  4. Version published to 10.1101/2020.04.21.053058v2 on bioRxiv
  5. Version published to 10.1101/2020.04.21.053058v1 on bioRxiv