Dual Inhibition of Vacuolar-ATPase and TMPRSS2 Is Required for Complete Blockade of SARS-CoV-2 Entry into Cells

  1. Simoun Icho
  2. Edurne Rujas
  3. Krithika Muthuraman
  4. John Tam
  5. Huazhu Liang
  6. Shelby Landreth
  7. Mingmin Liao
  8. Darryl Falzarano
  9. Jean-Philippe Julien
  10. Roman A. Melnyk

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Abstract

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively.

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  1. Version published to 10.1128/aac.00439-22
  2. ScreenIT

    SciScore for 10.1101/2022.03.11.484006: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Vero cells (ATCC) were seeded in 96-well clear CellBind plates (Sigma Aldrich) 24 hours prior to the experiment at a density of 40,000 cells/well in a 100 μL volume of complete DMEM supplemented with 10% inactive FBS and 1% of penicillin/streptomycin.
    Vero
    suggested: None
    Briefly, plasmids expressing a lentiviral backbone encoding the luciferase reporter gene (BEI NR52516), the HIV structural and regulatory proteins Tat (BEI NR52518), Gag-pol (BEI NR52517) and Rev (BEI NR52519), and the full-length SARS-CoV-2 S protein were co-transfected into human kidney HEK293T cells (ATCC, CRL-3216) using the BioT transfection reagent (Bioland Scientific) following the manufacturer’s instructions.
    HEK293T
    suggested: None
    Neutralization was determined in a single-cycle neutralization assay using HeLa cells expressing ACE2 (HeLa-ACE2), kindly provided by D.R. Burton; The Scripps Research Institute, and Vero E6 cells constitutively expressing the transmembrane protease, serine 2 (Vero-TMPRSS2), obtained from the Centre For AIDS Reagents (National Institute for Biological Standards and Control) [51, 52].
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Vero E6
    suggested: RRID:CVCL_XD71)
    HeLa-ACE2 and Vero-TMPRSS2 cells were prepared following the above-mentioned protocol, where 10,000 cells/well of pre-seeded cells were co-cultured with the same serial dilutions of the sample compounds at 37°C for 2 hours.
    HeLa-ACE2
    suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)
    Authentic SARS-CoV-2 Neutralization and Titer Assay: Authentic SARS-CoV-2 experiments were conducted using Vero’76 (ATCC, CRL-1587) or Calu-3 (ATCC, HTB-55) cells.
    Vero’76
    suggested: None
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    The assay was performed using Vero-TMPRSS2 cells following the above protocol.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Recombinant DNA
    SentencesResources
    Similarly, the wild-type S plasmid was substituted with plasmids codifying for the B.1.117 or B.1.351 SARS-CoV-2 S proteins (kindly provided by David Ho, Columbia University) or B.1.1.529 (synthesized and cloned by GeneArt, LifeTechnologies into pcDNA3.4 expression vector) to generate the corresponding SARS-CoV-2 pseudoviral particle (PsV) variants.
    pcDNA3.4
    suggested: None
    Software and Algorithms
    SentencesResources
    Data was plotted using Prism 9 (GraphPad).
    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2022.03.11.484006 on bioRxiv