Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

  1. Mizuki Yamamoto
  2. Jin Gohda
  3. Ayako Kobayashi
  4. Keiko Tomita
  5. Youko Hirayama
  6. Naohiko Koshikawa
  7. Motoharu Seiki
  8. Kentaro Semba
  9. Tetsu Akiyama
  10. Yasushi Kawaguchi
  11. Jun-ichiro Inoue

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Abstract

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

Author Summary

To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

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  1. ScreenIT

    SciScore for 10.1101/2021.12.14.472513: (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

    Antibodies
    SentencesResources
    The primary antibodies used were rabbit anti-ACE2 (1:1000, ab15348, Abcam)
    anti-ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    , rabbit anti-TACE (1:1000, 3976S, Cell Signaling Technology, MA, USA), rabbit anti-ADAM10 (1:1000, 14194S, Cell Signaling Technology), rabbit anti-Flag-tag (1:1000, PM020, MBL, MA, USA)
    anti-TACE
    suggested: (Leinco Technologies Cat# T460, RRID:AB_2831960)
    anti-ADAM10
    suggested: (LSBio (LifeSpan Cat# LS-C122598-1000, RRID:AB_10800328)
    anti-Flag-tag
    suggested: None
    , mouse anti-tubulin (1:1000, CP06, Millipore), and mouse anti-VSVM (1:1000, 23H12, Absolute antibody).
    anti-tubulin
    suggested: (LSBio (LifeSpan Cat# LS-C76623-1000, RRID:AB_10633290)
    CP06
    suggested: (Millipore Cat# CP06, RRID:AB_2617116)
    anti-VSVM
    suggested: None
    The Secondly antibodies used were HRP-linked donkey anti-rabbit IgG antibody (NA934; GE Healthcare, Piscataway, NJ, USA) and HRP-linked donkey anti-mouse IgG antibody (NA931V; GE Healthcare)
    anti-rabbit IgG
    suggested: (GE Healthcare Cat# NA934, RRID:AB_772206)
    HRP-linked donkey anti-mouse IgG
    suggested: None
    Cells were then incubated with anti-SARS-CoV-2 nucleocapsid (1:1000, GTX135357, GeneTex, CA, USA) primary antibody for 16 h at 4 °C and detected with anti-rabbit-Alexa488 (1:200, A11008, Invitrogen, CA, USA) secondary antibodies for 40 min at RT.
    anti-SARS-CoV-2 nucleocapsid ( 1:1000 , GTX135357
    suggested: None
    anti-rabbit-Alexa488
    suggested: None
    A11008
    suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)
    Experimental Models: Cell Lines
    SentencesResources
    A704, Calu-3, VeroE6, HEC50B, and Caco-2 cells were maintained in Eagle’s minimum essential medium (EMEM; 055-08975,
    Calu-3
    suggested: None
    Caco-2
    suggested: None
    OUMS-23, IGROV1, and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; 041-30081,
    293T
    suggested: None
    HEC50B cells infected with pseudotype viruses were selected with 300 μg/mL hygromycin for at least 1 week.
    HEC50B
    suggested: JCRB Cat# JCRB1145, RRID:CVCL_2929)
    For the DSP assay using 293FT cells, DSP8-11 expressing effector cells expressing S protein and DSP1-7 expressing target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10 cm cell culture plates (4 × 106 cells/10 mL) one day prior to the assay (S1a,b Fig).
    293FT
    suggested: None
    After an overnight incubation at 37 °C in 5% CO2, cells were treated with protease inhibitors for 1 h and added with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for HEC50B and HEC50B-TMPRSS2 cells, and MOI of 0.1 for VeroE6, Calu-3, and A704 cells.
    HEC50B-TMPRSS2
    suggested: None
    A704
    suggested: NCI-DTP Cat# A704, RRID:CVCL_1065)
    Experimental Models: Organisms/Strains
    SentencesResources
    OVTOKO (JCRB1048), OVISE (JCRB1043), HEC50B (JCRB1145), VeroE6-TMPRSS2 (JCRB1819)[67], and OUMS-23 (JCRB1022) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka,
    VeroE6-TMPRSS2 (JCRB1819)
    suggested: None
    VeroE6-TMPRSS2 (JCRB1819) cells were cultured in DMEM containing 10% FBS and 1 mg/mL G418.
    VeroE6-TMPRSS2
    suggested: None
    Recombinant DNA
    SentencesResources
    To establish stable cell lines expressing the S protein of SARS-CoV, SARS-CoV-2, or MERS-CoV, recombinant pseudotype lentiviruses were produced in 293T cells with psPAX2 packaging plasmid, vesicular stomatitis virus (VSV)-G-expressing plasmid and lentiviral transfer plasmid expressing S protein.
    VSV)-G-expressing
    suggested: None
    recombinant pseudotype lentivirus expressing TMPRSS2 was produced using 293T cells with psPAX2 packaging plasmid and VSV-G-expressing plasmid.
    psPAX2
    suggested: RRID:Addgene_12260)
    VSV-G-expressing
    suggested: None
    Software and Algorithms
    SentencesResources
    IGROV1 cells (SCC203) were purchased from Merck (Darmstadt, Germany) and Caco-2 cells (RCB0988) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).
    RIKEN BioResource
    suggested: (RIKEN BioResource Center, RRID:SCR_003250)
    Statistical analysis: Statistical analyses were performed in Microsoft Excel 2016 (
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    (Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA).
    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 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: 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

    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 10.1101/2021.12.14.472513 on bioRxiv