SPINT2 controls SARS-CoV-2 viral infection and is associated to disease severity

  1. Carlos Ramirez Alvarez
  2. Ashwini Kumar Sharma
  3. Carmon Kee
  4. Leonie Thomas
  5. Steeve Boulant
  6. Carl Herrmann

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Abstract

COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2 . Here, we explored the existence of a co-regulation between SPINT2 / TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.28.424029: (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
    Mouse monoclonal antibody against SARS-CoV-2 Nucleocapsid (NC) protein (Sino biologicals MM05) as primary antibody was diluted in 1% BSA-phosphate-buffered saline (PBS) and incubated for 1h at RT.
    antibody against SARS-CoV-2 Nucleocapsid ( NC ) protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell line and Viruses: Human lung adenocarcinoma cell lines Calu-3 (ATCC HTB-55) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Glutamax (Gibco), 10% fetal bovine serum and 1% penicillin/streptomycin while Vero E6 cells (ATCC CRL 1586) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco).
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Vero E6
    suggested: None
    Lentivirus production and selection of stable cell lines: HEK293T cells (ATCC CRL-3216) were seeded on 10 cm2 dishes and allowed to adhere for 36 hours.
    HEK293T
    suggested: None
    We performed Differential Expression Analysis using Seurat 65 (DEA) between Calu-3 and H1299 cells in non-infected mock cells at 4 hours of culture (z).
    H1299
    suggested: NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)
    Software and Algorithms
    SentencesResources
    These masks were used on CellProfiler 3.1.9 to measure the intensity of the conjugated secondary antibodies in each nucleus.
    CellProfiler
    suggested: None
    Next, we estimated the feature importance for each of the permissivity signature genes and performed enrichment analysis using enrichR 66 on the top 25% ranked genes.
    enrichR
    suggested: (Enrichr, RRID:SCR_001575)
    TFBS were visualised using the PlotTracks TOBIAS function and the network was built in Cytoscape 70.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)

    Results from OddPub: Thank you for sharing your code and data.

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.12.28.424029v1 on bioRxiv