The RNA sensor MDA5 detects SARS-CoV-2 infection

  1. Natalia G. Sampaio
  2. Lise Chauveau
  3. Jonny Hertzog
  4. Anne Bridgeman
  5. Gerissa Fowler
  6. Jurgen P. Moonen
  7. Maeva Dupont
  8. Rebecca A. Russell
  9. Marko Noerenberg
  10. Jan Rehwinkel

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Abstract

Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.

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  1. Version published to 10.1038/s41598-021-92940-3
  2. ScreenIT

    SciScore for 10.1101/2021.03.26.437180: (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 AuthenticationContamination: Cell culture and virus infection: Cells were cultured at 37°C and 5% CO2 and routinely screened for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies included: beta-actin-HRP (AC-15, Sigma Aldrich), pSTAT1 (Y701) (58D6, Cell Signaling Technology), STAT1 (42H3, Cell Signaling Technology), pSTAT2 (D3P2P, Cell Signaling Technology), STAT2 (D9J7L, Cell Signaling Technology), hcGAS (D1D3G, Cell Signaling Technology), hSTING (D2P2F, Cell Signaling Technology), MyD88 (D80F5, Cell Signaling Technology), TBK1 (D1B4, Cell Signaling Technology), MAVS (ALX-210-929-C100
    pSTAT1
    suggested: (Fluidigm Cat# 3153003, RRID:AB_2661824)
    Cells were incubated with antibodies against human MxA (clone M143, kind gift from G Kochs) and SARS-CoV-2 N protein (clone EY-2A, kind gift from Alain Townsend56; 1:200, 30 minutes, 4°C), and goat anti-mouse AlexaFluor 488 (Life Technologies, A11029) and anti-human AlexaFluor 647 (1:500, 30 minutes, 4°C; Life Technologies, A21445), and resuspended in CellFix (1:10 in water; BD, 340181).
    SARS-CoV-2 N protein
    suggested: (ABclonal Cat# A20021, RRID:AB_2862924)
    anti-mouse
    suggested: (Molecular Probes Cat# A-11029, RRID:AB_138404)
    anti-human AlexaFluor 647
    suggested: None
    A21445
    suggested: (Molecular Probes Cat# A-21445, RRID:AB_2535862)
    Experimental Models: Cell Lines
    SentencesResources
    Calu-3 cells (ATCC) were maintained in MEM (Gibco) supplemented with 10% v/v foetal calf serum (FCS, Gibco), 2 mM L-glutamine (Gibco), 1x sodium pyruvate (Gibco) and 1x non-essential amino acids (Gibco).
    Calu-3
    suggested: None
    THP1 cells (kind gift from V Cerundolo) were maintained in RPMI (Sigma Aldrich) supplemented with 10% v/v (FCS) and 2 mM L-glutamine (Gibco).
    THP1
    suggested: None
    All other cells (A549, kind gift from G Kochs; HEK293T, HEK293 and VERO E6, kind gifts from C Reis E Sousa; Huh7, kind gift from J McKeating) were maintained in DMEM (Sigma Aldrich) supplemented with 10% v/v FCS and 2 mM L-glutamine.
    A549
    suggested: None
    HEK293T
    suggested: None
    HEK293
    suggested: None
    Huh7
    suggested: None
    Briefly, virus was serially diluted ten-fold in DMEM with 1% FCS, and 100 μl of dilutions were added in quadruplicates to 24 well plates containing 2.5 x 105 Vero E6 cells in 500 μl of medium.
    Vero E6
    suggested: None
    0, ENZO Life Science), IRF3 (D6I4C, Cell Signaling Technology), MDA5 (generated in house55) and RIG-I (clone ALME-I, ProSci #PSI-36-102).
    MDA5
    suggested: None
    Software and Algorithms
    SentencesResources
    After removing the overlay and the medium, cells were washed in PBS, and fixed and stained using Amido Black stain for at least 30 minutes at room temperature.
    Amido
    suggested: None
    Lentiviral shRNA knockdown: Lentiviral plasmids encoding shRNAs targeting GFP (control; SHC005) and MAVS (06: TRCN0000149206; 45: TRCN0000148945) were obtained from the Sigma Mission library (Merck Darmstadt)
    Sigma Mission library
    suggested: None
    Cells were analysed by flow cytometry on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and data were analysed using FlowJo software (BD).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

    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.

  3. Version published to 10.1101/2021.03.26.437180v1 on bioRxiv