Type-I interferon signatures in SARS-CoV-2 infected Huh7 cells

  1. Xi Chen
  2. Elisa Saccon
  3. K. Sofia Appelberg
  4. Flora Mikaeloff
  5. Jimmy Esneider Rodriguez
  6. Beatriz Sá Vinhas
  7. Teresa Frisan
  8. Ákos Végvári
  9. Ali Mirazimi
  10. Ujjwal Neogi
  11. Soham Gupta

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-β production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce the susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV, and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon-stimulated genes and their role in the inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.

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  1. Version published to 10.1038/s41420-021-00487-z
  2. ScreenIT

    SciScore for 10.1101/2021.02.04.429738: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: Antibodies and their manufacturers were: rabbit anti-RIG-I clone D14G6 (1:1000; #3743), rabbit anti-MDA5 clone D74E4 (1:1000; #5321) from Cell-Signaling Technologies (Danvers, MA, USA), mouse anti-ISG15 (1:1000, sc-166755) from Santa-Cruz Biotechnology (santa Cruz, CA, USA), recombinant Anti-GAPDH clone EPR16891(1:10000, Ab181602) and rabbit anti TRIM25 clone EPR7315 (1:2000; ab167154) from Abcam (Cambridge, MA, USA).
    anti-RIG-I
    suggested: (Cell Signaling Technology Cat# 3743, RRID:AB_2269233)
    anti-MDA5
    suggested: None
    anti-ISG15
    suggested: (Santa Cruz Biotechnology Cat# sc-166755, RRID:AB_2126308)
    anti TRIM25
    suggested: (Santa Cruz Biotechnology Cat# sc-166755, RRID:AB_2126308)
    Subsequent antibody incubation was performed at 4°C overnight or for one hour at room temperature using Dako polyclonal goat anti-rabbit or anti-mouse immunoglobulins/HRP (Agilent Technologies, USA).
    anti-rabbit
    suggested: None
    anti-mouse immunoglobulins/HRP
    suggested: None
    The Western blot analysis was performed by using antibodies targeting RIG-I, MDA-5, TRIM25, ISG15, GAPDH.
    MDA-5
    suggested: None
    TRIM25
    suggested: None
    GAPDH
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and virus: The human hepatocyte-derived cellular carcinoma Huh7 cell line was obtained from Marburg Virology Lab, Germany and Caco2 was obtained from CLS cell line services, GmbH, Germany (#300137).
    Caco2
    suggested: None
    Etopside treatment: Huh7 cells were seeded in 6-well plates in DMEM supplemented with 10% heat-inactivated FBS.
    Huh7
    suggested: None
    The Western blot analysis was performed by using antibodies targeting RIG-I, MDA-5, TRIM25, ISG15, GAPDH.
    MDA-5
    suggested: None
    Software and Algorithms
    SentencesResources
    The Primers and probes used were E_Sarbeco_F1: 5’-ACAGGTACGTTAATAGTTAATAGCGT-3’, E_Sarbeco_R2: 5’-ATATTGCAGCAGTACGCACACA-3’ and Probe: [FAM] ACACTAGCCATCCTTACTGCGCTTCG [BBQ650].
    Probe
    suggested: (UniPROBE, RRID:SCR_005803)
    Proteins were searched against both SwissProt human and SARS-CoV/SARS-CoV2 databases using the search engine Mascot Server v2.5.1 (MatrixScience Ltd, UK) in Proteome Discoverer v2.4 (ThermoFisher Scientific) software allowing up to two missed cleavages.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    The raw mass spectrometric data was deposited to the ProteomeXhanger Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD023450.
    PRIDE
    suggested: (Pride-asap, RRID:SCR_012052)
    Statistical analysis: Statistical analyses for proteomics and transcriptomics were performed in R package LIMMA.
    LIMMA
    suggested: (LIMMA, RRID:SCR_010943)
    All other statistical calculations were performed in GraphPad Prism (Version 8.0.0) using unpaired t-test.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Significant proteins (proteomics data, LIMMA, FDR < 0.05) were represented as a network with Cytoscape ver 3.6.1.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Protein-protein interactions were retrieved from STRING Db (v5.0) (https://string-db.org/).
    STRING
    suggested: (STRING, RRID:SCR_005223)
    for Results from each comparison were retrieved and represented as volcano plot using ggplot2, Venn diagram using interactivenn (http://www.interactivenn.net/) and heatmap of fold changes using R package complexHeatmap.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    complexHeatmap
    suggested: (ComplexHeatmap, RRID:SCR_017270)

    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 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

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  3. Version published to 10.1101/2021.02.04.429738v1 on bioRxiv