Multi-level inhibition of coronavirus replication by chemical ER stress

  1. Mohammed Samer Shaban
  2. Christin Müller
  3. Christin Mayr-Buro
  4. Hendrik Weiser
  5. Johanna Meier-Soelch
  6. Benadict Vincent Albert
  7. Axel Weber
  8. Uwe Linne
  9. Torsten Hain
  10. Ilya Babayev
  11. Nadja Karl
  12. Nina Hofmann
  13. Stephan Becker
  14. Susanne Herold
  15. M. Lienhard Schmitz
  16. John Ziebuhr
  17. Michael Kracht

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Abstract

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

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  1. Version published to 10.1038/s41467-021-25551-1
  2. ScreenIT

    SciScore for 10.1101/2020.08.26.266304: (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: HuH7 and MRC-5 cells were confirmed to be free of mycoplasma using the Venor® GeM Classic kit (Minerva Biolabs).

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778)
    anti β-actin
    suggested: None
    , anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01)
    anti ATF3
    suggested: None
    anti HERPUD1
    suggested: None
    anti CTH antibody (Cruz, #sc-374249),
    anti CTH
    suggested: (LSBio (LifeSpan Cat# LS-C80809, RRID:AB_2087504)
    The following secondary antibodies were used: Dako P0447; polyclonal goat anti-mouse immunoglobulins/HRP, Dako P0448; polyclonal goat anti-rabbit immunoglobulins/HRP, Cy3-coupled anti rabbit (rb) IgG (dk, Merck Millipore, #AP182C), Dylight
    anti-mouse immunoglobulins/HRP
    suggested: (Agilent Cat# P0447, RRID:AB_2617137)
    anti-rabbit
    suggested: (Millipore Cat# AP182C, RRID:AB_92588)
    anti rabbit ( rb ) IgG
    suggested: None
    After immunoblotting (see below), membranes were stained with Coomassie brilliant blue and then hybridized with an anti puromycin antibody (Kerafast, #EQ0001) to detect puromycinylated polypetides.
    anti puromycin antibody ( Kerafast , #EQ0001
    suggested: None
    After 2x washing, cells were fixed with 4% paraformaldehyde in PBS (Santa Cruz, #281692) for 5 min, washed 3x 10 min with Hank’s BSS (PAN, #PO4-32505), blocked with 10% normal donkey serum (Jackson ImmunoResearch, #017-000-121) for 20 min and incubated with primary and secondary antibodies diluted in Hank’s BSS containing 0.005% saponin (Sigma-Aldrich, #S4521-10G) for 2 h at room temperature.
    normal donkey serum
    suggested: (Jackson ImmunoResearch Labs Cat# 017-000-121, RRID:AB_2337258)
    Following 3 washing steps with Hank’s BSS containing 0.005% saponin, Cy3-conjugated (Millipore, #AP182C, 1:100) and Dylight488-conjugated (ImmunoReagents #DkxMu-003D488NHSX, 1:100) secondary antibodies were used.
    #AP182C
    suggested: (Millipore Cat# AP182C, RRID:AB_92588)
    Experimental Models: Cell Lines
    SentencesResources
    MRC-5 human embryonic lung fibroblasts ((ATCC, CCL-171)) were maintained in DMEM containing 1.5 g / l (w / v) NaHCO3 and complemented with 10% fetal calf serum (FCS; PAN Biotech Cat No. 1502-P110704), 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin, 1% minimum essential medium nonessential amino acids (100x MEM NEAA; Gibco Cat No 11140-035) and 1 mM sodium pyruvate (100 mM; Gibco 11360-039)
    MRC-5 human embryonic lung fibroblasts
    suggested: None
    Virus infections and assessments of antiviral activity: To analyze the antiviral activity of thapsigargin, HuH7 cells (for HCoV-229E, MERS-CoV), MRC-5 cells (for HCoV-229E) and Vero E6 (for SARS-CoV-2) were infected at the indicated multiplicities of infection (MOI) and incubated at 33°C (for HCoV-229E and SARS-CoV-2) or 37°C (for MERS-CoV) in the presence or absence of thapsigargin, or with the appropriate volume of solvent control (DMSO) as indicated.
    Vero E6
    suggested: RRID:CVCL_XD71)
    In brief, 1.2 x 104 HuH7 or 1 x 104 MRC-5 cells were seeded in 96-well plates for 24 hours and thereafter treated with DMSO, thapsigargin, virus alone or virus plus thapsigargin for 24 hours as indicated in the figure legends.
    MRC-5
    suggested: None
    For the data shown in Fig. 4 and 5, raw data from 96 LC-MS/MS runs (representing two independent experiments and three technical replicates per sample) were mapped to Homo sapiens (uniprot ID UP000005640 for HuH7 cells)
    HuH7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Software and Algorithms
    SentencesResources
    SARS-CoV-2 genome sequencing data have been submitted to the NCBI Short Read Archive repository under bioproject PRJNA658242 (SRA accession number SRP278165).
    NCBI Short Read Archive
    suggested: None
    EC50 values were calculated by non-linear regression analysis using GraphPad Prism 5.0 or 8.4.3 (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Quality control of RNA-seq reads was performed using the FastQC command line tool version 0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Reads were aligned using STAR version 2.4.2a (Dobin et al, 2013) to an index based on the human genome version hg19.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    The resulting bam files were imported into R (Team, 2015) (https://www.R-project.org/) and gene-specific read counts based on hg19 UCSC gene annotations were extracted using FeatureCounts from the R subread package version 1.24.2 (Liao et al, 2013).
    FeatureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    subread
    suggested: (Subread, RRID:SCR_009803)
    Detection of differentially expressed genes was done using DESeq2 version 1.14.1 (Love et al, 2014).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    From the entire data set, only normalized read counts and ratio values for 166 gene IDs assigned to KEGG 04141 were extracted and further analyzed.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Data analysis was performed using MaxQuant with the Andromeda search engine and Uniprot databases were used for annotating and assigning protein identifiers (Tyanova et al, 2016a).
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    Uniprot
    suggested: (UniProtKB, RRID:SCR_004426)
    All subsequent filtering steps and heatmap representations were performed in Excel 2016 as described in the figure legends.
    Excel
    suggested: None
    Overrepresentation analyses were done using the gene IDs / gene names of differentially enriched proteins and Metascape software with the express settings (Zhou et al., 2019)
    Metascape
    suggested: (Metascape, RRID:SCR_016620)
    Protein network data were extracted from the most recent version of STRING (Szklarczyk et al., 2019) and visualized with Cytoscape 3.8.0 (Cline et al, 2007).
    STRING
    suggested: (STRING, RRID:SCR_005223)
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Mapping of ratio values on KEEG pathway 04141 was done with Pathview or Pathview Web software (Luo et al, 2017).
    Pathview
    suggested: (Pathview, RRID:SCR_002732)
    Quantification and statistical analysis: Statistical parameters (t-tests, standard variations, confidence intervals, Pearson correlations) were calculated using SigmaPlot 11, GraphPad Prism 5.0 or 8.4.3 or Microsoft Excel 2016 in addition to the software tools mentioned above.
    SigmaPlot
    suggested: (SigmaPlot, RRID:SCR_003210)
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    Results from OddPub: Thank you for sharing your 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 10.1101/2020.08.26.266304v1 on bioRxiv