Transcriptomics-inferred dynamics of SARS-CoV-2 interactions with host epithelial cells

  1. Lukas Adam
  2. Megan Stanifer
  3. Fabian Springer
  4. Jan Mathony
  5. Maik Brune
  6. Chiara Di Ponzio
  7. Roland Eils
  8. Steeve Boulant
  9. Dominik Niopek
  10. Stefan M. Kallenberger

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Virus-host interactions can reveal potentially effective and selective therapeutic targets for treating infection. Here, we performed an integrated analysis of the dynamics of virus replication and the host cell transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using human Caco-2 colon cancer cells as a model. Time-resolved RNA sequencing revealed that, upon infection, cells immediately transcriptionally activated genes associated with inflammatory pathways that mediate the antiviral response, which was followed by an increase in the expression of genes involved in ribosome and mitochondria function, thus suggesting rapid alterations in protein production and cellular energy supply. At later stages, between 24 and 48 hours after infection, the expression of genes involved in metabolic processes—in particular, those related to xenobiotic metabolism—was decreased. Mathematical modeling incorporating SARS-CoV-2 replication suggested that SARS-CoV-2 proteins inhibited the host antiviral response and that virus transcripts exceeded the translation capacity of the host cells. Targeting kinase-dependent pathways that exhibited increases in transcription in host cells was as effective as a virus-targeted inhibitor at repressing viral replication. Our findings in this model system delineate a sequence of SARS-CoV-2 virus-host interactions that may facilitate the identification of druggable host pathways to suppress infection.

Article activity feed

  1. Version published to 10.1126/scisignal.abl8266
  2. ScreenIT

    SciScore for 10.1101/2021.07.04.450986: (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 AuthenticationContamination: Cells have been tested negative for mycoplasma infection (MycoAlert Plus; Lonza, Basel, Switzerland).
    Authentication: Microscopy: To analyze SARS-CoV-2 N protein expression in Caco-2 cells by immunofluorescence, microscopic images were taken at 0, 4, 24 and 48 hpi using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokio, Japan).

    Table 2: Resources

    Antibodies
    SentencesResources
    Reagents: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics).
    GFP
    suggested: None
    For microscopy, the secondary antibody Alexa Fluor goat anti-mouse 568 (Thermo Fisher Scientific, Waltham, MA, USA) was applied.
    anti-mouse 568
    suggested: None
    A Horseradish peroxidase-conjugated anti-mouse antibody (Southern Biotech, Birmingham, AL, USA) was used for immunoblotting.
    anti-mouse
    suggested: None
    Secondary antibody (anti-mouse CW800) and DNA dye Draq5 (Abcam, Cambridge, UK) were diluted 1/10,000 in blocking buffer and incubated for 1 h at RT.
    anti-mouse CW800
    suggested: None
    Draq5
    suggested: (Biostatus Cat# DR50050, RRID:AB_2314341)
    Experimental Models: Cell Lines
    SentencesResources
    Caco-2, Vero E6 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin and streptomycin (100μg/ml; Invitrogen).
    Caco-2
    suggested: None
    Vero E6
    suggested: None
    HEK293T cells were transfected with X-tremeGENE HP (Roche Diagnostics, Rotkreuz, Switzerland) following the manufacturer’s instructions.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    To generate a construct for co-expressing mCherry and SARS-CoV-2 main protease (3CLpro), a fragment encoding 3CLpro was first obtained as a double-stranded DNA fragment (gBlock; IDT, San José, CA, USA) and cloned into pcDNA3.1(−) via unique NheI/NotI restriction sites.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    The 3CLpro encoding vector was then linearized 5’ of the 3CLpro start codon via NheI and the mCherry-P2A fragment was inserted via Gibson Assembly, hence yielding a vector encoding mCherry-P2A-3CLpro.
    mCherry-P2A-3CLpro
    suggested: None
    Software and Algorithms
    SentencesResources
    Paired-end sequencing of 2×150bp was performed at GeneWiz Inc. using an Illumina NovaSeq 6000 instrument (Illumina, San Diego CA, USA).
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Live-cell experiments were performed on a Nikon Ti inverted microscope, equipped with a CSU-22 Yokogawa confocal spinning disc slider (Yokogawa Electric Corporation, Tokyo, Japan), a 60× Plan Apo NA 1.4 objective lens (Nikon), a Hamamatsu C9100-02 EMCCD camera (Hamamatsu Photonics, Hamamatsu, Japan), and the Volocity software (PerkinElmer; Waltham, MA, USA).
    Volocity
    suggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)
    Microscopic images were evaluated with ImageJ software (NIH, Bethesda, MA, USA).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    For read alignment, the STAR software was used with default settings (Dobin et al, 2013).
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Subsequently, BAM files were split by their reference using the SAMtools software suite (Li et al, 2009) and counted separately using the featureCounts function from the Subread package (Liao et al, 2019).
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    Overrepresented GO terms were inferred using the ‘goana’ and ‘topGO’ function from the limma package (Ritchie et al, 2015).
    limma
    suggested: (LIMMA, RRID:SCR_010943)
    The MATLAB toolbox PottersWheel was used for model fitting (Maiwald & Timmer, 2008).
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)
    To this end, the following six model simplifications were tested: (1) description of A turnover by one turnover parameter instead of separate parameters for synthesis and degradation of A (Variant 4.0.1), (2) description of the synthesis of A by mass-action instead of Michaelis-Menten (MM) kinetics (Variant 4.0.2), (3) mass-action instead of MM-kinetics for describing synthesis of P (Variant 4.0.3), (4) mass-action kinetics for describing synthesis and degradation of A by single turnover parameter (Variant 4.0.4), (5) single parameter for describing turnover of A and mass-action instead of MM-kinetics for synthesis of P (Variant 4.0.5), (6) mass-action kinetics for synthesis of A as well as P.
    Variant
    suggested: (VARIANT, RRID:SCR_005194)

    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.

  3. Version published to 10.1101/2021.07.04.450986v1 on bioRxiv