Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

  1. James M. Burke
  2. Laura A. St Clair
  3. Rushika Perera
  4. Roy Parker

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Abstract

A key feature of the mammalian innate immune response to viral infection is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response and limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN -encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. In addition, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both activation of the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. These findings argue that inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response.

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    SciScore for 10.1101/2021.04.19.440452: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: All cell culture and plate preparation work were conducted under biosafety level 2 conditions, while all viral infections were conducted under biosafety level 3 conditions at Colorado State University.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were routinely tested for mycoplasma contamination by the cell culture core facility.

    Table 2: Resources

    Antibodies
    SentencesResources
    Expression of ACE2 was confirmed via immunoblot analysis (protocol described in Burke et al., 2019) using Anti-Angiotensin Converting Enzyme 2 antibody [EPR4435(2) (Abcam: ab108252) at 1:1000.
    Anti-Angiotensin Converting Enzyme 2
    suggested: (Abcam Cat# ab108252, RRID:AB_10864415)
    For immunofluorescence detection, cells were incubated with Rabbit polyclonal anti-PABP antibody (Abcam: ab21060) and Mouse monoclonal anti-G3BP antibody (Abcam: ab56574) primary antibodies at 1:1000 for two hours, washed three times, and then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) (Abcam: ab150079) and Goat Anti-Mouse IgG H&L (FITC) (Abcam; ab97022) at 1:2000 for one hour.
    anti-PABP
    suggested: (Abcam Cat# ab21060, RRID:AB_777008)
    anti-G3BP
    suggested: (Abcam Cat# ab56574, RRID:AB_941699)
    Anti-Rabbit IgG
    suggested: (Abcam Cat# ab150079, RRID:AB_2722623)
    Anti-Mouse IgG
    suggested: (Abcam Cat# ab97022, RRID:AB_10681023)
    Experimental Models: Cell Lines
    SentencesResources
    RL-KO) A549, U2-OS, and HEK293T cell lines are described in Burke et al., 2019.
    HEK293T
    suggested: None
    The virus was passaged in Vero E6 cells, and viral titer was determined via plaque assay on Vero E6 as previously described in (Dulbecco et al., 1953).
    Vero E6
    suggested: None
    A549 cells were infected with dengue virus serotype 2 16681 strain at an MOI of 0.1, and with influenza A/Udorn/72 virus strain at MOI of 0.5, as described in Burke et al., 2021. Generation of ACE2-expressing cell lines: HEK293T cells (T-25 flask; 80
    A549
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: The flag-Nsp1 and flag-Nsp15 vectors were generated by ligating a g-block [Integrated DNA Technologies (IDT)] encoding for the flag and ORF of Nsp1 or Nsp15 between the xho1 and xba1 sites in pcDNA3.1+.
    flag-Nsp15
    suggested: None
    pcDNA3.1+
    suggested: RRID:Addgene_117272)
    The pLEX307-ACE2-puro plasmid was a gift from Alejandro Chavez and Sho Iketani
    pLEX307-ACE2-puro
    suggested: None
    % confluent) were co-transfected with 2.4-ug of pLenti-pLex307-ACE lentiviral transfer plasmid (Addgene: 158448), 0.8-ug of pVSV-G, 0.8-ug of pRSV-Rev, and 1.4-ug of pMDLg-pRRE using 20-ul of lipofectamine 2000.
    pLenti-pLex307-ACE
    suggested: None
    pVSV-G
    suggested: RRID:Addgene_138479)
    pRSV-Rev
    suggested: RRID:Addgene_12253)
    pMDLg-pRRE
    suggested: None
    Software and Algorithms
    SentencesResources
    The probes were labeled with Atto-633 using ddUTP-Atto633 (Axxora: JBS-NU-1619-633), with ATTO-550 using 5-Propargylamino-ddUTP (Axxora; JBS-NU-1619-550), or ATTO-488 using 5-Propargylamino-ddUTP (Axxora; JBS-NU-1619-488) with terminal deoxynucleotidyl transferase (Thermo Fisher Scientific: EP0161) as described in (Gaspar et al., 2017).
    Thermo Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    Z-planes were stacked, and minimum and maximum display values were set in ImageJ for each channel to properly view fluorescence.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Imaris Image Analysis Software (Bitplane) (University of Colorado-Boulder, BioFrontiers Advanced Light Microscopy Core) was used to quantify smFISH foci in nucleus and cytoplasm.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04350671Enrolling by invitationInterferon Beta 1a in Hospitalized COVID-19 Patients
    NCT04388709WithdrawnInterferon Lambda Therapy for COVID-19
    NCT04552379RecruitingThe Containing Coronavirus Disease 19 (COVID-19) Trial

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 9, 12, 5 and 7. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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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  2. Version published to 10.1101/2021.04.19.440452 on bioRxiv