Host Cellular RNA Helicases Regulate SARS-CoV-2 Infection

  1. Yasuo Ariumi

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Abstract

SARS-CoV-2 has a large RNA genome, of approximately 30 kb. To regulate and maintain such a large viral RNA genome, host RNA helicases may be involved in SARS-CoV-2 replication.

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  1. Version published to 10.1128/jvi.00002-22
  2. ScreenIT

    SciScore for 10.1101/2021.06.29.450452: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Pre-cleared supernatants were incubated with 5 μg of anti-SARS-CoV-2 nucleocapsid antibody mixture (GTX135357 and GTX632269 [6H3]; GeneTex), anti-DDX21 (A300-627A; Bethyl Lab), anti-DDX6 (A300-460A; Bethyl Lab), or anti-MOV10 (A301-571A; Bethyl Lab) antibody at 4°C for 1 hour (h).
    anti-SARS-CoV-2 nucleocapsid
    suggested: None
    GTX632269
    suggested: (GeneTex Cat# GTX632269, RRID:AB_2888304)
    anti-DDX21
    suggested: (Bethyl Cat# A300-627A, RRID:AB_513601)
    anti-DDX6
    suggested: (Bethyl Cat# A300-460A, RRID:AB_420926)
    A301-571A; Bethyl Lab
    suggested: None
    The proteins were then subjected to SDS-PAGE, followed by immunoblot analysis using anti-SARS-CoV-2 nucleocapsid (GTX632269 [6H3]), anti-DDX21, anti-DDX6 or anti-MOV10 antibody.
    anti-SARS-CoV-2 nucleocapsid ( GTX632269
    suggested: None
    anti-MOV10
    suggested: None
    The cells were fixed in 3.6% formaldehyde in phosphate-buffered saline (PBS), permeabilized in 0.1% NP-40 in PBS at room temperature, and incubated with anti-HA antibody (3F10; F. Hoffmann-La Roche AG, Basel, Switzerland) at a 1:300 dilution in PBS containing 3% bovine serum albumin (BSA) at 37°C for 30 min.
    anti-HA
    suggested: None
    We also used anti-DDX6 (A300-460A; Bethyl Lab), anti-XRN1 (A300-443A; Bethyl Lab), anti-G3BP1 (A302-033A; Bethyl) anti-DDX21 (A300-627A; Bethyl Lab), anti-MOV10 (A301-571A; Bethyl Lab), anti-DDX5 (A300-523A; Bethyl Lab), or anti-SARS-CoV-2 nucleocapsid (ab273434 [6H3]; Abcam or GTX632269 [6H3]; GeneTex) antibodies as primary antibodies.
    anti-XRN1
    suggested: (Bethyl Cat# A300-443A, RRID:AB_2219047)
    anti-G3BP1
    suggested: (Bethyl Cat# A302-033A, RRID:AB_1576539)
    anti-DDX5
    suggested: (Bethyl Cat# A300-523A, RRID:AB_451048)
    Cells were then stained with Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 594 conjugate and/or Donkey anti-rabbit or anti-rat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate (Thermo Fisher Scientific Inc.
    anti-mouse IgG
    suggested: None
    anti-rabbit or anti-rat IgG
    suggested: None
    anti-rat
    suggested: None
    The SARS-CoV-2-infected cells were detected using anti-SARS-CoV-2 Spike (GTX632604 [1A9]; GeneTex, Irvine, CA, USA) or anti-SARS-CoV-2 nucleocapsid (ab273434 [6H3]; Abcam or GTX632269 [6H3]; GeneTex) antibodies.
    anti-SARS-CoV-2
    suggested: None
    anti-SARS-CoV-2 nucleocapsid (ab273434 [
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: 293T, HepG2, HEK293T ACE2 (SL221
    HepG2
    suggested: None
    HEK293T
    suggested: None
    Plasmid construction: To construct pcDNA3-HA-MOV10, a DNA fragment encoding MOV10 was amplified from HuH-7 cDNA by PCR using KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan) and the following pairs of primers: 5’-CGGGATCCAAGATGCCCAGTAAGTTCAGCTG-3’ (Forward), 5’-CCGCTCGAGTCAGAGCTCATTCCTCCACTCTG-3’ (Reverse).
    HuH-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    The lentiviral vector particles were produced by transient transfection of the second-generation packaging construct pCMVΔR8.74 (42, 43) and the VSV-G-envelope-expressing plasmid pMD2G as well as pRDI292 into 293T cells with TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA).
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    For infection experiments with SARS-CoV-2 virus, HEK293T ACE2 cells (2×105 cells/well) were plated onto 6-well plates and cultured for 24 h.
    HEK293T ACE2
    suggested: None
    Actually, 1μl of culture supernatants of SARS-CoV-2-infected VeroE6 TMPRSS2 cells (2.7×108 TCID50/ml) were inoculated.
    VeroE6 TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Recombinant DNA
    SentencesResources
    Plasmid construction: To construct pcDNA3-HA-MOV10, a DNA fragment encoding MOV10 was amplified from HuH-7 cDNA by PCR using KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan) and the following pairs of primers: 5’-CGGGATCCAAGATGCCCAGTAAGTTCAGCTG-3’ (Forward), 5’-CCGCTCGAGTCAGAGCTCATTCCTCCACTCTG-3’ (Reverse).
    pcDNA3-HA-MOV10
    suggested: None
    The obtained DNA fragments were subcloned into either the BamHI-NotI sites of the pcDNA3-HA vector, and the nucleotide sequences were determined by Sanger sequencing.
    pcDNA3-HA
    suggested: RRID:Addgene_78782)
    We previously described pHA-DDX3 (8–12), pcDNA3-HA-DDX1 and pcDNA3-HA-DDX6 (9).
    pHA-DDX3
    suggested: None
    pcDNA3-HA-DDX1
    suggested: None
    pcDNA3-HA-DDX6
    suggested: None
    pcDNA3.1 SARS-CoV-2 N was a gift from Dr. Jeremy Luban
    pcDNA3.1 SARS-CoV-2
    suggested: RRID:Addgene_158080)
    Addgene plasmid #158079; http://n2t.net/addgene:158079; RRID:Addgene_158079) (38).
    detected: RRID:Addgene_158079)
    The oligonucleotides above were annealed and subcloned into the BglII-HindIII site, downstream from an RNA polymerase III promoter of pSUPER (39), to generate pSUPER-DDX1, pSUPER-DDX21, and pSUPER-MOV10, respectively.
    pSUPER-DDX1
    suggested: None
    pSUPER-DDX21
    suggested: None
    pSUPER-MOV10
    suggested: None
    To construct pLV-shDDX1, pLV-shDDX21, or pLV-shMOV10, the BamHI-SalI fragments of the corresponding pSUPER plasmids were subcloned into the BamHI-SalI site of pRDI292, an HIV-1-derived self-inactivating lentiviral vector containing a puromycin resistance marker allowing for the selection of transduced cells (40).
    pLV-shDDX1
    suggested: None
    pLV-shDDX21
    suggested: None
    pLV-shMOV10
    suggested: None
    pSUPER
    suggested: RRID:Addgene_26210)
    pRDI292
    suggested: None
    We previously described pLV-shDDX3, pLV-shDDX5, and pLV-shDDX6 (11, 12, 41)
    pLV-shDDX3
    suggested: None
    pLV-shDDX5
    suggested: None
    pLV-shDDX6
    suggested: None
    The lentiviral vector particles were produced by transient transfection of the second-generation packaging construct pCMVΔR8.74 (42, 43) and the VSV-G-envelope-expressing plasmid pMD2G as well as pRDI292 into 293T cells with TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA).
    pCMVΔR8.74
    suggested: None
    VSV-G-envelope-expressing
    suggested: None
    pMD2G
    suggested: None
    Software and Algorithms
    SentencesResources
    CACO-2 human colon carcinoma cells (RCB0988; RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan) were cultured in DMEM supplemented with 20% FBS.
    RIKEN BioResource
    suggested: (RIKEN BioResource Center, RRID:SCR_003250)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56, 53, 54 and 55. 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.

    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 10.1101/2021.06.29.450452v1 on bioRxiv