SARS-CoV-2 Triggers an MDA-5-Dependent Interferon Response Which Is Unable To Control Replication in Lung Epithelial Cells

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1. Version published to 10.1128/jvi.02415-20

   Mar 25, 2021
2. 

   ScreenIT

   Oct 30, 2020

   SciScore for 10.1101/2020.10.28.358945: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   An overnight incubation at 4°C with mab J2 (Scicons) followed by incubation in a secondary anti-mouse antibody conjugated to Alexa Fluor 546 and in Alexa Fluor 488 Phalloidin (Thermofisher Scientific) for 2h at RT were used to visualise dsRNA and F-actin, respectively.	F-actin suggested: None
   Cells were incubated on ice for 30-45 min in FACS buffer (PBS1X-5% FCS) containing a 1/250 dilution of Alexa 488-conjugated anti-Spike antibody (GTX632604 conjugated …

   SciScore for 10.1101/2020.10.28.358945: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   An overnight incubation at 4°C with mab J2 (Scicons) followed by incubation in a secondary anti-mouse antibody conjugated to Alexa Fluor 546 and in Alexa Fluor 488 Phalloidin (Thermofisher Scientific) for 2h at RT were used to visualise dsRNA and F-actin, respectively.	F-actin suggested: None
   Cells were incubated on ice for 30-45 min in FACS buffer (PBS1X-5% FCS) containing a 1/250 dilution of Alexa 488-conjugated anti-Spike antibody (GTX632604 conjugated using the Zenon Alexa Fluor 488 Mouse IgG Labeling Kit, ThermoFisher) and washed 4 times in FACS buffer.	anti-Spike suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
   , RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).	RIG-I suggested: None MDA-5 suggested: (Cell Signaling Technology Cat# 5321, RRID:AB_10694490) anti-mouse suggested: None anti-rabbit immunoglobulin suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Caco-2 and Calu-3 cells were obtained from American Type Culture Collection (ATCC); HEK-Blue™ IFN-α/β and IFN-γ cells were obtained from InvivoGen; 293T, MDCK, A549, Vero E6 cells were gifts from Michael Malim’s lab, Wendy Barclay’s lab, and from the CEMIPAI facility, respectively.	Caco-2 suggested: None MDCK suggested: None Vero E6 suggested: RRID:CVCL_XD71)
   A549 and Caco-2 cells stably expressing ACE2 and TMPRSS2 were generated by transduction with either RRL.sin.cPPT.SFFV/IRES-puro.WPRE, RRL.sin.cPPT.SFFV/IRES-neo.WPRE, RRL.sin.cPPT.SFFV/IRES-hygro.WPRE or RRL.sin.cPPT.SFFV.	A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Cas9 activity was checked using the XPR_047 assay (a gift from David Root, Addgene 107145) and was 79.5% and >83.4%, respectively, for Cas9-expressing A549-ACE2 and Calu-3 cells.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   Lentiviral production and infection: Lentiviral vector stocks were obtained by polyethylenimine (PEI; for LentiGuides) or Lipofectamine 3000 (Thermo Scientific; for ACE2 and TMPRSS2 lentiviral vectors)-mediated multiple transfection of 293T cells in 6-well plates with vectors expressing Gag-Pol, the miniviral genome, the Env glycoprotein at a ratio of 1:1:0.5.	293T suggested: None
   , RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).	MDA-5 suggested: None
   Software and Algorithms
   Sentences	Resources
   Post-processing of RAW Airyscan images was performed using the Zen Black software.	Zen Black suggested: (Black Zen software, RRID:SCR_018163)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.

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3. 

   Version published to 10.1101/2020.10.28.358945v1 on bioRxiv

   Oct 28, 2020