ORF6 protein of SARS-CoV-2 inhibits TRIM25 mediated RIG-I ubiquitination to mitigate type I IFN induction

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   ScreenIT

   May 10, 2022

   SciScore for 10.1101/2022.05.05.490850: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Secondary antibody incubation was performed at room temperature for 2 hours using Goat Anti-Mouse IgG H&L (31430, Thermo) or Goat Anti-Rabbit IgG H&L (31460, Thermo).	Anti-Mouse IgG suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307) Anti-Rabbit IgG suggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341)
   Experimental Models: Cell Lines
   Sentences	Resources
   ORF6 variant mutants were constructed by subcloning the construct in a TA backbone, followed by substituting the …

   SciScore for 10.1101/2022.05.05.490850: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Secondary antibody incubation was performed at room temperature for 2 hours using Goat Anti-Mouse IgG H&L (31430, Thermo) or Goat Anti-Rabbit IgG H&L (31460, Thermo).	Anti-Mouse IgG suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307) Anti-Rabbit IgG suggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341)
   Experimental Models: Cell Lines
   Sentences	Resources
   ORF6 variant mutants were constructed by subcloning the construct in a TA backbone, followed by substituting the residues as described before (Edelheit et al, 2009) and cloning in the pCAGGS backbone using EcoRI and XhoI. Cell Lines and cell culture: Human embryonic kidney 293T (HEK293T), A549 lung adenocarcinoma, and HeLa cells were purchased from National Centre for Cell Science (NCCS), and HEK-ACE2, Vero cells were procured from the America Type Culture Collection (ATCC, Bethesda, MD).	HEK293T suggested: None A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Viruses and infection: Infection: SARS-CoV2 (Isolate Hong Kong/VM20001061/2020, NR-52282, BEI Resources, NIAID, NIH) was propagated and titered by plaque assay in Vero E6 cells as described before (Case et al, 2020).	Vero E6 suggested: None
   Plasmid transfection: HEK-293T cells (0.1 X 106 cells/well) were seeded in a 24 well plate pre-coated with 0.1 mg/mL poly-L-lysine (P9155-5MG, Sigma-Aldrich) and 24 hours later used for transfection.	HEK-293T suggested: None
   Similarly, Vero cells were seeded on coverslips in 24 well plates (0.1 X 106 cells/well) for overnight incubation.	Vero suggested: None
   Similarly, HeLa cells were transfected with 250 ng of RIG I Flag or MAVS Flag and 250 ng of ORF6 strep for 24 hours.	HeLa suggested: None
   Recombinant DNA
   Sentences	Resources
   For constructing the deletion mutants, the desired sequence was PCR amplified from pLVX-EF1alpha-SARS-CoV-2-ORF6-2xStrep-IRES-Puro plasmid, followed by cloning in the pCAGGS backbone using EcoRI (ER0271, Thermo scientific) and XhoI (ER0691, Thermo scientific) restriction enzyme including the full-length ORF6.	pLVX-EF1alpha-SARS-CoV-2-ORF6-2xStrep-IRES-Puro suggested: RRID:Addgene_141387) pCAGGS suggested: RRID:Addgene_127347)
   Luciferase Reporter Assay: For the IFN induction assay, HEK-293T cells were co-transfected, in duplicates, with 50 ng of IFNβ-luc firefly luciferase reporter plasmid, and 20 ng of pRL-TK Renilla luciferase reporter plasmid along with 500 ng of SARS-CoV-2 protein expression plasmid or empty vector.	pRL-TK Renilla luciferase reporter suggested: RRID:Addgene_12179)
   They were then co-transfected with 500 ng of IRF3-GFP and 500 ng of SARS-CoV-2 protein-expressing plasmid using Lipofectamine 2000 reagent (11668019, Invitrogen).	SARS-CoV-2 protein-expressing suggested: None
   Software and Algorithms
   Sentences	Resources
   Graphical representations and statistical analysis: All numerical data of luciferase assays and qRt-PCR were analysed and plotted using GraphPad Prism v8.0.2.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The model diagram of ORF6 action (Fig 7) and 3-D structure (Sup Fig 3C) were made by using Biorender	Biorender suggested: (Biorender, RRID:SCR_018361)

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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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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.05.05.490850 on bioRxiv

   May 6, 2022