Antiviral immune responses, cellular metabolism and adhesion are differentially modulated by SARS-CoV-2 ORF7a or ORF7b

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   ScreenIT

   Jun 3, 2022

   SciScore for 10.1101/2022.06.01.494101: (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
   Protein samples were resolved by SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA), followed by blocking for 1 h with 5% nonfat milk in Tris-buffered saline-Tween 20 buffer and probing with antibodies against Strep Tag (SAB2702215, Sigma-Aldrich), γ-catenin (sc-514115, Santa Cruz Biotechnology) and GAPDH (A00084, GenScript) (Supplementary Table 6).	γ-catenin suggested: (Immunological …

   SciScore for 10.1101/2022.06.01.494101: (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
   Protein samples were resolved by SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA), followed by blocking for 1 h with 5% nonfat milk in Tris-buffered saline-Tween 20 buffer and probing with antibodies against Strep Tag (SAB2702215, Sigma-Aldrich), γ-catenin (sc-514115, Santa Cruz Biotechnology) and GAPDH (A00084, GenScript) (Supplementary Table 6).	γ-catenin suggested: (Immunological Sciences Cat# AB-90215, RRID:AB_2892157) sc-514115, Santa Cruz Biotechnology suggested: None GAPDH suggested: None
   The washed membranes were incubated with secondary antibody StarBright Blue 700 Goat anti-mouse IgG (Bio-rad).	anti-mouse IgG suggested: None
   Coverslips were washed three times with PBS before secondary anti-mouse antibodies incubation (1:1000 dilution).	anti-mouse suggested: None
   For staining, cells were resuspended in PBS 0.1% BSA 0.01% NaN3 containing the monoclonal antibody PE mouse anti-human CD54 (BD Pharmingen) or its isotype control at a concentration of 1/500.	anti-human CD54 suggested: None
   The primary anti-ZO-1 antibody (ref. 61-7300, Invitrogen) was diluted at 1:100 in PBS containing 1% BSA and incubated for 3 h at RT.	anti-ZO-1 suggested: (Innovative Research Cat# 61-7300, RRID:AB_138452)
   The secondary antibody (anti-rabbit IgG-FITC, ref. 9887, Sigma) was diluted 1:200 in PBS and incubated for 1 h at RT.	anti-rabbit suggested: (Sigma-Aldrich Cat# F9887, RRID:AB_259816) anti-rabbit IgG-FITC suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, VSV-G pseudotyped ORF7a or ORF7b lentivirus was produced by co-transfection of HEK293T cells with the pLVX-ORF7a or pLVX-ORF7b plasmids, pCMV-Gag-Pol and pCMV-VSV-G using Lipofectamine 2000 Reagent (Thermo Fisher Scientific) as per manufacturer instructions.	HEK293T suggested: None
   RNA isolation and sequencing: WT A549, A549-ORF7a and A549-ORF7b cells were seeded (3×10E5) in 6-well plates and lysed using RLT buffer for RNA isolation (RNeasy mini kit, Qiagen).	A549-ORF7b suggested: None
   After 24 h incubation, A549 cells were labeled with calcein-AM.	A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Recombinant DNA
   Sentences	Resources
   Lentivirus production, cell culture and transduction: ORF7a or ORF7b coding sequences (codon-optimized for mammalian expression) were cloned into pLVX-EF1α-IRES-Puro Cloning and Expression Lentivector (System Biosciences) to generate pseudotyped lentiviral particles encoding the ORF7a or ORF7b accessory proteins of SARS-CoV-2 (Wuhan-Hu-1 isolate) at the CNIC (Centro Nacional de Investigaciones Cardiovasculares) Viral Vector Unit (ViVU).	pLVX-EF1α-IRES-Puro suggested: None
   Briefly, VSV-G pseudotyped ORF7a or ORF7b lentivirus was produced by co-transfection of HEK293T cells with the pLVX-ORF7a or pLVX-ORF7b plasmids, pCMV-Gag-Pol and pCMV-VSV-G using Lipofectamine 2000 Reagent (Thermo Fisher Scientific) as per manufacturer instructions.	pLVX-ORF7a suggested: None pLVX-ORF7b suggested: None pCMV-Gag-Pol suggested: None pCMV-VSV-G suggested: RRID:Addgene_8454)
   Software and Algorithms
   Sentences	Resources
   Raw counts were transformed with the vst function in the DESeq2 package (Love et al., 2014) of the R software version 3.6.3 (R Core Team, 2020), and subsequent PCA was performed with the prcomp function.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Finally, the PCA graph was made with Graphad Prism software.	Graphad Prism suggested: None
   All sequencing data sets are available in the NCBI BioProject database under accession number PRJNA841835.	BioProject suggested: (NCBI BioProject, RRID:SCR_004801)
   Enrichment analyses were carried out by selecting the genomics sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, and CORUM.	GO Biological suggested: None
   Relative expression results were calculated using GenEx6 Pro software (MultiD-Göteborg, Sweden), based on the Cq values obtained.	GenEx6 Pro suggested: None
   For these experiments, a CytoFLEX flow cytometer (Beckman Coulter) was used and data was analyzed using FlowJo v10 (BD Biosciences).	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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   Results from OddPub: Thank you for sharing your data.

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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 10.1101/2022.06.01.494101v1 on bioRxiv

   Jun 1, 2022