SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein

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1. Version published to 10.1371/journal.ppat.1010811

   Sep 12, 2022
2. 

   ScreenIT

   Jan 7, 2022

   SciScore for 10.1101/2022.01.04.474979: (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	Contamination: Cells were frequently tested for mycoplasma contamination and consistently tested negative.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 15 additional minutes, cells were incubated in blocking buffer (permeabilization buffer supplemented with 1 % BSA), and further incubated in blocking buffer containing anti-Strep mouse antibody (1:1000 dilution), and anti-Sirt5 rabbit antibody (1:1000 dilution).	anti-Strep suggested: None anti-Sirt5 suggested: None
   The next day, the cells were washed with PBS three times and incubated in the …

   SciScore for 10.1101/2022.01.04.474979: (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	Contamination: Cells were frequently tested for mycoplasma contamination and consistently tested negative.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 15 additional minutes, cells were incubated in blocking buffer (permeabilization buffer supplemented with 1 % BSA), and further incubated in blocking buffer containing anti-Strep mouse antibody (1:1000 dilution), and anti-Sirt5 rabbit antibody (1:1000 dilution).	anti-Strep suggested: None anti-Sirt5 suggested: None
   The next day, the cells were washed with PBS three times and incubated in the blocking buffer containing anti-mouse IgG donkey antibody conjugated with Alexa 488 (1:500 dilution, Thermo Fisher)	anti-mouse IgG suggested: None
   anti-rabbit IgG donkey antibody conjugated with Alexa 555 (1:500 dilution, Thermo Fisher), and for counter-staining, DAPI (1 μg/ml	anti-rabbit IgG donkey antibody suggested: None anti-rabbit IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   A549 cells stably expressing ACE2 (A549-ACE2) were a gift from O. Schwartz (Pasteur Institute, Paris).	A549 suggested: None
   Early passage HEK293T cells were transfected with 1.5 μg of dCas9-KRAB-MeCP2 repressor plasmid (Addgene #110824) and 0.5 μg of Piggyback Transposase (gift from Maxim Greenberg), using PEI 25K transfection reagent (Polysciences Inc, Cat. 23966-1), according to the manufacturer’s instructions.	HEK293T suggested: None
   We combined 10 pmol of Streptococcus pyogenes NLS-Sp.Cas9-NLS (SpCas9) nuclease (Aldevron, USA, Cat. 9212) with 30 pmol of total synthetic sgRNA (10 pmol each sgRNA) to form ribonucleoproteins (RNPs) in 20 μL of total volume with SE Buffer for A549-ACE2 cells.	A549-ACE2 suggested: None
   Transfection, Strep affinity purification, and Flag immunoprecipitation in HEK-293T cells: HEK-293T cells were plated in six-well plates or 10-cm dishes.	HEK-293T suggested: None
   Sample Preparation for Proteomic Analysis: HEK-293T Sirt5-KD cells were transfected with plasmids expressing Nsp14-strep in the presence or absence of SIRT5 with 3 biological replicates for each condition.	Sirt5-KD suggested: None
   SARS-CoV-2 stocks were propagated in Vero-E6 cells, and their sequences were verified by next-generation sequencing.	Vero-E6 suggested: None
   For infection experiments, A549-ACE2 or Calu3 cells were seeded into 12- or 24-well plates and rested for at least 24 hours prior to infection.	Calu3 suggested: RRID:CVCL_EQ19)
   Infections of HCT-8 cells with HCoV-OC43 were performed similarly.	HCT-8 suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmids expressing GFP and Nsp14 proteins (from SARS-CoV-2, SARS-CoV and HCoV-OC43) with a C-terminus strep tag were a gift from Nevan Krogan (1, 2), and are also available on Addgene (pLVX-EF1alpha-SARS-CoV-2-nsp14-2xStrep-IRES-Puro, Addgene #141380).	pLVX-EF1alpha-SARS-CoV-2-nsp14-2xStrep-IRES-Puro suggested: None
   Mammalian expression plasmids for SIRT5 and SIRT5-H158Y with a myc-his tag in a pCDNA 3.1 vector were available in the Verdin lab (26).	pCDNA 3.1 suggested: RRID:Addgene_20407)
   Early passage HEK293T cells were transfected with 1.5 μg of dCas9-KRAB-MeCP2 repressor plasmid (Addgene #110824) and 0.5 μg of Piggyback Transposase (gift from Maxim Greenberg), using PEI 25K transfection reagent (Polysciences Inc, Cat. 23966-1), according to the manufacturer’s instructions.	dCas9-KRAB-MeCP2 suggested: RRID:Addgene_110821)
   Nsp14 is cytotoxic, and we used 0.5 μg of Nsp14-strep plasmid for a six-well plate and 4 μg for a 10-cm dish.	Nsp14-strep suggested: None
   Other co-transfecting plasmids, such as pcDNA-SIRT5, were used at the same concentration except when specifically mentioned.	pcDNA-SIRT5 suggested: None
   Protein purification and enzymatic assays: Nsp10 and Nsp14 proteins from the Wuhan strain of SARS-CoV-2 (NC_045512.2) were codon-optimized, ordered as Gblocks (IDT), and cloned into a pVFT1S expression vector using a HiFi DNA Assembly kit (NEB)	pVFT1S suggested: None
   Software and Algorithms
   Sentences	Resources
   Methyltransferase assays were performed in reaction buffer (50 mM Hepes, pH 7.0, 6 mM KCl, 2 mM DTT, 1 mM MgCl2, and 0.1 mg/ml BSA) in presence of 0.1 mM NAD+ and 10 μM SAM.	SAM suggested: (SAM, RRID:SCR_010951)
   Analyses were run using GraphPad Prism version 9.1.2 for macOS (GraphPad Software, USA, www.graphpad.com).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   A genome index was constructed using GRCh38 genome build with Gencode v38 annotation of the transcriptome, and Genbank MT246667.1 for the sequence of SARS-CoV-2, USA/WA-1/2020 isolate.	Gencode suggested: (GENCODE, RRID:SCR_014966)
   Differential gene expression analysis was done with DEseq2, which was also used to generate normalized gene counts (71).	DEseq2 suggested: (DESeq2, RRID:SCR_015687)
   Over-representation of biological gene sets in the gene clusters was investigated using the R clusterProfiler package and enricher function (72).	clusterProfiler suggested: (clusterProfiler, RRID:SCR_016884)

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   Results from OddPub: Thank you for sharing your code and 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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3. 

   Version published to 10.1101/2022.01.04.474979v1 on bioRxiv

   Jan 5, 2022