Nonstructural protein 1 of SARS-CoV-2 is a potent pathogenicity factor redirecting host protein synthesis machinery toward viral RNA

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1. 

   ScreenIT

   Aug 14, 2020

   SciScore for 10.1101/2020.08.09.243451: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Then antigen-specific antibodies with specific dilutions were added into cells and incubated for 30 min on ice.	antigen-specific suggested: None
   Antibody used: anti-cleaved Caspase-3(Asp175) (Sigma, 9669s, 1:200).	anti-cleaved Caspase-3 suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869) 9669s …

   SciScore for 10.1101/2020.08.09.243451: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Then antigen-specific antibodies with specific dilutions were added into cells and incubated for 30 min on ice.	antigen-specific suggested: None
   Antibody used: anti-cleaved Caspase-3(Asp175) (Sigma, 9669s, 1:200).	anti-cleaved Caspase-3 suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869) 9669s suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869)
   Experimental Models: Cell Lines
   Sentences	Resources
   D) Bar plot of firefly luciferase reporter measurement of viability effects of SARSCoV-2 ORFs in H1299-PL cells, at 24, 48 and 72 hours post ORF introduction (n = 3 replicates).	H1299-PL suggested: None
   D) Bar plot of firefly luciferase reporter measurement of viability effects of SARSCoV-2 ORFs in Vero E6-PL cells, at 24, 48 and 72 hours post ORF introduction (n = 3 replicates).	Vero E6-PL suggested: None
   Generation of stable cell lines Lentivirus was produced by transfection of co-transgene plasmid (Lenti-Fluc-Puro) and packaging plasmids (psPAX2, pMD2.G) into HEK293FT cells, followed by supernatant harvesting, filtering and concentration with Amicon filters (Sigma).	HEK293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   H1299 and Vero E6 cells were infected with Lenti-Fluc-Puro lentivirus.	H1299 suggested: NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)
   Mammalian cell culture ’ H1299, H1299-PL, Vero E6, Vero E6-PL cell lines were cultured in Dulbecco s ’ modified Eagle s medium (DMEM; Thermo fisher) supplemented with 10% Fetal bovine serum (FBS, Hyclone),1% penicillin-streptomycin (Gibco), named as D10 medium.	Vero E6 suggested: None
   Cells were transferred into 100 µl NucleocuvetteTM Vessel and NCI-H1299 [H1299] cell specific protocol were utilized according to the manufacturer’s protocol (4D-NucleofectorTM X Unit, Lonza).	NCI-H1299 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Nsp1 mutant4 has N128 and K129 were converted to S128 and E129 (N128S/K129E).	Nsp1 suggested: None
   Software and Algorithms
   Sentences	Resources
   DAVID pathway analysis of Nsp1 differentially expressed gene sets Sup table 4.1 Functional clustering of Nsp1 vs Vector Control highly Sup table 4.2 Functional clustering of Nsp1 vs Nsp1 Mutant highly Sup table 4.3 Functional clustering of Nsp1 vs Vector Control highly upregulated genes (q < 1e-30) Sup table 4.4 Functional clustering of Nsp1 vs Nsp1 Mutant highly upregulated genes (q < 1e-30) Sup table 4.5 Biological processes enrichment of Nsp1 vs Vector Control highly Sup table 4.6 Biological processes enrichment of Nsp1 vs Nsp1 Mutant highly Sup table 4.7 Biological processes enrichment of Nsp1 vs Vector Control highly upregulated genes (q < 1e-30) Sup table 4.8 Biological processes enrichment of Nsp1 vs Nsp1 Mutant highly upregulated genes (q < 1e-30) Sup table 4.9 Gene list of Nsp1 vs Vector Control highly downregulated genes (q < 1e-30) Sup table 4.10 Gene list enrichment of Nsp1 vs Nsp1 Mutant highly Sup table 4.11 Gene list enrichment of Nsp1 vs Vector Control highly upregulated genes (q < 1e-30) Sup table 4.12 Gene list enrichment of Nsp1 vs Nsp1 Mutant highly upregulated genes (q < 1e-30) Sup table 4.13 Gene list of Nsp1 vs Vector Control all downregulated genes (q < 0.01) Sup table 4.14 Gene list of Nsp1 vs Vector Control all upregulated genes (q < 0.01) STAR Methods.	STAR suggested: (STAR, RRID:SCR_015899)
   Differentially upregulated and downregulated genes were subjected to pathway analysis by DAVID (Huang et al., 2007) and/or GSEA (Subramanian et al., 2005).	DAVID suggested: (DAVID, RRID:SCR_001881) GSEA suggested: (SeqGSEA, RRID:SCR_005724)
   Data analysis was performed using GraphPad Prism v.8. and/or RStudio.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) RStudio suggested: (RStudio, RRID:SCR_000432)
   Standard in vitro transcription protocol was used for IRES RNA synthesis and purification (MEGAscript™ T7 Transcription Kit, Ambion, USA).	MEGAscript™ suggested: None
   Automated data collection was performed using SerialEM (Mastronarde, 2005).	SerialEM suggested: (SerialEM, RRID:SCR_017293)
   The model of Nsp1 C-terminal domain was manually built in COOT (Emsley et al., 2010).	COOT suggested: (Coot, RRID:SCR_014222)
   All structural figures were generated using PyMol (Schrodinger, 2015) and Chimera (Pettersen et al.,	PyMol suggested: (PyMOL, RRID:SCR_000305)

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

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 42, 48, 51 and 54. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

   Read the original source
2. 

   ScreenIT

   Aug 11, 2020

   SciScore for 10.1101/2020.08.09.243451: (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
   Then antigen-specific antibodies with specific dilutions were added into cells and incubated for 30 min on ice.	antigen-specific suggested: None
   Antibody used: anti-cleaved Caspase-3(Asp175) (Sigma, 9669s, 1:200).	anti-cleaved Caspase-3 suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869) 9669s suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869)
   Experimental Models: Cell Lines
   Sentences	Resources
   Generation of stable cell lines: Lentivirus …

   SciScore for 10.1101/2020.08.09.243451: (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
   Then antigen-specific antibodies with specific dilutions were added into cells and incubated for 30 min on ice.	antigen-specific suggested: None
   Antibody used: anti-cleaved Caspase-3(Asp175) (Sigma, 9669s, 1:200).	anti-cleaved Caspase-3 suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869) 9669s suggested: (Cell Signaling Technology Cat# 9669, RRID:AB_2069869)
   Experimental Models: Cell Lines
   Sentences	Resources
   Generation of stable cell lines: Lentivirus was produced by transfection of co-transgene plasmid (Lenti-Fluc-Puro) and packaging plasmids (psPAX2, pMD2.G) into HEK293FT cells, followed by supernatant harvesting, filtering and concentration with Amicon filters (Sigma).	HEK293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   Luc expressing H1299 and Vero E6 that with puromycin resistance cell lines were obtained and named as H1299-PL and Vero E6-PL (Vero E6-PL for short) respectively.	Vero E6 suggested: None
   Mammalian cell culture: H1299, H1299-PL, Vero E6, Vero E6-PL cell lines were cultured in Dulbecco’s modified Eagle’ s medium (DMEM; Thermo fisher) supplemented with 10% Fetal bovine serum (FBS, Hyclone),1% penicillin-streptomycin (Gibco), named as D10 medium.	H1299 suggested: None Vero E6-PL suggested: None
   Cells were transferred into 100 μl NucleocuvetteTM Vessel and NCI-H1299 [H1299] cell specific protocol were utilized according to the manufacturer’s protocol (4D-NucleofectorTM X Unit, Lonza).	NCI-H1299 suggested: None
   Gene expression analysis by mRNA sequencing (mRNA-seq, RNA-seq): For H1299-PL cells electroporated with Nsp1 or Nsp1 mutant, mRNA-seq libraries were prepared following next-generation sequencing (NGS) protocols.	H1299-PL suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Nsp1 mutant4 has N128 and K129 were converted to S128 and E129 (N128S/K129E).	Nsp1 suggested: None
   Software and Algorithms
   Sentences	Resources
   Differentially upregulated and downregulated genes were subjected to pathway analysis by DAVID (Huang et al., 2007) and/or GSEA (Subramanian et al., 2005).	DAVID suggested: (DAVID, RRID:SCR_001881) GSEA suggested: (SeqGSEA, RRID:SCR_005724)
   Data analysis was performed using GraphPad Prism v.8. and/or RStudio.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) RStudio suggested: (RStudio, RRID:SCR_000432)
   Standard in vitro transcription protocol was used for IRES RNA synthesis and purification (MEGAscript™ T7 Transcription Kit, Ambion, USA).	MEGAscript™ suggested: None
   Automated data collection was performed using SerialEM (Mastronarde, 2005).	SerialEM suggested: (SerialEM, RRID:SCR_017293)
   The model of Nsp1 C-terminal domain was manually built in COOT (Emsley et al., 2010).	COOT suggested: (Coot, RRID:SCR_014222)
   The structures of Nsp1-40S ribosome complex and Nsp1-IRES-40S ribosome complex were refined with phenix.real_space_refine module in PHENIX (Adams et al., 2010).	PHENIX suggested: (Phenix, RRID:SCR_014224)
   All structural figures were generated using PyMol (Schrodinger, 2015) and Chimera (Pettersen et al., 2004).	PyMol suggested: (PyMOL, RRID:SCR_000305)

   ---

   Results from OddPub: Thank you for sharing your data.

   ---

   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 did not find any issues relating to the usage of bar graphs.

   ---

   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 48, 42, 51 and 54. 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.

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   Read the original source
3. 

   Version published to 10.1101/2020.08.09.243451 on bioRxiv

   Aug 10, 2020