Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen

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1. Version published to 10.1128/jvi.02216-21

   Mar 23, 2022
2. 

   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.11.04.467291: (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
   The SARS-CoV-2 Nsp1 antibody (PA5-116941) was purchased from Themo Fisher Scientific.	Nsp1 suggested: None
   The β-actin antibody (GTX109639) was purchased from Gentex.	β-actin suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and reagents: The human kidney epithelial cell line Lenti-X 293T was purchased from Takara.	Lenti-X 293T suggested: RRID:CVCL_4401)
   The human liver cell line Huh7.5.1 was provided by Dr. Francis Chisari (Scripps Research Institute).	Huh7.5.1 suggested: RRID:CVCL_E049)
   The baby hamster kidney fibroblast cell line BHK21 (CCL-10), African green monkey kidney …

   SciScore for 10.1101/2021.11.04.467291: (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
   The SARS-CoV-2 Nsp1 antibody (PA5-116941) was purchased from Themo Fisher Scientific.	Nsp1 suggested: None
   The β-actin antibody (GTX109639) was purchased from Gentex.	β-actin suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and reagents: The human kidney epithelial cell line Lenti-X 293T was purchased from Takara.	Lenti-X 293T suggested: RRID:CVCL_4401)
   The human liver cell line Huh7.5.1 was provided by Dr. Francis Chisari (Scripps Research Institute).	Huh7.5.1 suggested: RRID:CVCL_E049)
   The baby hamster kidney fibroblast cell line BHK21 (CCL-10), African green monkey kidney epithelial cells (Vero E6; CRL-1586), Caco-2 (HTB-37), Calu-3 (HTB-55) and A549 (CCL-185) were purchased from the American Type Culture Collection.	BHK21 suggested: None A549 suggested: None
   Plasmid Construction: Doxycycline-inducible expression of SARS-CoV-2 NP was established in Vero E6, Huh7.5.1, and BHK-21 using TripZ-NP plasmid.	Vero E6 suggested: None
   RNA Electroporation: Forty-eight hours post doxycycline treatment, BHK-21-NPDox-ON cells were washed with phosphate buffered saline (PBS), trypsinized, and resuspended in complete growth medium.	BHK-21-NPDox-ON suggested: None
   For validation, A549-hACE2, Caco-2 or Calu-3 cells were seeded in 96-well plates at a density of 104 cells/well.	Caco-2 suggested: None Calu-3 suggested: None
   Recombinant DNA
   Sentences	Resources
   NP cDNA was subcloned into pTripZ (AgeI/MluI) using the following primers: TripZ-NPf: 5’-ATATAGACCGGTCCACCATGTCTGATAATGGACCCCA- 3’, TripZ-NPr: 5’- ATATAGACGCGTTTAGGCCTGAGTTGAGTCAG-3’	pTripZ suggested: RRID:Addgene_127696)
   Upon receipt, fragment 4 was subsequently subcloned into a low-copy plasmid pSMART LCAmp (Lucigen) to increase stability.	pSMART suggested: RRID:Addgene_102283)
   To introduce Nsp1 R124S/K125E, N128S/K129E and K164A/H165A mutations into the SARS-CoV-2-Rep-NanoLuc-Neo replicon, puc57-CoV2-F1 plasmids containing mutated Nsp1 were first created by using overlap PCR method with the following primers: PCR fragments were digested by Bgl II/Nhe I and ligated into Bgl II/Nhe I digested F1 plasmid.	puc57-CoV2-F1 suggested: None
   To generate standard plasmids, the cDNAs of SARS-CoV-2 ORF1ab gene, NanoLuc gene sgmRNA and neomycin phosphotransferase gene sgmRNA were cloned into a pCR2.1-TOPO plasmid respectively.	pCR2.1-TOPO suggested: None
   Software and Algorithms
   Sentences	Resources
   CC50 values were calculated using a nonlinear regression curve fit in Prism Software version 9 (GraphPad).	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 29. 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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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • No funding statement was detected.
   • 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 10.1101/2021.11.04.467291v1 on bioRxiv

   Nov 8, 2021