Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

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1. 

   ScreenIT

   Jan 4, 2022

   SciScore for 10.1101/2021.12.29.474471: (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
   A chimeric neutralizing antibody specific against SARS-CoV-2 S protein receptor binding domain (NR-55410) was provided by ACROBiosystems through BEI resources.	SARS-CoV-2 S protein receptor binding domain suggested: None
   Rabbit monoclonal anti-N antibody (Sino Biologicals #40143-R001) was diluted 1:5,000 in 0.1% Tween-20 in PBS (PBS-T), and incubated on samples overnight at 4 °C.	anti-N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Huh7.5-hAT cells (provided by Gregory …

   SciScore for 10.1101/2021.12.29.474471: (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
   A chimeric neutralizing antibody specific against SARS-CoV-2 S protein receptor binding domain (NR-55410) was provided by ACROBiosystems through BEI resources.	SARS-CoV-2 S protein receptor binding domain suggested: None
   Rabbit monoclonal anti-N antibody (Sino Biologicals #40143-R001) was diluted 1:5,000 in 0.1% Tween-20 in PBS (PBS-T), and incubated on samples overnight at 4 °C.	anti-N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Huh7.5-hAT cells (provided by Gregory Melikian and Mariana Marin), have been modified to stably express human ACE2 and human TMPRSS2, to enhance infection by SARS-CoV-2.	Huh7.5-hAT suggested: None
   A549 (ATCC) are derived from human adenocarcinoma cells.	A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Human embryonic kidney (HEK) 293T cells stably express the SV40 large T antigen, are resistant to neomycin, and were selected for their high transfectability (ATCC).	HEK suggested: None
   293T-hAT cells (provided by Bruce Torbett) have been modified to stably express human ACE2 and human TMPRSS2, to permit infection by SARS-CoV-2	293T-hAT suggested: None
   Chinese hamster ovary (CHO)-K1 cells (ATCC) are a clonal derivative of the parental CHO cell line and were cultured in humidified conditions at 37 °C in 5% CO2 in F12 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin and 100 μg/ml streptomycin.	CHO)-K1 suggested: None CHO suggested: None
   Both Caco2 and Calu3 cells were cultured in humidified conditions at 37 °C in 5% CO2 in Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS, 2 mM L-glutamine, NEAA, and 100 U/ml penicillin and 100 μg/ml streptomycin.	Caco2 suggested: None Calu3 suggested: RRID:CVCL_EQ19)
   Construction of the SARS-CoV-2 subgenomic replicon (SARS-2R): DNA fragments of SARS-CoV-2 replicon were either synthesized from Integrated DNA Technologies Inc or amplified by reverse transcription and PCR (RT-PCR) with viral RNA template that was extracted from the supernatants of SARS-CoV-2-infected Vero cells (SARS-CoV strain 2019-nCoV/USA_WA1/2020 (WA1).	Vero suggested: None
   Cells were pulsed once at 280 V for BHK-21 cells and 250 V for 293T cells with Ingenio EZporator MIR51000 electroporation system in low voltage (LV) mode (150 Ω internal resistance and 1050 μF capacitance).	293T suggested: None
   Determination of Replicon EC50 and EC90 values: BHK21 cells seeded in 6-well plate were transfected with SARS-2R using jetPRIME transfection reagent (Polyplus transfection).	BHK21 suggested: ECACC Cat# 05062302, RRID:CVCL_6F52)
   Generation of cells that carry SARS-2R replicon: BHK-21 cells were seeded into a 6-well plate, then transfected with replicon SARS-2R_NG_NeoR_NL using jetPRIME transfection reagent following the manufacturer’s instructions.	BHK-21 suggested: None
   qPCR to determine levels of SARS-CoV-2 nucleic acid: RNA and DNA was harvested from the BHK-SARS-2R_GFP_NeoR_NL cell line using the RNeasy mini kit (Qiagen) and the QIAamp genomic DNA mini kit (Qiagen), respectively.	BHK-SARS-2R_GFP_NeoR_NL suggested: None
   To titer viral stocks, VeroE6 cells were seeded in a 96-well plate at 20,000 cells per well.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Target cells were seeded at 4 × 104 cells/well (293T, 293T-hAT) or 2 × 104 cells/well (Huh-7.5, Huh7.5-hAT) and incubated at 37 °C for 24 h.	Huh-7.5 suggested: RRID:CVCL_7927)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Production of SARS-CoV-2 stocks: SARS-CoV-2 stocks were produced by infection of VeroE6 with SARS-CoV strain 2019-nCoV/USA_WA1/2020 or SARS2 hCoV-19/England/204820464/2020 (B.1.1.7).	SARS-CoV-2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmids, antivirals and chemicals: Plasmids pLVX-EF1a-IRES-Puro SARS-CoV-2 E_NR-52967 and pLVX-EF1a-IRES-Puro SARS-CoV-2 M_NR-52968 (BEI) were gifts from Nevan Krogan (University of California, San Francisco) (71).	pLVX-EF1a-IRES-Puro suggested: None
   SARS-S in pCAGGS was a gift from Paul Bates (University of Pennsylvania) (72).	pCAGGS suggested: RRID:Addgene_127347)
   SARS-CoV-2 S was synthesized (Genscript) and cloned into pcDNA3.1 between two PmeI sites using NEBuilder.	pcDNA3.1 suggested: RRID:Addgene_79663)
   Multi-step cloning strategy was used, whereby the fragments named SARS-CoV-2/1 to SARS-CoV-2/9 were sequentially cloned into pBeloBAC11 vector to generate pBAC-SARS-CoV-2-REP.	pBeloBAC11 suggested: RRID:Addgene_60342) pBAC-SARS-CoV-2-REP suggested: None
   Each well was transfected with 1 μg replicon SARS-2R_GFP_NeoR_NL, and 0.25 μg each of N, E, and M expression vectors, and 0.25 μg of expression vectors for one of SARS-CoV S, SARS-CoV-2 S, VSV-G or an empty vector, with 4 μl jetPRIME transfection reagent (Polyplus).	VSV-G suggested: RRID:Addgene_138479)
   Software and Algorithms
   Sentences	Resources
   293T-hAT cells (provided by Bruce Torbett) have been modified to stably express human ACE2 and human TMPRSS2, to permit infection by SARS-CoV-2	SARS-CoV-2 suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
   Images were captured as a 5×4 matrix and GFP positive cells were enumerated using Gen5 software (Biotek).	Gen5 suggested: (Gen5, RRID:SCR_017317)
   The number of infected cells at each compound concentration was determined by high content microscopy and dose response curves were plotted and EC50s calculated with Prism software.	Prism suggested: (PRISM, RRID:SCR_005375)
   A theoretically perfect assay would have Z’ = 1. Statistics: Data were tabulated and graphs plotted using Excel (Microsoft) or Prism (Graphpad).	Excel suggested: None Graphpad suggested: (GraphPad, RRID:SCR_000306)

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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: We detected the following sentences addressing limitations in the study:

   A primary limitation in SARS-CoV-2 research is the high risk associated with working with a pathogenic respiratory pathogen, in terms of both the availability of suitable facilities and of suitably trained researchers. Here we present a range of approaches to facilitate research into all facets of SARS-CoV-2 replication, including cell entry, nucleic acid transcription and replication, and virus assembly and release; collectively these systems are tools to identify and characterize inhibitors at any point in SARS-CoV-2 replication. These systems are based on subgenomic SARS-CoV-2 replicons carrying reporter genes, reducing biological hazards and providing means to easily measure viral replication. The most popular strategy for generating replicons from positive-strand RNA virus is to transcribe RNA in vitro that can be electroporated into target cells to initiate replication. To facilitate use of the assay, particularly in the context of large-scale screening, we eliminated the need for in vitro transcription by cloning cDNA for the SARS-CoV-2 replicons into a BAC under control of a CMV promoter. The replicon comprised all replication-essential proteins and sequences but omitted the structural proteins S, E, and M; additionally, reporter genes were included as a separate ORF using the TRS-B of M to drive production of the sgRNA expressing the reporter cassette. Placing the reporter cassette in a sgRNA ensured that formation of a functional RTC was essential for reporter gene ...

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04960202	Recruiting	EPIC-HR: Study of Oral PF-07321332/Ritonavir Compared With P…

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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: 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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   Read the original source
2. 

   Version published to 10.1101/2021.12.29.474471v1 on bioRxiv

   Dec 30, 2021