The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants

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1. Version published to 10.1128/jvi.01301-22

   Oct 12, 2022
2. 

   ScreenIT

   May 25, 2022

   SciScore for 10.1101/2022.05.20.492832: (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: All the cell lines were cultured at 37°C under a 5% CO2 atmosphere, and were routinely screened for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The membrane was blocked with 5% dried milk in PBS Tween 0.1 %, before incubation with three primary antibodies (S1, S2, and p24 Gag or actin) for 1h at room temperature (RT), followed by 3 washes in PBS Tween 0.1 %, and incubation with secondary antibodies for 30 min at RT.	S1 suggested: None S2 suggested: None Gag or actin suggested: None
   Primary antibodies consisted in two …

   SciScore for 10.1101/2022.05.20.492832: (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: All the cell lines were cultured at 37°C under a 5% CO2 atmosphere, and were routinely screened for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The membrane was blocked with 5% dried milk in PBS Tween 0.1 %, before incubation with three primary antibodies (S1, S2, and p24 Gag or actin) for 1h at room temperature (RT), followed by 3 washes in PBS Tween 0.1 %, and incubation with secondary antibodies for 30 min at RT.	S1 suggested: None S2 suggested: None Gag or actin suggested: None
   Primary antibodies consisted in two anti-spike antibodies (rabbit anti-S1 Genetex #GTX135356, 1:1000 and mouse anti-S2 Genetex #GTX632604, 1:1000), and one normalization antibody: mouse anti-p24 Gag (R&D Systems # MAB7360; 1:1000) or mouse anti-actin (Cell Signaling #8H10D10, 1:2000).	anti-spike suggested: None anti-p24 suggested: None anti-actin (Cell Signaling #8H10D10 suggested: None
   Anti-rabbit and anti-mouse IgG secondary antibodies, conjugated to DyLight-800 (Bethyl Laboratories, #A80-304D8) and DyLight-680 (ThermoFisher, #SA5-35521), respectively, were used at a 1:5000 dilution each.	Anti-rabbit suggested: None anti-mouse IgG suggested: (Thermo Fisher Scientific Cat# SA5-35521, RRID:AB_2556774)
   After two PBS washes, cells were stained with a Goat anti-human AF647 antibody (ThermoFisher, #A21445) at 1:500 for 30 min at 4°C.	anti-human AF647 suggested: None
   To evaluate ACE2 and TMPRSS2 expression in the target cell lines used, cells were surface-labelled with the goat anti-ACE2 antibody (R&D #AF933) at 5 mg/mL for 30 min at 4°C.	anti-ACE2 suggested: None
   After washing, cells were incubated with the secondary antibody donkey anti-goat-AF647 (ThermoFisher #A21447) at a 1:500 dilution.	anti-goat-AF647 suggested: None
   Cells were intracellularly stained with a rabbit anti-TMPRSS2 antibody (Atlas antibodies, HPA035787) at 0.3 mg/mL.	anti-TMPRSS2 suggested: (Sigma-Aldrich Cat# HPA035787, RRID:AB_2674782)
   After washing, the secondary staining was done with a donkey anti-rabbit-AF555 antibody (Fisher #16229260) at a 1:500 dilution.	anti-rabbit-AF555 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: HEK 293Tn (purchased from SBI Biosciences) and Calu-3 (ATCC	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   HEK 293T-hACE2-TMPRSS2 cells (called herein HEK-ACE2-TMPRSS2) were induced for TPMRSS2 expression by addition of doxycycline (0.5 μg/mL, Sigma) and were maintained in DMEMc with blasticidin (10 μg/mL, InvivoGen) and puromycin (1 μg/mL, Alfa Aesar).	HEK 293T-hACE2-TMPRSS2 suggested: None
   U2OS cells expressing hACE2 and GFP1-10 or hACE2, TMPRSS2, and GFP1-10 were maintained in DMEMc supplemented with blasticidin (10 μg/mL, InvivoGen), puromycin (1 μg/mL	U2OS suggested: None
   HEK 293T and Vero-E6 cells expressing GFP1-10 and GFP11 were maintained in DMEMc supplemented with 1 μg/mL and 4 μg/mL of puromycin, respectively.	HEK 293T suggested: None Vero-E6 suggested: None
   Production of spike-pseudotyped lentivectors: GFP lentiviral particles pseudotyped with the SARS-CoV-2 spike were prepared by transfection of HEK 293Tn cells using the CaCl2 method.	HEK 293Tn suggested: RRID:CVCL_UL49)
   Infection with spike-pseudotyped lentivectors: The day before infection with spike-pseudotyped GFP-lentivectors, 100,000 HEK-ACE2-TMPRSS2 cells were plated in 96-well plates, and TMPRSS2 was induced when needed by the addition of doxycycline.	HEK-ACE2-TMPRSS2 suggested: None
   HEK and U2OS were infected with 0.125 μg of p24 Gag equivalent, while Calu-3 were infected with 1 μg of p24 Gag equivalent, all in a final volume of 100 μL.	HEK suggested: None
   For infection, 50 μL of medium containing 0.065 μg of p24 Gag equivalent was added onto HEK ACE2 +/− TMPRSS2 and U2OS ACE2 +/− TMPRSS2, while 0.5 μg of p24 Gag equivalent in 50 μl added onto Calu-3 cells, resulting in a 2x dilution of the protease inhibitors.	HEK ACE2 suggested: None
   After 30 min, transfected cells were washed in DMEMna and spun at 500 g for 5 min before being mixed at a 1:1 ratio with Vero GFP11 cells in DMEMna.	Vero GFP11 suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmids: All the spike mutations were inserted into a codon-optimized version of the Wuhan-Hu-1 SARS-CoV-2 spike (GenBank: QHD43416.1) cloned into a phCMV backbone ( GenBank: AJ318514).	phCMV suggested: RRID:Addgene_15802)
   Plasmids used to produce GFP-lentiviruses were the lentivector backbone pCDH-EF1α-GFP (System Biosciences), the packaging plasmid psPAXII (Addgene), and the pRev plasmid (a gift from P. Charneau).	pCDH-EF1α-GFP suggested: None psPAXII suggested: None pRev suggested: None
   Transfection of HEK 293Tn cells with spike vectors: Transfection was performed with Lipofectamine 2000 (ThermoFisher, #11668019), using 125 ng of phCMV-Spike plasmid diluted in OptiMem medium in a final volume of 25 μL.	phCMV-Spike suggested: None
   The lentiviral vector pCDH-EF1a-GFP, the packaging plasmid psPAXII, the spike expression vector phCMV-Spike and the pRev plasmid were mixed at a 2:2:1:1 ratio, with a total DNA amount of 252 μg used per 175 cm2 flask.	pCDH-EF1a-GFP suggested: None
   The pQCXIP-empty plasmid was used to generate a spike-negative control.	pQCXIP-empty suggested: None
   Luciferase lentiviral particles were produced according to the same protocol, but with different plasmid ratios: the lentiviral backbone pHAGE-CMV-Luc2-IRES-ZsGreen, the packaging plasmid pHDM-Hgpm2, the Tat and Rev plasmids pHDM-tat1b and pRC-CMV-rev1b, and the spike plasmid phCMV-Spike were used at ratio 4.4:1:1:1:1.5 to transfect a 175 cm2 flask.	pHDM-Hgpm2 suggested: RRID:Addgene_164441) pHDM-tat1b suggested: RRID:Addgene_164442) pRC-CMV-rev1b suggested: RRID:Addgene_164443)
   Software and Algorithms
   Sentences	Resources
   Data were analyzed with the FlowJo software v10.7.1 (Becton Dickinson).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: All statistical analyses were carried out with the GraphPad Prism software (v9).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   All the graphs were generated in GraphPad Prism, except for the spider plots, which were made using Microsoft Excel (v16.16.27).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798) Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137)

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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 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 10.1101/2022.05.20.492832v1 on bioRxiv

   May 20, 2022