SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

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   ScreenIT

   Oct 14, 2021

   SciScore for 10.1101/2021.10.11.463956: (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	The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
   Blinding	The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
   Power Analysis	No statistical methods were used to predetermine sample size.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Cell Lines: 293T (or HEK293T) cells, 293T-ACE2, and Expi293F cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, 2 mM L-glutamine, and in the …

   SciScore for 10.1101/2021.10.11.463956: (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	The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
   Blinding	The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
   Power Analysis	No statistical methods were used to predetermine sample size.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Cell Lines: 293T (or HEK293T) cells, 293T-ACE2, and Expi293F cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, 2 mM L-glutamine, and in the presence of 5% CO2.	HEK293T suggested: None Expi293F suggested: RRID:CVCL_D615)
   293T-ACE2 cells, derived from 293T, stably express human ACE2 71.	293T-ACE2 suggested: None
   Expi293F cells, derived from the 293 cells, were purchased from ThermoFisher Scientific (cat # A14528; RRID: CVCL_D615).	Expi293F detected: ( RRID:CVCL_D615)
   Viral infectivity measurements: The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72,73. 293T cells were transfected at 60–80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311).	293T suggested: None
   Recombinant DNA
   Sentences	Resources
   Untagged and double-tagged SARS-CoV-2 spike variants were generated based on a template full-length pCMV3-SARS-CoV-2 Spike (codon-optimized, Sino Biological, cat # VG40589-UT) plasmid that has translated amino acid sequence identical to QHD43416.1 (GenBank).	pCMV3-SARS-CoV-2 suggested: None
   D614G point mutation was introduced into both untagged full-length pCMV3 SD614 and double-tagged SD614 Q3/A4 constructs to generate both untagged and tagged SG614 variants.	pCMV3 suggested: RRID:Addgene_161029)
   E484K point mutation was introduced into both untagged and tagged pcDNA3.1 SAlpha by site-specific mutagenesis.	pcDNA3.1 suggested: RRID:Addgene_79663)
   Viral infectivity measurements: The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72,73. 293T cells were transfected at 60–80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311).	pCMV suggested: RRID:Addgene_20783)
   Briefly, DNA sequence encoding monomeric hACE2 followed by an HRV3C cleavage site, monomeric Fc tag, and 8xHisTag at the 3’-end were synthesized and cloned into the pVRC8400 vector.	pVRC8400 suggested: RRID:Addgene_63164)
   Software and Algorithms
   Sentences	Resources
   Where indicated, the conformational effects of hACE2 on S proteins were conducted by pre-incubating fluorescently labeled viruses with 200 μg/ml hACE2 for 90 mins at room temperature before imaging and smFRET imaging data were taken in the continued presence of 200 μg/ml hACE2. smFRET quantification and statistical analysis: The analysis of smFRET data was performed using a MATLAB-based customized SPARTAN software package 78.	SPARTAN suggested: (SPARTAN, RRID:SCR_014901)
   Based on visual inspection of fluorescence and FRET traces that revealed direct observations of state-to-state (conformation-to-conformation) transitions, FRET histograms were fitted into the sum of four Gaussian distributions using the least-squares fitting algorithm in MATLAB.	MATLAB suggested: (MATLAB, RRID:SCR_001622)

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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 pages 20 and 22. 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.
   • 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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   Version published to 10.1101/2021.10.11.463956v1 on bioRxiv

   Oct 12, 2021