Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

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1. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.07.04.187757: (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

   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293 cells (ATCC CRL-1573), and HEK-293T cells (CRL-3216 or CRL-11268) were cultured in DMEM supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 20 mM GlutaMAX, 1×	HEK293 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   Calu3 cells (ATCC HTB-55) were maintained in EMEM supplemented with 10% FBS.	Calu3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Caco2 cells (ATCC HTB-37) were maintained in EMEM supplemented with 20% FBS.	Caco2 suggested: ATCC Cat# HTB-37, RRID:CVCL_0025)
   SUP-T1 [VB] cells (ATCC CRL-1942) were cultured in RPMI supplemented with 10% heat-inactivated FBS, 1mM …

   SciScore for 10.1101/2020.07.04.187757: (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

   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293 cells (ATCC CRL-1573), and HEK-293T cells (CRL-3216 or CRL-11268) were cultured in DMEM supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 20 mM GlutaMAX, 1×	HEK293 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   Calu3 cells (ATCC HTB-55) were maintained in EMEM supplemented with 10% FBS.	Calu3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Caco2 cells (ATCC HTB-37) were maintained in EMEM supplemented with 20% FBS.	Caco2 suggested: ATCC Cat# HTB-37, RRID:CVCL_0025)
   SUP-T1 [VB] cells (ATCC CRL-1942) were cultured in RPMI supplemented with 10% heat-inactivated FBS, 1mM sodium pyruvate	SUP-T1 suggested: None
   Vero cells (ATCC CCL-81) were cultured in DMEM high glucose media containing 10% heat-inactivated fetal bovine serum, and 1X Penicillin/Streptomycin/L-Glutamine.	Vero suggested: None
   Virus production: 24 hrs prior to transfection, 6 × 105 HEK-293 cells were plated per well in 6 well plates.	HEK-293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   TMPRSS2 expressing SupT1 cells were then transduced with a second vector expressing ACE2, followed by puromycin selection at 1 ug/mL.	SupT1 suggested: BCRC Cat# 60191, RRID:CVCL_1714)
   HEK293T cells were seeded overnight in DMEM high glucose media (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), and Penicillin/-Streptomycin-L-Glutamine (Life Technologies).	HEK293T suggested: None
   Production of SARS-CoV-2 virus-like particles (VLPs): HEK-293T cells were cultured in DMEM supplemented with 10% heat-inactivated bovine serum, and transfected with pcDNA3.1 plasmids encoding the SARS-CoV-2 M, E, N, and S proteins, in different combinations, as indicated.	HEK-293T suggested: None
   Software and Algorithms
   Sentences	Resources
   These sequences were processed using a script importing biopython (Cock et al., 2009) to remove any gaps introduced by the alignment process and translate the sequence to protein space.	biopython suggested: (Biopython, RRID:SCR_007173)
   The bresulting values were rendered as plots using matplotlib (Hunter, 2007).	matplotlib suggested: (MatPlotLib, RRID:SCR_008624)
   The script for analyzing and plotting D614G variant frequency is available via GitHub: https://github.com/broadinstitute/sc2-variation-scripts The diversity of SNPs and their functional effects based on the same GISAID sequences and MAFFT alignment used to plot the frequency of D614G over time, with the 5’ and 3’ ends not masked.	MAFFT suggested: (MAFFT, RRID:SCR_011811)
   The VCF file with SNPs was annotated for functional effects using SnpEff (Cingolani et al., 2012).	SnpEff suggested: (SnpEff, RRID:SCR_005191)
   Data was analyzed using FlowJo 10.5 (FlowJo, LLC, Ashland, OR)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The steady state analysis was performed using Scrubber software and the KD value was determined.	Scrubber suggested: (Scrubber2, RRID:SCR_015745)
   Local resolution was estimated using cryoSPARC to extend from 3Å to 6 Å	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Model building: Atomic models were prepared with Coot based on the resolved structure of D614 SARS-CoV-2 Spike (PDB: 6vxx and 6vsb).	Coot suggested: (Coot, RRID:SCR_014222)
   Real-space refinements were performed using PHENIX with secondary structure restraints.	PHENIX suggested: (Phenix, RRID:SCR_014224)
   MolProbity was used to evaluate the geometries of the structural model.	MolProbity suggested: (MolProbity, RRID:SCR_014226)

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   Results from OddPub: Thank you for sharing your code and 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 TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04425629	Recruiting	Safety, Tolerability, and Efficacy of Anti-Spike (S) SARS-Co…
   NCT04426695	Recruiting	Safety, Tolerability, and Efficacy of Anti-Spike (S) SARS-Co…

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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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2. Version published to 10.1016/j.cell.2020.09.032

   Oct 1, 2020
3. 

   NCRC

   Aug 3, 2020

   Our take

   This analysis of over 52,000 SARS-CoV-2 genomes, available as a preprint and thus not yet peer reviewed, shows that a genetic variant with a single amino acid change in the spike protein (D614G) has become the dominant infection globally since its discovery in late January 2020. Experiments in human cell cultures indicate that the variant is more infectious than the wildtype (D614), although human antibodies are still capable of neutralizing both spike variants. Additional studies in animal models will be necessary to determine if the D614G variant produces higher viral loads and is more transmissible.

   Study design

   ecological;other

   Study population and setting

   The authors analyzed SARS-CoV-2 genomes available on GISAID (https://www.gisaid.org/) as of June 25, 2020 (n = 52,292), representing …

   Our take

   This analysis of over 52,000 SARS-CoV-2 genomes, available as a preprint and thus not yet peer reviewed, shows that a genetic variant with a single amino acid change in the spike protein (D614G) has become the dominant infection globally since its discovery in late January 2020. Experiments in human cell cultures indicate that the variant is more infectious than the wildtype (D614), although human antibodies are still capable of neutralizing both spike variants. Additional studies in animal models will be necessary to determine if the D614G variant produces higher viral loads and is more transmissible.

   Study design

   ecological;other

   Study population and setting

   The authors analyzed SARS-CoV-2 genomes available on GISAID (https://www.gisaid.org/) as of June 25, 2020 (n = 52,292), representing infections in Africa, South America, North America, Europe, Oceania, and Asia. They analyzed the frequency of a viral variant with an amino acid change (D to G) at position 614 of the spike protein over time, both globally and within geographic regions. They compared the relative infectivity of the D614G variant virus versus the wildtype (D614) using HIV-1 vectors expressing either variant of the SARS-CoV-2 spike protein. Relative infectivity was compared in human lung, colon, and embryonic kidney cells or human T lymphocytes expressing angiotensin-converting enzyme 2 (ACE2; SARS-CoV-2 uses ACE2 as a receptor to enter human cells) and transmembrane protease, serine 2 (TMPRSS2; SARS-CoV-2 spike protein is primed by TMPRSS2). The authors also compared the relative infectivity of D614G variant in human embryonic kidney cells expressing different ACE2 orthologues from human, pangolin, pig, cat, dog, rat, and mouse. To determine the possible effect of D614G on human immunity, the authors tested the ability of four monoclonal antibodies designed to target and neutralize SARS-CoV-2 spike protein. They tested each antibody alone or in combinations in a neutralization assay against wildtype D14 spike variant and the D614G variant.

   Summary of main findings

   The D614G variant was first detected in sequenced genomes in late January 2020 in China and Germany, and has since increased in frequency so that it is the dominant variant globally (>97% of published sequences). Even for Africa and South America where early cases had a higher frequency of D614G, suggesting a founder effect, the frequency has continued to increase. In all human cell types tested, the D614G variant was 4-6 times more infectious than the D614 variant. In embryonic kidney cells or T lymphocytes expressing ACE2 and TMPRSS2, the D614G variant was 9 times more infectious than D614. Similarly, the D614G variant was 2-3 times more infectious than D614 in human embryonic kidney cells expressing human, pangolin, pig, cat, and dog orthologues of ACE2; infection of cells with mouse or rat ACE2 with D614 or D614G was not detectable. All monoclonal antibodies, and combinations thereof, were equally capable of neutralizing D614 and D614G spike protein variants.

   Study strengths

   Compared to previous studies, this study uses many more sequences to assess the change in frequency of the D614G variant. It also provides evidence that the D614G is potentially more infectious (at least in cell cultures) than the wildtype D614 variant.

   Limitations

   While the selective advantage of the D614G variant suggested by this study is an important finding, there are other neutral factors (founder effects, bottlenecks in transmission) that may have contributed to the spread of the D614G variant. Modeling efforts that account for selection and neutral effects will be needed to ascertain the relative effects of each. Additionally, the authors cannot tell the exact geographical origin of D614G, because of the few viral genomes that were sequenced early in the outbreak. Since the sequences used in this study represent only a miniscule fraction of the total cases (0.5% of almost 10 million cases as of 25 June 2020), more sequencing from banked samples may provide more resolution. Lastly, the experimental model, wherein lentiviral particles pseudotyped with SARS-CoV-2 spike protein are used to infect human cell cultures, cannot fully represent the complexity of SARS-CoV-2 infection in humans. Additional experiments in an animal model with D614 and D614G variants of actual SARS-CoV-2 that demonstrate higher viral loads for the D614G variant (and possibly increased transmissability between animals), or human outbreak data that demonstrates greater transmissability in cases with the D614G variant, will be a necessary next step to verifying any selective advantage claimed from population-level frequency data.

   Value added

   This study supports earlier reports that the D614G variant of SARS-CoV-2 has increased in frequency during the pandemic and now represents the dominant genetic variant globally, confirms that the D614G variant is more infective in human cell cultures, and demonstrates that human antibodies are still capable of neutralizing the D614G variant as well as the wildtype.

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4. 

   Version published to 10.1101/2020.07.04.187757v2 on bioRxiv

   Jul 16, 2020
5. 

   ScreenIT

   Jul 5, 2020

   SciScore for 10.1101/2020.07.04.187757: (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

   Cell culture All cells were cultured in humidified incubators with 5 % CO2 at 37º C , and monitored for mycoplasma contamination using the Mycoplasma Detection kit ( Lonza LT07-318)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   To determine if this is the case , the neutralization potency was assessed of four monoclonal antibodies that target the SARS-CoV-2 Spike protein receptor binding domain .	SARS-CoV-2 Spike protein receptor binding domain . suggested: None …

   SciScore for 10.1101/2020.07.04.187757: (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

   Cell culture All cells were cultured in humidified incubators with 5 % CO2 at 37º C , and monitored for mycoplasma contamination using the Mycoplasma Detection kit ( Lonza LT07-318)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   To determine if this is the case , the neutralization potency was assessed of four monoclonal antibodies that target the SARS-CoV-2 Spike protein receptor binding domain .	SARS-CoV-2 Spike protein receptor binding domain . suggested: None
   As all four of these monoclonal antibodies bind epitopes within the Spike protein receptor binding domain , it remains important to determine whether the D614G variants changes neutralization sensitivity to other classes of anti-Spike monoclonal antibodies .	anti-Spike suggested: None
   Vero cells were challenged with pVSV-SARS-CoV-2-S-mNeon pseudoparticles encoding either ( A ) 614D or ( B ) 614G spike variants , in the presence of serial dilutions of the indicated anti-Spike monoclonal antibodies or IgG1 isotype control .	IgG1 isotype control . suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   When Calu-3 human lung epithelial cells were used as targets , challenge with lentivirus bearing D614G resulted in 6-fold more GFP-positive cells , or 5-fold more bulk luciferase activity , than did particles bearing D614 S protein ( Figure 2) .	Calu-3 suggested: BCRJ Cat# 0264, CVCL_0609
   When Caco-2 human colon epithelial cells were used as the target cells 4-fold higher infectivity was observed with D614G ( Figure 2) .	Caco-2 suggested: CVCL_YP12
   Additionally , when HEK-293 cells or SupT1 cells had been rendered infectable by stable expression of exogenous ACE2 and TMPRSS2 , D614G was 9-fold more infectious than D614 ( Figure 2) .	HEK-293 suggested: None
   SARS-CoV-2 D614G S protein variant enhances infectivity Lentiviral virions bearing either GFP or Luciferase transgenes , and pseudotyped with either SARS-CoV-2 D614 or D614G S proteins , were produced by transfection of HEK293 cells , and used to transduce human Calu3 lung cells , Caco2 colon cells , and either HEK293 or SupT1 cells stably expressing ACE2 and TMPRSS2 .	HEK293 suggested: None <div style="margin-bottom:8px"> <div><b>SupT1</b></div> <div>suggested: ICLC Cat# HTL96007, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_1714">CVCL_1714</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In a heterologous expression system on HeLa cells , human , civet , horse-shoe bat ( Rhinolophus sinicus) , and pig orthologs conferred susceptibility to infection , while the mouse ortholog did not ( Zhou et al. , 2020b) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>HeLa</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293 cells ( ATCC CRL-1573) , and HEK-293T cells ( CRL-3216 or CRL-3216 ) were cultured in DMEM supplemented with 10 % heat-inactivated FBS , 1 mM sodium pyruvate , 20 mM GlutaMAX , 1×</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>HEK-293T</b></div> <div>suggested: ATCC Cat# CRL-3216, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu3 cells ( ATCC HTB-55 ) were maintained in EMEM supplemented with 10 % FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Calu3</b></div> <div>suggested: ATCC Cat# HTB-55, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Caco2 cells ( ATCC HTB-37 ) were maintained in EMEM supplemented with 20 % FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Caco2</b></div> <div>suggested: ATCC Cat# HTB-37, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0025">CVCL_0025</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SUP-T1 [ VB ] cells ( ATCC CRL-1942 ) were cultured in RPMI supplemented with 10 % heat-inactivated FBS , 1mM sodium pyruvate</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>SUP-T1</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were seeded overnight in DMEM high glucose media ( Life Technologies ) containing 10 % heat-inactivated fetal bovine serum ( Life Technologies) , and Penicillin/- Streptomycin-L-Glutamine ( Life Technologies) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>HEK293T</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody dilutions were mixed 1:1 with pseudoparticles for 30 minutes at room temperature prior to addition onto Vero cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Vero</b></div> <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned to the ancestral reference sequence ( NCBI GenBank accession NC_045512.2 ) using mafft v7.464 ( Katoh and Standley , 2013 ) with the " --keeplength " and " --addfragments " parameters , which preserve the coordinate space of the reference sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>mafft</b></div> <div>suggested: (MAFFT, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011811">SCR_011811</a>)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These sequences were processed using a script importing biopython ( Cock et al. , 2009 ) to remove any gaps introduced by the alignment process and translate the sequence to protein space.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>biopython</b></div> <div>suggested: (Biopython, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007173">SCR_007173</a>)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting values were rendered as plots using matplotlib ( Hunter , 2007) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>matplotlib</b></div> <div>suggested: (MatPlotLib, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008624">SCR_008624</a>)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data was analyzed using FlowJo 10.5 ( FlowJo , LLC , Ashland , OR)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>FlowJo</b></div> <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div> </div> </td></tr></table> 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 OddPub: Thank you for sharing your code. 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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

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6. 

   Version published to 10.1101/2020.07.04.187757v1 on bioRxiv

   Jul 4, 2020