Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

  1. Tyler N. Starr
  2. Allison J. Greaney
  3. Sarah K. Hilton
  4. Daniel Ellis
  5. Katharine H.D. Crawford
  6. Adam S. Dingens
  7. Mary Jane Navarro
  8. John E. Bowen
  9. M. Alejandra Tortorici
  10. Alexandra C. Walls
  11. Neil P. King
  12. David Veesler
  13. Jesse D. Bloom

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  1. ScreenIT

    SciScore for 10.1101/2020.06.17.157982: (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
    SentencesResources
    Following overnight equilibration of ACE2 binding at room temperature, cells were washed in ice-cold PBS-BSA, and resuspended in PBS-BSA containing 1:200 diluted FITC-conjugated anti c-Myc antibody (Immunology Consultants Lab, CMYC-45F) to label for RBD surface expression via a C-terminal c-Myc epitope tag, and 1:200 diluted PE-conjugated streptavidin (Thermo Fisher S866) to detect bound biotinylated ACE2 ligand.
    anti c-Myc
    suggested: None
    c-Myc epitope tag ,
    suggested: None
    For library expression experiments, 45 OD units yeast were washed twice with PBS-BSA and labeled in 3mL 1:100 diluted anti-Myc-FITC antibody for 1hr at 4°C with gentle mixing.
    anti-Myc-FITC
    suggested: (Sigma-Aldrich Cat# SAB4700448, RRID:AB_10896411)
    Antibody epitopes were mapped from crystal structures 6W41 (Yuan et al., 2020b), 6WAQ (Wrapp et al., 2020b), 2DD8 (Prabakaran et al., 2006), 3BGF (Pak et al., 2009), 2GHW (Hwang et al., 2006), 7BZ5 (Wu et al., 2020), and cryo-EM structures 6NB6 and 6NB7 (Walls et al., 2019), and 6WPS (Pinto et al., 2020).
    6NB7
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, 2.5e5 293T cells per well were seeded in 12-well plates in 1 mL D10 growth media (DMEM with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin).
    293T
    suggested: None
    Media was removed from the 293T-ACE2 cells and replaced with fresh D10 containing 50 μL of pseudovirus supernatant in a final volume of 150 μL.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Plasmids were transfected into 150mL suspension expi293F or HEK293F cells at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm and harvested 3 days later.
    HEK293F
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Alignment and phylogeny: We used the curated RBD sequence set from Letko et al. (Letko et al., 2020), adding newly described RBD sequences from sarbecovirus strains RaTG13 (Zhou et al., 2020b), RmYN02 (Zhou et al., 2020a), GD-Pangolin and GX-Pangolin (Lam et al., 2020), and the additional non-Asian bat sarbecovirus isolate BtKY72 (Tong et al., 2009).
    RaTG13
    suggested: None
    Software and Algorithms
    SentencesResources
    For one bin in which the number of HiSeq reads was less than the number of cells sorted into a bin, we re-amplified PCR product from a newly purified plasmid aliquot, and obtained reads via a single lane of MiSeq 50bp single end sequencing.
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Data visualization: The interactive heatmap of mutational effects shown at https://jbloomlab.github.io/SARS-CoV-2-RBD_DMS/ was made using the altair (VanderPlas et al., 2018) Python package.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Structural images were rendered in PyMol.
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)
    RBD nucleotide sequences were aligned via mafft with a gap opening penalty of 4.5, and the maximum likelihood phylogeny was inferred in RAxML (Stamatakis, 2014) under the GTR model with 4 gamma-distributed discrete categories of among-site rate variation.
    RAxML
    suggested: (RAxML, RRID:SCR_006086)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    To some degree, these caveats are universal of experimental studies, as even sophisticated animal models are imperfect proxies for true fitness (Louz et al., 2013)—but they are especially true for basic biochemical phenotypes like the ones we measure. However, on a hopeful note, our measurements correlate well with cellular entry by spike-pseudotyped viral particles expressing sarbecovirus RBD homologs (Figures 1D) and single mutants of the SARS-CoV-2 RBD (Figure 4E). Furthermore, fitness ultimately arises from the concerted action of biochemical phenotypes, which are in turn determined by genotype (Dean and Thornton, 2007; Harms and Thornton, 2013; Russell et al., 2014). By making the first link from mutations to biochemical phenotypes, we have taken a step towards enabling better interpretation of viral genetic variation. One important area where our maps do have clear relevance is assessing the potential for SARS-CoV-2 to undergo antigenic drift by fixing mutations at sites targeted by antibodies, as occurs for some other viruses such as influenza (Smith et al., 2004). The RBD is the dominant target of neutralizing antibodies (Cao et al., 2020; Ju et al., 2020; Pinto et al., 2020; Rogers et al., 2020; Seydoux et al., 2020; Shi et al., 2020; Wu et al., 2020; Zost et al., 2020), and so any antigenic drift will be constrained by its mutational tolerance. Our results show that many mutations to the RBD are well-tolerated with respect to both protein folding and ACE2 binding. H...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    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.

    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 checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1016/j.cell.2020.08.012
  3. ScreenIT

    SciScore for 10.1101/2020.06.17.157982: (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
    SentencesResources
    We cloned the RBDs into a vector for yeast cell surface display, induced RBD expression, and incubated with a fluorescent antibody targeting a C-terminal epitope tag and varying concentrations of fluorescently labeled human ACE2 (Figure 1B).
    ACE2
    suggested: None
    Following overnight equilibration of ACE2 binding at room temperature , cells were washed in icecold PBS-BSA , and resuspended in PBS-BSA containing 1:200 diluted FITC-conjugated anti c-Myc antibody ( Immunology Consultants Lab , CMYC-45F ) to label for RBD surface expression via a C-terminal c-Myc epitope tag , and 1:200 diluted PEconjugated streptavidin ( Thermo Fisher S866 ) to detect bound biotinylated ACE2 ligand.
    anti c-Myc
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>c-Myc epitope tag ,</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For library expression experiments , 45 OD units yeast were washed twice with PBS-BSA and labeled in 3mL 1:100 diluted anti-Myc-FITC antibody for 1hr at 4°C with gentle mixing .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-Myc-FITC</b></div>
        <div>suggested: (Sigma-Aldrich Cat# SAB4700448, <a href="https://scicrunch.org/resources/Any/search?q=AB_10896411">AB_10896411</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody epitopes were mapped from crystal structures 6W41 ( Yuan et al. , 2020b) , 6WAQ ( Wrapp et al. , 2020b) , 2DD8 ( Prabakaran et al. , 2006) , 3BGF ( Pak et al. , 2009) , 2GHW ( Hwang et al. , 2006) , 7BZ5 ( Wu et al. , 2020) , and cryo-EM structures 6NB6 and 6NB7 ( Walls et al. , 2019) , and 6WPS ( Pinto et al. , 2020) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>6NB7</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</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">Briefly , 2.5e5 293T cells per well were seeded in 12-well plates in 1 mL D10 growth media ( DMEM with 10 % heat-inactivated FBS , 2 mM lglutamine , 100 U/mL penicillin , and 100 μg/mL streptomycin) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Media was removed from the 293TACE2 cells and replaced with fresh D10 containing 50 μL of pseudovirus supernatant in a final volume of 150 μL .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293TACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids were transfected into 150mL suspension expi293F or HEK293F cells at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm and harvested 3 days later.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293F</b></div>
        <div>suggested: None</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">Methods Data and Code Availability We provide all data and code in the following ways: ● Raw data tables of our replicate functional scores at the level of single mutations ( Supplemental File 3 , and GitHub: https://github.com/jbloomlab/SARS-CoV-2-RBD_DMS/blob/master/results/single_mut_effects/single_mut_effects.csv ) ● Raw data tables of our replicate functional scores among sarbecovirus homologs ( Supplemental File 1 and GitHub: https://github.com/jbloomlab/SARS-CoV-2-RBD_DMS/blob/master/results/single_mut_effects/homolog_effects.csv ) ● Illumina sequencing counts for each barcode among FACS bins ( https://github.com/jbloomlab/SARS-CoV-2RBD_DMS/blob/master/results/counts/variant_counts.csv ) ● The complete variant:barcode lookup table ( https://github.com/jbloomlab/SARS-CoV-2RBD_DMS/blob/master/results/variants/codon_variant_table.csv ) ● The complete computational workflow to generate and analyze these data , including reproducible code within a programmatically constructed computational environment ( https://github.com/jbloomlab/SARS-CoV-2-RBD_DMS ) ● A Markdown summary of the organization of analysis steps , with links to key data files and Markdown summaries of each step in the analysis pipeline ( https://github.com/jbloomlab/SARS-CoV-2RBD_DMS/blob/master/results/summary/summary.md) , with specific Markdown summaries linked in the relevant Methods sections below ● All raw sequencing data are uploaded to the NCBI Short Read Archive ( BioProject PRJNA639956)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>NCBI Short Read Archive</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>BioProject</b></div>
        <div>suggested: (NCBI BioProject, <a href="https://scicrunch.org/resources/Any/search?q=SCR_004801">SCR_004801</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For one bin in which the number of HiSeq reads was less than the number of cells sorted into a bin , we re-amplified PCR product from a newly purified plasmid aliquot , and obtained reads via a single lane of MiSeq 50bp single end sequencing .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MiSeq</b></div>
        <div>suggested: (A5-miseq, <a href="https://scicrunch.org/resources/Any/search?q=SCR_012148">SCR_012148</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data visualization The interactive heatmap of mutational effects shown at https://jbloomlab.github.io/SARS-CoV-2-RBD_DMS/ was made using the altair ( VanderPlas et al. , 2018 ) Python package.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Python</b></div>
        <div>suggested: (IPython, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001658">SCR_001658</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural images were rendered in PyMol .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PyMol</b></div>
        <div>suggested: (PyMOL, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000305">SCR_000305</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD nucleotide sequences were aligned via mafft with a gap opening penalty of 4.5 , and the maximum likelihood phylogeny was inferred in RAxML ( Stamatakis , 2014 ) under the GTR model with 4 gamma-distributed discrete categories of among-site rate variation .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>RAxML</b></div>
        <div>suggested: (RAxML, <a href="https://scicrunch.org/resources/Any/search?q=SCR_006086">SCR_006086</a>)</div>
      </div>
    </td></tr></table>

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    • . To some degree, these caveats are universal of experimental studies, as even sophisticated animal models are imperfect proxies for true fitness

    Results from OddPub: Thank you for sharing your code and data.

    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.

  4. Version published to 10.1101/2020.06.17.157982v1 on bioRxiv