Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution

  1. Tyler N. Starr
  2. Allison J. Greaney
  3. William W. Hannon
  4. Andrea N. Loes
  5. Kevin Hauser
  6. Josh R. Dillen
  7. Elena Ferri
  8. Ariana Ghez Farrell
  9. Bernadeta Dadonaite
  10. Matthew McCallum
  11. Kenneth A. Matreyek
  12. Davide Corti
  13. David Veesler
  14. Gyorgy Snell
  15. Jesse D. Bloom

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved variants with substitutions in the spike receptor-binding domain (RBD) that affect its affinity for angiotensin-converting enzyme 2 (ACE2) receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites—a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single–amino acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently Asn 501 →Tyr (N501Y), cause epistatic shifts in the effects of mutations at other sites. These epistatic shifts shape subsequent evolutionary change—for example, enabling many of the antibody-escape substitutions in the Omicron RBD. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.

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  1. Version published to 10.1126/science.abo7896
  2. ScreenIT

    SciScore for 10.1101/2022.02.24.481899: (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
    To quantify ACE2 expression levels, HEK-293T cells expressing high, low or no ACE2 were resuspended in FACS buffer (PBS+2% BSA) and incubated for 1 h with 1:500 rabbit anti-ACE2 antibody (Abcam ab272500).
    ACE2
    suggested: (LSBio (LifeSpan Cat# LS-C349-500, RRID:AB_1271970)
    Experimental Models: Cell Lines
    SentencesResources
    Entry titers of spike-pseudotyped lentiviral particles were determined on HEK-293T cell lines expressing high levels of human ACE2 (BEI Resources NR-52511) (34) and low levels of human ACE2.
    HEK-293T
    suggested: RRID:CVCL_A7UK)
    For titering, ACE2-high and ACE2-low cells were seeded at 1.2e4 cells per well in poly-L-lysine-coated 96-well plates (Greiner 655930) in 50 uL D10 media (plus 1 μg/mL doxycycline for the ACE2-low cell line).
    ACE2-low
    suggested: None
    Software and Algorithms
    SentencesResources
    Library generation: Site-saturation mutagenesis libraries spanning all 201 positions in variant RBDs were created as described for the Beta library by Greaney et al. (14).
    Beta
    suggested: (BETA, RRID:SCR_007556)
    Raw CCS reads are available on the NCBI Sequence Read Archive, BioProject PRJNA770094
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Nucleotide mutations annotated on the tree were converted to amino acid mutations using matUtils (version 0.4.8) (20), using the Wuhan-Hu-1 genome (NCBI RefSeq NC_045512.2) as a reference.
    matUtils
    suggested: None
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    Cell culture supernatant was collected five days after transfection and supplemented to a final concentration of 80 mM Tris-HCl pH 8.0, 100 mM NaCl, and then incubated with BioLock (IBA GmbH) solution.
    BioLock
    suggested: None
    Several subsequent rounds of model building and refinement were performed using Coot (38), ISOLDE (39), Refmac5 (40), Phenix (41) and MOE (https://www.chemcomp.com), to arrive at a final model of the quaternary complex.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    The following force fields were used: Amber ff14SB for the protein (44), GLYCAM_06j-1 for glycans (45), TIP3P for water (46) and for the neutralizing 0.15 M of NaCl Joung & Cheatham parameters (47) were used, and the Li parameters (48) for Zn and divalent ions.
    Amber
    suggested: (AMBER, RRID:SCR_016151)
    Figures were generated using matplotlib.
    matplotlib
    suggested: (MatPlotLib, RRID:SCR_008624)
    Structures were aligned in PyMol to minimize RBD structure RMSD.
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)

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

    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 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.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2022.02.24.481899 on bioRxiv