SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

  1. Matteo Castelli
  2. Luigi Scietti
  3. Nicola Clementi
  4. Mattia Cavallaro
  5. Silvia Faravelli
  6. Alberta Pinnola
  7. Elena Criscuolo
  8. Roberta Antonia Diotti
  9. Massimo Clementi
  10. Federico Forneris
  11. Nicasio Mancini

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Abstract

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 – the closest bat relative – spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

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  1. Version published to 10.1101/2021.08.11.455960v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.08.11.455960: (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
    SentencesResources
    Recombinant Protein Expression and Purification: Recombinant hACE2 ectodomain, affiACE2 ectodomain, SARS-CoV-2 RBD, and RaTG13 RBD were produced using HEK293-F cells (Invitrogen) cultivated in suspension using Freestyle medium (Invitrogen) as described in (35).
    HEK293-F
    suggested: RRID:CVCL_6642)
    Recombinant DNA
    SentencesResources
    Generation of plasmid vectors for recombinant protein production: The pCAGGS plasmids for production of the C-terminal His-tagged SARS-CoV-2 S protein (#NR_52310) and RBD (#NR_52310), were obtained from BEIRESOURCES (NY, USA).
    pCAGGS
    suggested: RRID:Addgene_18926)
    The designed codon-optimized sequence encoding for RaTG13 RBD and the cDNA for affiACE2 ectodomain (residue 19-615, based on the sequence deposited on Genbank under the ID MT394225.1) were synthesized by Genewiz with flanking 5’-BamHI and 3’-NotI restriction sites and sub-cloned into pUPE.
    pUPE
    suggested: None
    Software and Algorithms
    SentencesResources
    RaTG13-associated affiACE2 allele identification: R. affinis ACE2 known coding sequences (GenBank database accession IDs MT394203 to MT394225) were fed to Sequence Read Archive (SRA) Nucleotide BLAST to retrieve ACE2 Illumina reads from the SRA dataset with accession ID SRX7724752.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    The designed codon-optimized sequence encoding for RaTG13 RBD and the cDNA for affiACE2 ectodomain (residue 19-615, based on the sequence deposited on Genbank under the ID MT394225.1) were synthesized by Genewiz with flanking 5’-BamHI and 3’-NotI restriction sites and sub-cloned into pUPE.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Final 3D models were generated using iterations of automatic refinement using low resolution protocols in phenix.refine using non crystallographic symmetry restraints as well as external restraints based on individual chains from PDB ID 6VW1, alternated with manual adjustments using COOT (40).
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Assessment of final structure quality was carried out with the Molprobity server and with the RCSB PDB Validation Server (39, 41).
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Unbiased Spike Simulations: Deglycosylated spike ectodomains (residues 14-1146) were completed, reverted to the wild- type when needed or mutated with Modeller using the following templates: PDB ID 6ZGF for RaTG13 closed, PDB ID 6ZGG for all cleaved, 1-up systems, PDB ID 6ZGI for all cleaved, closed systems, PDB ID 6VYB for all uncleaved, 1-up system and PDB ID 6ZGE for all uncleaved, closed systems (42).
    Modeller
    suggested: (MODELLER, RRID:SCR_008395)
    All simulations were performed using the GROMACS 5.1.4 code (44).
    GROMACS
    suggested: (GROMACS, RRID:SCR_014565)

    Results from OddPub: Thank you for sharing your 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 29 and 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2021.08.11.455960v1 on bioRxiv