Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

  1. Huihui Mou
  2. Brian D. Quinlan
  3. Haiyong Peng
  4. Guanqun Liu
  5. Yan Guo
  6. Shoujiao Peng
  7. Lizhou Zhang
  8. Meredith E. Davis-Gardner
  9. Matthew R. Gardner
  10. Gogce Crynen
  11. Lindsey B. DeVaux
  12. Zhi Xiang Voo
  13. Charles C. Bailey
  14. Michael D. Alpert
  15. Christoph Rader
  16. Michaela U. Gack
  17. Hyeryun Choe
  18. Michael Farzan

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus ) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

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  1. Version published to 10.1371/journal.ppat.1009501
  2. ScreenIT

    SciScore for 10.1101/2020.06.29.178459: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    hACE2 expression was confirmed by immunofluorescence staining using mouse monoclonal antibody against c-Myc antibody 9E10 (Thermo Fisher) and goat-anti-mouse FITC (Jackson ImmunoResearch Laboratories, Inc).
    c-Myc
    suggested: None
    FITC
    suggested: (S. Britt; University of Texas at Austin Cat# anti-Rh6 FITC, RRID:AB_2736995)
    Expression of ACE2 orthologs were detected using mouse monoclonal antibody against c-Myc antibody 9E10 (Thermo Fisher) and Goat-anti-mouse FITC (Jackson ImmunoResearch Laboratories, Inc).
    Goat-anti-mouse FITC
    suggested: None
    Briefly, a CM5 sensor chip was immobilized with a mouse anti-human IgG CH2 mAb using reagents and instructions supplied with the Human Antibody Capture Kit (GE Healthcare) in order to capture RBD-Fc or ACE2-Fc.
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expi293 FTM cell (Thermo Fisher) was cultured in Expi293 FTM expression medium (Thermo Fisher) at 37°C in a shaking incubator at 125 rpm and 8% CO2.
    Expi293
    suggested: RRID:CVCL_D615)
    Parental 293T cells were transduced with generated MLV virus, and the 293T-hACE2 cell lines were selected and maintained with medium containing puromycin (Sigma).
    293T
    suggested: None
    293T-hACE2
    suggested: None
    293T-human ACE2 stable cells were maintained in growth media including 3 μg/ml puromycin for selection of stably transduced cells.
    ACE2
    suggested: RRID:CVCL_DR94)
    Flow cytometry to test the binding of coronavirus RBD-Fc proteins to receptors: HEK293T cells were transfected with plasmids encoding ACE2 orthologs or human DPP4 using PEI 40K (Polysciences) according to manufacturer’s instructions.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were analyzed by flow cytometry (BD Accuri C6 Flow Cytometry) and data was analyzed using FlowJo (FlowJo, LLC)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Computational analysis: IC50 analysis was performed after concentration was log10 transformed, using default settings for log(inhibitor) vs. response Variable slope method in GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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).

    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 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).

    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

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  3. Version published to 10.1101/2020.06.29.178459v1 on bioRxiv