Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains

  1. Sarah M. Roelle
  2. Nidhi Shukla
  3. Anh T. Pham
  4. Anna M. Bruchez
  5. Kenneth A. Matreyek

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Abstract

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV) and SARS-CoV-2 enter cells via engagement of angiotensin converting enzyme-2 (ACE2), while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clades 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from Rhinolophus alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

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  1. Version published to 10.1371/journal.pbio.3001738
  2. ScreenIT

    SciScore for 10.1101/2021.12.25.474149: (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
    (3F10 antibody from Roche) or anti-β-actin antibody (Santa Cruz, #S47778).
    3F10
    suggested: None
    anti-β-actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The lentiviral vectors were produced by transfecting 1.5 million HEK 293T cells in a single well of a 6-well plate, using PEI-Max MW 40,000 (PolySciences, CAS Number: 49553-93-7) mixed with 600 ng of PsPax2 (Addgene # 12260), 600 ng of the lentiviral transfer vector pLenti_CMV-EGFP-2A-mNeonGreen (Addgene # 171599), and 600 ng of various viral envelope plasmids.
    HEK 293T
    suggested: None
    Recombinant DNA
    SentencesResources
    (Addgene plasmid #171595), and AttB_ACE2[dEcto]_IRES-mCherry-H2A-P2A-PuroR (Addgene plasmid #171596). psPAX2 and pMD2.
    pMD2
    suggested: None
    (Addgene plasmid #12260; http://n2t.net/addgene:12260; RRID: Addgene_12260) and (Addgene plasmid # 12259; http://n2t.net/addgene:12259; RRID:Addgene_12259), respectively.
    detected: RRID:Addgene_12260)
    detected: RRID:Addgene_12259)
    The lentiviral vectors were produced by transfecting 1.5 million HEK 293T cells in a single well of a 6-well plate, using PEI-Max MW 40,000 (PolySciences, CAS Number: 49553-93-7) mixed with 600 ng of PsPax2 (Addgene # 12260), 600 ng of the lentiviral transfer vector pLenti_CMV-EGFP-2A-mNeonGreen (Addgene # 171599), and 600 ng of various viral envelope plasmids.
    pLenti_CMV-EGFP-2A-mNeonGreen
    suggested: RRID:Addgene_171599)
    For comparing the singleplex and duplex infection assays, 50,000 cells stably modified with the attB-ACE2(dEcto)_IRES-mCherry-H2A-2A-PuroR plasmid, 50,000 cells stably modified with the attB-ACE2_IRES-iRFP670-H2A-2A-PuroR plasmid, or various combinations of both cells were plated into individual wells of a 24-well plate.
    dEcto)_IRES-mCherry-H2A-2A-PuroR
    suggested: None
    attB-ACE2_IRES-iRFP670-H2A-2A-PuroR
    suggested: None
    Software and Algorithms
    SentencesResources
    BLAST searches and protein sequence alignments: The receptor binding domains of SARS-CoV, WIV1, and SARS-CoV-2 spikes were used as initial query sequences for National Center for Biotechnology Information (NCBI) BLASTp searches.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    All RBD fragments were manually curated and aligned using Clustal Omega[58].
    Clustal Omega[58
    suggested: None
    The aligned sequences were used as the input for a custom python script that performed calculations of amino acid identity at each position for any given pair of RBD sequences.
    python
    suggested: (IPython, RRID:SCR_001658)
    To perform a comprehensive search for clade 2 RBD spike sequences to gain a near-complete sampling of their sequence diversity, we first performed an NCBI BLASTp search using the YN2013 RBD amino acid sequence as the query.
    BLASTp
    suggested: (BLASTP, RRID:SCR_001010)
    The captured TIFF files were analyzed with a custom Python script utilizing the numpy, scipy, cv2, skimage, and PIL packages.
    numpy
    suggested: (NumPy, RRID:SCR_008633)
    scipy
    suggested: (SciPy, RRID:SCR_008058)
    Data Analysis and statistics: Data analysis was performed using version 1.4.1717 of RStudio, with the exception of flow cytometry data, which was first analyzed using version 10.8.0 of FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Modeling of Bat ACE2 three-dimensional structures: The HHpred web server was used to perform homology alignment of various Bat ACE2 sequences with human ACE2 structures (pdb: 6m17 and 6m18) [62, 63].
    HHpred
    suggested: (HHpred, RRID:SCR_010276)
    A structural model was then built with the MODELLER web server [64], and the ACE2 models were each aligned to the SARS-CoV-2 RBD in PDB:6m17 using the default alignment settings in PyMol.
    MODELLER
    suggested: (MODELLER, RRID:SCR_008395)
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)

    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 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.

    About SciScore

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  3. Version published to 10.1101/2021.12.25.474149v1 on bioRxiv