The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

  1. Pei Li
  2. Ruixuan Guo
  3. Yan Liu
  4. Yintgtao Zhang
  5. Jiaxin Hu
  6. Xiuyuan Ou
  7. Dan Mi
  8. Ting Chen
  9. Zhixia Mu
  10. Yelin Han
  11. Zhewei Cui
  12. Leiliang Zhang
  13. Xinquan Wang
  14. Zhiqiang Wu
  15. Jianwei Wang
  16. Qi Jin
  17. Zhaohui Qian

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Abstract

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with SARS-CoV-2 among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although RaACE2 binding to the receptor binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but limited for mouse, ACE2s enter cells. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.16.385849: (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
    Antibodies: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against SARS-CoV-2 RBD antibodies(#40592-T62), rabbit polyclonal against SARS-CoV-2 S2 antibodies(#40590-T62), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RB01) were purchased from Sino Biological Inc. (Beijing, China).
    Antibodies
    suggested: (Sino Biological Cat# 40150-MM02, RRID:AB_2860459)
    SARS
    suggested: (Sino Biological Cat# 40150-MM02, RRID:AB_2860459)
    #40150-MM02
    suggested: (Sino Biological Cat# 40150-MM02, RRID:AB_2860459)
    SARS-CoV-2 RBD antibodies(#40592-T62),
    suggested: None
    SARS-CoV-2
    suggested: None
    S2
    suggested: None
    antibodies(#40590-T62)
    suggested: None
    HIV-1
    suggested: None
    11695-RB01
    suggested: None
    Mouse monoclonal anti-FLAG M2 antibody and Mouse monoclonal anti-β-Actin antibody were purchased from Sigma-Aldrich.
    anti-β-Actin
    suggested: None
    After washing three times with PBS with 2% FBS, cells were incubated with rabbit polyclonal anti-6xHis antibody (1:200 dilution) (Shanghai Enzyme-Linked Biotechnology Co., Shanghai, China), followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200).
    anti-6xHis
    suggested: None
    anti-rabbit IgG
    suggested: None
    The primary antibodies used for blotting were polyclonal goat anti-MHV S antibody AO4 (1:2000), polyclonal anti-SARS S1 antibodies T62 (1:2000) (Sinobiological Inc, Beijing, China), mouse monoclonal against SARS S1 antibody MM02 (1:1000) (Sinobiological Inc, Beijing, China), rabbit polycolonal anti-SARS-CoV-2 RBS antibodies (1:1000) (Sinobiological Inc, Beijing, China), rabbit polycolonal anti-SARS-CoV-2 S2 antibodies (1:1000) (Sinobiological Inc, Beijing, China) and anti-FLAG M2 antibody (1:1000) (Sigma, St. Louis, MO, USA), respectively.
    anti-MHV S
    suggested: None
    anti-SARS S1
    suggested: None
    anti-SARS-CoV-2 RBS
    suggested: None
    anti-SARS-CoV-2 S2
    suggested: (BioLegend Cat# 943201, RRID:AB_2888742)
    anti-FLAG
    suggested: (Advanced Targeting Systems Cat# AB-450-1000, RRID:AB_10584603)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Human embryonic kidney cell lines 293 (#CRL-1573) and 293T expressing the SV40 T-antigen (#CRL-3216) were obtained from ATCC (Manassas, VA, USA), HEK239 cells stably expressing recombinant human ACE2 (293/hACE2), baby hamster kidney fibroblasts stably expressing recombinant human APN (BHK/hAPN), HEK239 cells stably expressing recombinant human DPP4 (293/hDPP4), HEK-293 cells stably expressing murine CEACAM1a (293/mCEACAM1a)were established in our lab.
    HEK239
    suggested: None
    Soluble RBD binding assay: HEK293 cells were transfected with plasmids encoding different ACE2 orthologs (Table S1) by polyetherimide (PEI) (Sigma, St Louis, MO, USA).
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Pseudovirion production and transduction: For pseudotyped virion production, HEK-293 cells were transfected with psPAX2, pLenti-GFP, and plasmids encoding either SARS-CoV-2 S, SARS-CoV S, RaTG13 S, or ZC45 S protein at equal molar ratios by PEI.
    HEK-293
    suggested: None
    Detection of S protein by western blot: Briefly, HEK293T cells transfected with plasmids encoding either SARS-CoV, SARS-CoV-2, bat SL-CoV RaTG13, or bat SL-CoV ZC45 S proteins were lysed at 40 hrs post transfection by RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP40, 1× protease inhibitor cocktail).
    HEK293T
    suggested: RRID:CVCL_ZC45)
    Software and Algorithms
    SentencesResources
    After 4 hrs of incubation, images of syncytia were captured with a Nikon TE2000 epifluorescence microscope running MetaMorph software (Molecular Devices).
    MetaMorph
    suggested: None
    Docking poses were viewed, aligned, and analyzed with PyMOL software.
    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.

    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 10.1101/2020.11.16.385849 on bioRxiv