Spike mutation T403R allows bat coronavirus RaTG13 to use human ACE2

  1. Fabian Zech
  2. Daniel Schniertshauer
  3. Christoph Jung
  4. Alexandra Herrmann
  5. Qinya Xie
  6. Rayhane Nchioua
  7. Caterina Prelli Bozzo
  8. Meta Volcic
  9. Lennart Koepke
  10. Jana Krüger
  11. Sandra Heller
  12. Alexander Kleger
  13. Timo Jacob
  14. Karl-Klaus Conzelmann
  15. Armin Ensser
  16. Konstantin M.J. Sparrer
  17. Frank Kirchhoff

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats 1 . A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor 2 . Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.31.446386: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After the transfer, the membrane was blocked in 1% Casein in PBS (Thermo Scientific) and stained using primary antibodies directed against SARS-CoV-2 S (1:1,000, Biozol, 1A9, #GTX632604), ACE2 (1:1,000
    SARS-CoV-2 S
    suggested: None
    ACE2
    suggested: None
    , VSV-M (1:2,000, Absolute Antibody, 23H12, #Ab01404-2.0), V5-tag (1:1,000
    V5-tag
    suggested: None
    , Cell Signaling, #13202), GAPDH (1:1,000, BioLegend, #631401) and Infrared Dye labelled secondary antibodies (1:20,000, LI-CORIRDye).
    GAPDH
    suggested: (BioLegend Cat# 631401, RRID:AB_2247301)
    Experimental Models: Cell Lines
    SentencesResources
    Human embryonic kidney 293T cells purchased from American type culture collection (ATCC: #CRL3216) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (PANBiotech), 100 μg/ml streptomycin (PANBiotech) and 100 U/ml penicillin (PANBiotech).
    293T
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    Pseudoparticle production: To produce pseudotyped VSVΔG-GFP particles, 6*106 HEK 293 T cells were seeded 18 hours before transfection in 10 cm dishes.
    HEK 293
    suggested: None
    Stem Cell Culture and Intestinal Differentiation: Human embryonic stem cell line HUES8 (Harvard University, Cambridge, MA) was used with permission from the Robert Koch Institute according to the “79.
    HUES8
    suggested: RRID:CVCL_B207)
    α5β5 integrin blocking: Caco-2 cells were preincubated with the indicated amounts of α5β5 integrin Inhibitor ATN-161 (Sigma) for two hours and infected with 100 μl freshly produced VSVΔG-GFP pseudo particles.
    Caco-2
    suggested: None
    Calu-3 cells were preincubated with the indicated amounts of ATN-161 (Sigma) for two hours and infected with SARS-CoV-2 Viral isolate BetaCoV/France/IDF0372/2020 (MOI 0.05, six hours).
    Calu-3
    suggested: None
    Recombinant DNA
    SentencesResources
    Expression constructs: pCG_SARS-CoV-2-Spike-IRES_eGFP, coding the spike protein of SARS-CoV-2 isolate Wuhan-Hu-1, NCBI reference Sequence YP_009724390.1, was kindly provided by Stefan Pöhlmann (German Primate Center, 473 Göttingen, Germany).
    pCG_SARS-CoV-2-Spike-IRES_eGFP
    suggested: None
    pCG_SARS-CoV-2-Spike C-V5-IRES_eGFP and RaTG13-S (synthesized by Baseclear) was PCR amplified and subcloned into a pCG-IRES_eGFP expression construct using the restriction enzymes XbaI and MluI (New England Biolabs).
    pCG_SARS-CoV-2-Spike C-V5-IRES_eGFP
    suggested: None
    pCG-IRES_eGFP
    suggested: None
    The resulting amplicons were assembled with a modified pBeloBAC11 backbone, containing CMV and T7 promotors as well as the HDV ribozyme and bGH polyA signal, using the NEBuilder HiFi DNA Assembly Cloning Kit.
    pBeloBAC11
    suggested: RRID:Addgene_60342)
    Assembled DNA was electroporated into E. coli GS1783 strain and resulting clones of pBelo-SARSARS-CoV-2 were confirmed by restriction digestion and next generation sequencing.
    pBelo-SARSARS-CoV-2
    suggested: None
    SARS-CoV-2 ΔS replicon system: HEK293 T cells were seeded in six well format and transfected with 3 μg pBelo-SARSCoV-2-dSpike-GLuc-K2 or pBelo-SARSCoV-2-dSpike-EGFP and 0.25 μg of each expression construct pLVX-EF1alpha-SARS-CoV2-N-2xStrep-IRES-Puro, pCG-ACE2, pCAG-T7-RNA-polymerase and one pCG-vector encoding the spike protein of SARS-CoV-2, RaTG13 or the indicated mutant S respectively.
    pBelo-SARSCoV-2-dSpike-GLuc-K2
    suggested: None
    pBelo-SARSCoV-2-dSpike-EGFP
    suggested: None
    pLVX-EF1alpha-SARS-CoV2-N-2xStrep-IRES-Puro
    suggested: None
    pCG-ACE2
    suggested: None
    pCAG-T7-RNA-polymerase
    suggested: None
    pCG-vector
    suggested: None
    Software and Algorithms
    SentencesResources
    The Organoids were imaged using the Cytation 3 cell imaging system and processed with Gen 5 and ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Sequence logos were generated using R packages ggplot2 and ggseqlogo34.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Statistics: Statistical analyses were performed using GraphPad PRISM 8 (GraphPad Software).
    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 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 page 28. 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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  2. Version published to 10.1101/2021.05.31.446386v1 on bioRxiv