Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

  1. Wenlin Ren
  2. Jun Lan
  3. Xiaohui Ju
  4. Mingli Gong
  5. Quanxin Long
  6. Zihui Zhu
  7. Yanying Yu
  8. Jianping Wu
  9. Jin Zhong
  10. Rong Zhang
  11. Shilong Fan
  12. Guocai Zhong
  13. Ailong Huang
  14. Xinquan Wang
  15. Qiang Ding

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Abstract

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as “Cluster 5” that contained mutations in the spike protein. However, the functional properties of these “Cluster 5” mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

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  1. Version published to 10.1371/journal.ppat.1010053
  2. ScreenIT

    SciScore for 10.1101/2021.08.24.457448: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Data collection and refinement: Diffraction data were collected at 100 K and at a wavelength of 0.9796 Å on the BL18U1 beam line of the Shanghai Synchrotron Research Facility.
    IRB: This study was approved by the Institution Review Board of Tsinghua University (20210040)
    Sex as a biological variableEight were male, and 1 was female.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were tested routinely and found to be free of mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    The blots were exposed to primary antibodies anti-β-Tubulin (CW0098, CWBIO), or anti-FLAG (F7425, Sigma) in 5% nonfat milk in 1× PBS containing 0.1% Tween 20 for 2 h.
    anti-β-Tubulin ( CW0098
    suggested: None
    anti-FLAG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T (American Tissue Culture Collection, ATCC, Manassas, VA, CRL-3216), A549 (ATCC
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    #CCL-185), Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HeLa (ATCC #CCL-2) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY, USA) supplemented with 10% (vol/vol)
    Vero E6
    suggested: None
    HeLa
    suggested: None
    Production of SARS-CoV-2 pseudotyped virus: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G, Addgene #12259) using VigoFect (
    HEK293T
    suggested: None
    ACE2-lg, a recombinant Fc fusion protein of soluble human ACE2 (residues Gln18-Ser740), was expressed in 293F cells and purified using protein A affinity chromatography as described in our previous study36.
    293F
    suggested: None
    Virus-serum mixtures were incubated at 37°C for 30min, then added to A549-hACE2 cells in 96-well plates and incubated at 37°C.
    A549-hACE2
    suggested: RRID:CVCL_A5KB)
    Recombinant DNA
    SentencesResources
    Plasmids: The cDNAs encoding human or mink ACE2 were synthesized by GenScript and cloned into pLVX-IRES-zsGreen1 vectors (Catalog No. 632187, Clontech Laboratories, Inc) with a C-terminal FLAG tag.
    pLVX-IRES-zsGreen1
    suggested: None
    (Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells.
    psPAX2
    suggested: RRID:Addgene_12260)
    Production of SARS-CoV-2 pseudotyped virus: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G, Addgene #12259) using VigoFect (
    pTG-MLV-Fluc
    suggested: None
    pTG-MLV-Gag-pol
    suggested: None
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    VSV-G
    suggested: RRID:Addgene_138479)
    pMD2 . G
    suggested: None
    Protein crystallization: The gene encoding 333-527aa of SARS-CoV-2 RBD (WT or Y453F mutant) was cloned into the pFastbac-dual vector using the BamH1 and Hind3 restriction enzyme.
    pFastbac-dual
    suggested: RRID:Addgene_135584)
    Software and Algorithms
    SentencesResources
    The two structures were determined using the molecular replacement method with Molrep in the CCP4 suite37, using the model of the human ACE2 and SARS-CoV-2 complex21.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    Subsequent model building and refinement were performed using COOT and PHENIX, respectively38,39.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)

    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 34 and 30. 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.

    About SciScore

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  3. Version published to 10.1101/2021.08.24.457448 on bioRxiv