Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS‐CoV‐2 variants

  1. Qiong Wang
  2. Sheng‐Bao Ye
  3. Zhi‐Jian Zhou
  4. Jin‐Yan Li
  5. Ji‐Zhou Lv
  6. Bodan Hu
  7. Shuofeng Yuan
  8. Ye Qiu
  9. Xing‐Yi Ge

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Abstract

Increasing evidence supports inter‐species transmission of SARS‐CoV‐2 variants from humans to domestic or wild animals during the ongoing COVID‐19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS‐CoV‐2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS‐CoV‐2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, to evaluate the impact of S mutations, we tested 27 pseudoviruses of SARS‐CoV‐2 carrying different spike mutants by infecting Hela cells expressing different angiotensin‐converting enzyme 2 (ACE2) orthologs from 20 animals. Of these 27 pseudoviruses, 20 bear single mutation and the other 7 were cloned from emerging SARS‐CoV‐2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (B.1.429), and Mu (B.1.621). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS‐CoV‐2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS‐CoV‐2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammal ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS‐CoV‐2, potentially contributing to the spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS‐CoV‐2 variants with these two mutations.

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  1. Version published to 10.1002/jmv.28116
  2. ScreenIT

    SciScore for 10.1101/2022.04.11.487828: (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
    For detection of S protein, the membrane was incubated with anti-HA tag mouse monoclonal antibody (bimake, USA,1:2000), and the bound antibodies were detected by Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG (Abbkine, China, 1:5,000).
    anti-HA
    suggested: None
    For detection of HIV-1 p24 in supernatants, monoclonal antibody against HIV p24 (p24 MAb) was used as the primary antibody at a dilution of 1:8,000, followed by incubation with HRP-conjugated goat anti-mouse IgG at the same dilution.
    HIV-1
    suggested: None
    HIV
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and plasmid construction: HEK293T and Hela cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin and 0.1 mg/ml of streptomycin in 5% CO2 at 37 °C.
    Hela
    suggested: None
    In brief, one day prior to transfection for virus production, HEK293T cells were digested and adjusted to an amount of 7×106 cells in a 10cm culture medium and incubated overnight in an incubator at 37 °C with 5% CO2.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    pcDNA3.1-S2 plasmid was used as the template to generate the plasmid with mutagenesis in S gene.
    pcDNA3.1-S2
    suggested: None
    When cells reached 80%-90% confluence, HEK293T cells were co-transfected with a luciferase-expressing HIV-1 plasmid (pNL4-3.
    HIV-1
    suggested: RRID:Addgene_115809)
    pNL4-3
    suggested: None
    The P24 gene of HIV virus was cloned into the vector pCDNA3.1(+) as a plasmid standard, with the viral copy number calculated accordingly.
    pCDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    Phylogenetic analysis and sequence alignment: The ACE2 aa sequences were aligned by MAFFT v7.149 in BioAider.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    BioAider
    suggested: None
    Then we constructed the maximum likelihood phylogenetic tree of ACE2 by IQ-tree v1.6.10 program with 10,000 ultrafast bootstraps (https://academic.oup.com/mbe/article/32/1/268/2925592), and the most appropriate evolutionary model was JTTDCMut+G4 which calculated using ModelFinder according to the bayesian information criterions.
    IQ-tree
    suggested: (IQ-TREE, RRID:SCR_017254)
    The mutations in the models were aligned, and the interactions between the SARS-CoV-2 S and ACE2 proteins were compared in PyMOL.
    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 10.1101/2022.04.11.487828 on bioRxiv