SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

  1. Seiya Ozono
  2. Yanzhao Zhang
  3. Hirotaka Ode
  4. Kaori Sano
  5. Toong Seng Tan
  6. Kazuo Imai
  7. Kazuyasu Miyoshi
  8. Satoshi Kishigami
  9. Takamasa Ueno
  10. Yasumasa Iwatani
  11. Tadaki Suzuki
  12. Kenzo Tokunaga

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Abstract

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.

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  1. Version published to 10.1038/s41467-021-21118-2
  2. Version published to 10.1101/2020.06.15.151779v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.15.151779: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Neutralization assays: Experiments using human samples were approved by the Medical Research Ethics Committee of the National Institute of Infectious Diseases, Japan.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: Cells were originally obtained from the ATCC and routinely tested negative for mycoplasma contamination (PCR Mycoplasma Detection Set, Takara)

    Table 2: Resources

    Antibodies
    SentencesResources
    Pelleted virions were resuspended in SDS-sample buffer and subjected to immunoblot analysis using an anti-SARS-CoV-2 spike mouse monoclonal antibody (1:1,000, GeneTex, GTX632604) and an anti-p24 monoclonal antibody (1:1,000; Nu24 (33)).
    anti-SARS-CoV-2
    suggested: None
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    anti-p24
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell maintenance and stable cell establishment: 293T and HepG2 cells were maintained under standard conditions.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    HepG2
    suggested: CLS Cat# 300198/p2277_Hep-G2, RRID:CVCL_0027)
    Software and Algorithms
    SentencesResources
    Structural modeling of SARS-CoV-2 S proteins: The structure-based complex models of ACE2 and the ectodomains (ECs) of SARS-CoV-2 S proteins were built by homology modeling with the Modeller 9v8 (34).
    Modeller
    suggested: (MODELLER, RRID:SCR_008395)
    The structural figures were generated by using PyMOL ver.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Statistical analyses of the data were performed by using GraphPad Prism version 8.04.
    GraphPad Prism
    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: 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

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  4. Version published to 10.1101/2020.06.15.151779v1 on bioRxiv