Interaction between Spike Protein of SARS-CoV-2 and Human Virus Receptor ACE2 Using Two-Color Fluorescence Cross-Correlation Spectroscopy

  1. Ai Fujimoto
  2. Yidan Lyu
  3. Masataka Kinjo
  4. Akira Kitamura

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Abstract

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is initiated by the interaction between a receptor protein, angiotensin-converting enzyme type 2 (ACE2) on the cell surface, and the viral spike (S) protein. This interaction is similar to the mechanism in SARS-CoV, a close relative of SARS-CoV-2, which was identified in 2003. Drugs and antibodies that inhibit the interaction between ACE2 and S proteins could be key therapeutic methods for preventing viral infection and replication in COVID-19. Here, we demonstrate the interaction between human ACE2 and a fragment of the S protein (S1 subunit) derived from SARS-CoV-2 and SARS-CoV using two-color fluorescence cross-correlation spectroscopy (FCCS), which can detect the interaction of fluorescently labeled proteins. The S1 subunit of SARS-CoV-2 interacted in solution with soluble ACE2, which lacks a transmembrane region, more strongly than that of SARS-CoV. Furthermore, one-to-one stoichiometry of the two proteins during the interaction was indicated. Thus, we propose that this FCCS-based interaction detection system can be used to analyze the interaction strengths of various mutants of the S1 subunit that have evolved during the worldwide pandemic, and also offers the opportunity to screen and evaluate the performance of drugs and antibodies that inhibit the interaction.

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  1. Version published to 10.3390/app112210697
  2. ScreenIT

    SciScore for 10.1101/2021.08.18.456769: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    The antibodies used were anti-GFP HRP-DirecT (#598-7, MBL, Nagano, Japan), anti-polyhistidine-tag (#PM032, MBL), and anti-mCherry (#Z2496N, TaKaRa, Shiga, Japan)
    anti-GFP
    suggested: (MBL International Cat# 598-7, RRID:AB_10597267)
    #598-7 , MBL , Nagano , Japan) , anti-polyhistidine-tag
    suggested: None
    anti-mCherry
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For observation of lysosome localization, mCherry-tagged protein-expressing Neuro2a cells were incubated in a medium containing 0.5 μM Lysotrascker green DND-26 (Thermo Fisher) for 30 min before image acquisition.
    Neuro2a
    suggested: None
    Recombinant DNA
    SentencesResources
    To construct an expression plasmid, cytoplasmic region- and transmembrane-lacking hACE2 tagged with a monomeric variant of eGFP carrying the A206K mutation (hACE2-eGFP), the fragment encoding hACE2, was inserted into pmeGFP-N1 with NheI and EcoRI.
    hACE2
    suggested: RRID:Addgene_1786)
    pmeGFP-N1
    suggested: RRID:Addgene_27766)
    A synthetic oligonucleotide for the ER-targeting signal peptide was inserted into pmCherry-N1 (pER-mCherry) with NheI and AgeI.
    pmCherry-N1
    suggested: RRID:Addgene_87327)
    pER-mCherry
    suggested: None
    The fragments were inserted into pER-mCherry-N1 with XhoI and BamHI (pER-mCherry-S1 and pER-mCherry-S1-2, respectively).
    pER-mCherry-N1
    suggested: None
    pER-mCherry-S1-2
    suggested: None
    The whole sequences of the coding regions of pER-mCherry-S1 and pER-mCherry-S1-2 are presented in Supplemental Figure.
    pER-mCherry-S1
    suggested: None
    Plasmids encoding hACE2-eGFP, ER-mCherry-S1, or ER-mCherry-S1-2 (16 μg) and sonicated salmon sperm DNA (14 μg) were transfected into the cells with 240 μL of polyethyleneimine (#43896,
    hACE2-eGFP
    suggested: None

    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.

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  3. Version published to 10.1101/2021.08.18.456769v1 on bioRxiv