Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity

  1. Sang Ho Park
  2. Haley Siddiqi
  3. Daniela V. Castro
  4. Anna A. De Angelis
  5. Aaron L. Oom
  6. Charlotte A. Stoneham
  7. Mary K. Lewinski
  8. Alex E. Clark
  9. Ben A. Croker
  10. Aaron F. Carlin
  11. John Guatelli
  12. Stanley J. Opella

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Abstract

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8–43) and a short cytoplasmic helix (residues 53–60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6–18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5’ position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

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  1. Version published to 10.1371/journal.ppat.1009519
  2. ScreenIT

    SciScore for 10.1101/2021.04.06.438579: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Formaldehyde fixed cells were washed with phosphate buffered saline and permeabilized for immunofluorescence using 0.1% Triton X-100 in PBS with 1% bovine serum albumin (BSA) fraction V (www.emdmillipore.com) and stained for SARS-CoV-2 with a primary anti-Nucleocapsid antibody (www.genetex.com GTX135357) labeled with AlexaFluor 594.
    anti-Nucleocapsid
    suggested: (GeneTex Cat# GTX135357, RRID:AB_2868464)
    Proteins in VLP and cell lysates were separated on 10% SDS-PAGE gels, transferred to PVDF membranes, and immunoblotted with the following antibodies (Fig 7A): mouse monoclonal anti-V5 tag (www.thermofisher.com, #R960-25), rabbit polyclonal anti-SARS M (generous gift of C.
    anti-V5
    suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)
    anti-SARS
    suggested: None
    Primary antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (www.bio-rad.com) or HRP-donkey anti-rabbit IgG (www.bio-rad.com) and Western Clarity detection reagent (www.bio-rad.com).
    anti-mouse IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 antiviral test of amilorides: Vero E6 and Caco-2 were obtained from ATCC and grown in DMEM (www.corning.com) with 10% FBS, 10mM HEPES, and Penicillin-Streptomycin (www.thermofisher.com).
    Caco-2
    suggested: None
    SARS-CoV-2 isolate USA-WA1/2020 (www.beiresources.org) was propagated on Caco-2 cells and infectious units quantified by focus forming assay using Vero E6 (ATCC) cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    HEK293T cells were cultured in complete Dulbecco’s modified Eagle medium containing 10% Fetal Bovine Serum and penicillin-streptomycin.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    IC50 and CC50 were determined using the nonlinear regression analysis in GraphPad Prism 9 with the bottom and top parameters constrained to 0 and 100, respectively.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Chemiluminescence was detected using a Bio-Rad Chemi Doc imaging system and analyzed using Bio-Rad Image Lab v5.1 software.
    Bio-Rad Image
    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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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  3. Version published to 10.1101/2021.04.06.438579v1 on bioRxiv