A mechanistic understanding of the modes of Ca ion binding to the SARS-CoV-1 fusion peptide and their role in the dynamics of host membrane penetration

  1. Juliana Debrito Carten
  2. George Khelashvili
  3. Miya K. Bidon
  4. Marco R. Straus
  5. Tiffany Tang
  6. Javier A. Jaimes
  7. Harel Weinstein
  8. Gary R. Whittaker
  9. Susan Daniel

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Abstract

The SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP) segment that mediates fusion of the viral and host cell membranes. Calcium ions are thought to position the FP optimally for membrane insertion by interacting with negatively charged residues in this segment (E801, D802, D812, E821, D825, and D830); however, which residues bind to calcium and in what combinations supportive of membrane insertion are unknown. Using biological assays and molecular dynamics studies, we have determined the functional configurations of FP-Ca +2 binding which promote membrane insertion. We first mutated the negatively charged residues in the SARS CoV-1 FP to assay their role in cell entry and syncytia formation, finding that charge loss in the D802A or D830A mutants reduced syncytia formation and pseudoparticle transduction. Interestingly, the D812A mutation led to increased pseudoparticle transduction, indicating the Ca 2+ effect depends on binding at specific FP sites. To interpret mechanistically these results and learn how specific modes of FP-Ca 2+ binding modulate membrane insertion, we performed molecular dynamics simulations. Preferred residue pairs for Ca 2+ binding were identified (E801/D802; E801/D830; D812/E821) which promote FP membrane insertion. In contrast, binding to residues E821/D825 inhibited FP membrane insertion, which is also supported by our biological assays. Our findings show that Ca 2+ binding to SARS-CoV-1 FP residue pairs E801/D802 and D812/E821 facilitates membrane insertion, whereas binding to the E801/D802 and D821/D825 pairs is detrimental. These conclusions provide an improved and nuanced mechanistic understanding of calcium binding modes to FP residues and their dynamic effects on host cell entry.

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  1. Version published to 10.1101/2022.03.03.482731v3 on bioRxiv
  2. Version published to 10.1101/2022.03.03.482731v2 on bioRxiv
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    SciScore for 10.1101/2022.03.03.482731: (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 SARS-CoV S protein was detected using the SARS-CoV S rabbit polyclonal primary antibody (NR-4569, BEI resources) and Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (Invitrogen).
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, plasmids, and reagents: Human embryonic kidney 293 (HEK293T) and African green monkey kidney epithelial (VeroE6) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Pseudoparticles were prepared by transfecting HEK293T cells with 600 µg of their respective SARS WT or mutant S plasmids, 800 µg of pTG-Luc, and 600 µg of pCMV-MLV gagpol using polyethylenimine (PEI) as the transfection reagent.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    The plasmids used for generating pseudoparticles were the pCMV-MLV gag-pol murine leukemia virus (MLV) packaging construct, the pTG-Luc transfer vector encoding the luciferase reporter gene, and the pCAGGS-VSVG plasmids were provided by Jean Dubuisson (Lille Pasteur Institute, Lille, France) and co-transfected as previously described [27].
    pCMV-MLV
    suggested: None
    pTG-Luc
    suggested: None
    pCAGGS-VSVG
    suggested: None
    The plasmid encoding the C9-tagged SARS-CoV spike protein (pcDNA3.1-C9-SARS-CoV S) was provided by Dr. Michael Farzan from the New England Primate Research Center.
    pcDNA3.1-C9-SARS-CoV
    suggested: None
    Site-directed mutagenesis: Site-directed mutagenesis was performed on the SARS-CoV spike protein vector, pcDNA3.1-SARS-CoV-S, via the QuikChange Lightning site-directed mutagenesis kit (Aligent).
    pcDNA3.1-SARS-CoV-S
    suggested: None
    Software and Algorithms
    SentencesResources
    The average number of nuclei per syncytium and standard deviation were calculated and visualized using Microsoft Excel and GraphPad Prism 7.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The sequence of the SARS-CoV S protein (GenBank accession no. AAT74874.1) was aligned to the PDB 5XLR SARS-CoV structure sequence using Geneious software (v.2020.1.1).
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    Images were created using Adobe Illustrator CC (v.24.03).
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    The system was equilibrated with NAMD version 2.13 [44] following a multi-step protocol during which the backbone atoms of the SARS-CoV FP as well as Ca2+ ions in the solution were first harmonically constrained and subsequently gradually released in four steps (totaling ∼3ns), changing the restrain force constants kF from 1, to 0.5, to 0.1 kcal/ (mol Å2), and 0 kcal/ (mol Å2).
    NAMD
    suggested: (NAMD, RRID:SCR_014894)
    After this phase, the velocities of all atoms of the system were reset, and ensemble MD runs were initiated with OpenMM version 7.4.
    OpenMM
    suggested: (OpenMM, RRID:SCR_000436)

    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: We detected the following sentences addressing limitations in the study:
    However, due to the limitations and variability of the syncytia assay, we chose to use SARS-CoV2 pseudoparticles to mimic a more in vivo-like system to examine the functionality of the various FP mutants. Successful pseudoparticle entry into host cells results in the integration of the luciferase reporter gene into the cellular genome. Luminescence can therefore be used as a readout of pseudoparticle infectivity. We first confirmed the incorporation of the WT and single-charged-to-alanine mutant FPs into the pseudoparticles (Fig 4A). Nearly all FP mutants generated (E801A, D802A, D812A, E821A, D825A, and D830A) were incorporated in the SARS-CoV2 pseudoparticles; the E801A mutant was not detected. We then infected VeroE6 cells using the SARS-CoV2 pseudoparticles we had generated and measured luminescence as a readout of infectivity and a proxy for viral entry. Introduction of WT pseudoparticles into VeroE6 cells results in a robust luminescence signal, indicating successful viral entry and fusion competency of the viral particles when calcium levels are unperturbed (Fig 4B and C). Infections with E821A and D825A-containing pseudoparticles at physiological levels of calcium also resulted in luminescence levels comparable to WT-containing pseudoparticles, suggesting that these residues are not required for FP function. To the contrary, pseudoparticles containing the D802A, D812A, or D830A mutations were unable to infect VeroE6 cells, resulting in aa significant drop in luminesce...

    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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  4. Version published to 10.1101/2022.03.03.482731v1 on bioRxiv