Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

  1. Alex L. Lai
  2. Jack H. Freed

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Abstract

Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV (“SARS-1”), SARS-CoV-2 (“SARS-2”) and MERS-CoV (“MERS”). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2’ cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca 2+ dependent fashion. In this study, we want to know which residues are involved in this Ca 2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca 2+ ions: both FP1 and FP2 bind one Ca 2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca 2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca 2+ , the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca 2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca 2+ channel to combat the ongoing COVID-19 pandemic.

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    SciScore for 10.1101/2021.11.03.467161: (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

    Experimental Models: Cell Lines
    SentencesResources
    Pseudotyped virus particle – SUV docking experiments: The murine leukemia virus-based SARS-1-CoV S-pseudotyped particles, harboring wild-type (wt) SARS-CoV S protein, were generated using a replication deficient murine leukemia virus core in HEK-293T cell line as previously described 59 and provided by Gary Whittaker and Susan Daniel Labs.
    HEK-293T
    suggested: None
    Software and Algorithms
    SentencesResources
    The data were analyzed with Origin.
    Origin
    suggested: (Origin, RRID:SCR_014212)

    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, the limitation of the NMR and the MD simulation studies is that there is only one FP in their system: one FP in a bicelles in the NMR study23; or one FP in a membrane patch in the MD simulation27. These environments exclude the possibility of inter-molecular interaction between the FPs. However, this is possible in our ESR study system as the FPs bind to liposomal membranes which are large enough to harbor multiple FPs. Considering in the “real biological scenario”, the three FPs are in a close position as they are in the whole S protein trimer. Thus, they are likely to form trimers as well. It is even possible that a higher number N-mer can be formed if multiple S protein trimers accumulate near a fusion site. It has been shown that in the case of influenza, 7-9 trimers accumulate in the fusion site53, 54. HIV FP is known to aggregate in the membrane and are supposed to aggregate in the context of whole gp41 timer during the membrane fusion as well55. However, we need a system that simulates the “real biological scenario” to test. That is why we develop a PP-SUV system. Does the separate FP work the same fashion as the FP on the whole S protein trimer?: It has long been questioned whether separate FPs function the same as in the whole protein. Our PP-SUV methodology monitors the membrane ordering induced by the pre-assembled S protein on the surface of the PP in real time. This provides several advantages. First, the FP is now a part of the entire protein. Second, t...

    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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  2. Version published to 10.1101/2021.11.03.467161v1 on bioRxiv