Characterization of various remdesivir-resistant mutations of SARS-CoV-2 by mathematical modeling and molecular dynamics simulation

  1. Shiho Torii
  2. Kwang Su Kim
  3. Jun Koseki
  4. Rigel Suzuki
  5. Shoya Iwanami
  6. Yasuhisa Fujita
  7. Yong Dam Jeong
  8. Yoshiharu Matsuura
  9. Teppei Shimamura
  10. Shingo Iwami
  11. Takasuke Fukuhara

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Abstract

Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection.

Significance Statement

Considering the emerging Omicron strain, quick characterization of SARS-CoV-2 mutations is important. However, owing to the difficulties in genetically modifying SARS-CoV-2, limited groups have produced multiple mutant viruses. Our cutting-edge reverse genetics technique enabled construction of eight reporter-carrying mutant SARS-CoV-2 in this study. We developed a mathematical model taking into account sequential changes and identified antiviral effects against mutant viruses with differing propagation capacities and lethal effects on cells. In addition to identifying the positions of mutations, we analyzed the structural changes in SARS-CoV-2 NSP12 by computer simulation to understand the mechanism of resistance. This multidisciplinary approach promotes the evaluation of future resistance mutations.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.22.481436: (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
    To detect SARS-CoV-2-infected cells, mouse monoclonal antibody against SARS-CoV-2 NP (Clone# S2N4-1242) was kindly provided by Bio Matrix research.
    SARS-CoV-2 NP
    suggested: None
    Alexa Fluor 488-conjugated anti-mouse antibodies were purchased from Life Technologies.
    anti-mouse
    suggested: None
    After washing with PBS three times, the cells were incubated with a 1:1,000 dilution of goat anti-mouse IgG Alexa Fluor 488- conjugated secondary antibody (Thermo Fisher Scientific) in PBS for 1 hour at room temperature.
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: HEK293-C34 cells were previously established and a different clone than HEK293-3P6C33 cell, both of which were IFNAR1 deficient, with expression of human ACE2 and TMPRSS2 induced by doxycycline hydrochloride (21).
    HEK293-3P6C33
    suggested: None
    TMPRSS2-expressing Vero E6 (VeroE6/TMPRSS2) cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB1819) and maintained in DMEM containing 10% FBS and G418 (Nacalai Tesque).
    Vero E6
    suggested: None
    Both HEK293-C34 cells and VeroE6/TMPRSS2 cells were cultured at 37°C in 5% CO2.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Time course analyses of infectious virus production: HEK293-C34 cells were prepared in 96-well plates in media containing 1 μg/ml doxycycline hydrochloride.
    HEK293-C34
    suggested: None
    Software and Algorithms
    SentencesResources
    The EC50 was calculated using the drc package (v3.0-1; R Project for Statistical Computing).
    R Project for Statistical
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)
    Statistical significances were determined by the one-way ANOVA with Dunnett’s test or the Kruskal–Wallis test with the two stage linear step-up procedure of Benjamini, Kreiger, and Yekutieli, which was performed using GraphPad Prism (Software ver.
    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.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2022.02.22.481436 on bioRxiv