Unraveling the Enzymatic Mechanism of the SARS-CoV-2 RNA-Dependent-RNA-Polymerase. An Unusual Active Site Leading to High Replication Rates

  1. Emmanuelle Bignon
  2. Antonio Monari

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Abstract

Viral infection relies on the hijacking of cellular machineries to enforce the reproduction of the infecting virus and its subsequent diffusion. In this context the replication of the viral genome is a key step performed by specific enzymes, i.e. polymerases. The replication of SARS-CoV-2, the causative agent of the COVID-19 pandemics, is based on the duplication of its RNA genome, an action performed by the viral RNA-dependent-RNA polymerase. In this contribution, for the first time and by using two-dimensional enhanced sampling quantum mechanics/ molecular mechanics, we have determined the chemical mechanisms leading to the inclusion of a nucleotide in the nascent viral RNA strand. We prove the high efficiency of the polymerase, which lowers the activation free energy to less than 10 kcal/mol. Furthermore, the SARS-CoV-2 polymerase active site is slightly different from those found usually found in other similar enzymes, and particularly it lacks the possibility to enforce a proton shuttle via a nearby histidine. Our simulations show that this absence is partially compensate by lysine, whose proton assist the reaction opening up an alternative, but highly efficient, reactive channel. Our results present the first mechanistic resolution of SARS-CoV-2 genome replication and shed light on unusual enzymatic reactivity paving the way for future rational design of antivirals targeting emerging RNA viruses.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.02.478873: (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

    Software and Algorithms
    SentencesResources
    All classical molecular dynamics (MD) simulations were carried on using NAMD.37,38 QM/MM simulations have been performed using the Terachem code39 interfaced with Amber 16.40 Visualization, rendering of MD trajectories and plots were performed using the VMD,41 Gnuplot and RStudio softwares.
    RStudio
    suggested: (RStudio, RRID:SCR_000432)
    Structural analysis was performed using the cpptraj module of AMBER and the Curves+ program.
    Curves+
    suggested: None
    Enhanced Sampling and Free Energy calculation: To allow for the exploration of chemical reactivity we resorted to enhanced sampling and more specific umbrella sampling (US) protocol as implemented in AMBER, thus obtaining a relevant free energy profile.
    AMBER
    suggested: (AMBER, RRID:SCR_016151)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 16. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

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  2. Version published to 10.1101/2022.02.02.478873v1 on bioRxiv