A dynamic regulatory interface on SARS-CoV-2 RNA polymerase

  1. Wei Shi
  2. Ming Chen
  3. Yang Yang
  4. Wei Zhou
  5. Shiyun Chen
  6. Yi Yang
  7. Yangbo Hu
  8. Bin Liu

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Abstract

The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design.

Author summary

Since it was first discovered and reported in late 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by highly contagious SARS-CoV-2 virus is wreaking havoc around the world. Currently, no highly effective and specific antiviral drug is available for clinical treatment. Therefore, the threat of COVID-19 transmission necessitates the discovery of more effective antiviral strategies. Viral RNA-dependent RNA polymerase (RdRp) is an important antiviral drug target. Here, our cryo-EM structure of a SARS-CoV-2 RdRp/RNA replicating complex reveals a previously uncharacterized overall shift of the cofactor nsp8(I)/nsp7 interface, leading to its rearrangement. Through in vitro functional test, we found that the specific interactions on the interface are important to the efficient RNA polymerase activity of SARS-CoV-2 RdRp. These observations let us to suggest this interface as a potential new drug intervention site, outside of the canonical polymerase active center, in RdRp for antiviral design. Our findings would provide new insights into regulatory mechanism of this novel SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and benefit the global public health.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.30.229187: (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
    Image processing: Cryo-EM data were processed using cryoSPARC v2.15 [32], and the procedure is outlined in S1A Fig.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Model building and refinement: The initial model was generated by docking the individual domains of the apo RdRp of SARS-CoV-2 (PDB ID 6M71) into the cryo-EM density map using Chimera [36] and COOT [37].
    COOT
    suggested: (Coot, RRID:SCR_014222)
    The intact model was then refined using real_space_refine in Phenix [38].
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    The final model has good stereochemistry as evaluated by MolProbity [39].
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    All figures were created using ChimeraX [42].
    ChimeraX
    suggested: (UCSF ChimeraX, RRID:SCR_015872)

    Results from OddPub: Thank you for sharing your data.

    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 32. 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.
    • 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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  2. Version published to 10.1101/2020.07.30.229187v1 on bioRxiv