Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

  1. Ruchi Yadav
  2. Valentine V. Courouble
  3. Sanjay K. Dey
  4. Jerry Joe E. K. Harrison
  5. Jennifer Timm
  6. Jesse B. Hopkins
  7. Ryan L. Slack
  8. Stefan G. Sarafianos
  9. Francesc X. Ruiz
  10. Patrick R. Griffin
  11. Eddy Arnold

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Abstract

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-11 polyprotein, whose mature products include cofactors of the viral replicase, and identified the order of cleavages. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and x-ray scattering yielded a nsp7-11 structural ensemble, demonstrating shared secondary structural elements with individual nsps. The pattern of cross-links and HDX footprint of the C145A Mpro and nsp7-11 complex demonstrate preferential binding of the enzyme active site to the polyprotein junction sites and additional transient contacts to help orient the enzyme on its substrate for cleavage. Last, proteolysis assays were used to characterize the effect of inhibitors/binders on Mpro processing/inhibition using the nsp7-11 polyprotein as substrate.

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  1. Version published to 10.1126/sciadv.add2191
  2. ScreenIT

    SciScore for 10.1101/2022.05.27.493767: (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

    Recombinant DNA
    SentencesResources
    The pGEX-6P-1-nsp5 (or Mpro) plasmid was a kind gift from Dr. Martin Walsh, Diamond Light Source. pGBWm4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon-optimized, with an initial Met and a cleavable C- terminal TEV 6x-His tag was a gift from Ginkgo Bioworks (Addgene plasmid 145611; http://n2t.net/ addgene:145611; RRID: Addgene_145611).
    pGEX-6P-1-nsp5
    suggested: None
    pGBWm4046979
    suggested: None
    detected: RRID:Addgene_145611)
    pGBWm4046852 (coding for full- length nsp8, NCBI Reference Sequence: YP_009725304.1, codon-optimized, with an initial Met and a cleavable C-terminal TEV 6x-His tag) was a gift from Ginkgo Bioworks (Addgene plasmid 145584; http://n2t.net/ addgene:145584; RRID: Addgene_145584).
    pGBWm4046852
    suggested: None
    detected: RRID:Addgene_145584)
    The gene encoding SARS-CoV-2 nsp10 was cloned into the pGEX-6P-1 vector to generate an expression construct containing an N-terminal GST tag and an HRV 3C protease cleavage site (GST3CNsp10).
    pGEX-6P-1
    suggested: None
    Plasmids for codon-optimized pET-28a- His6-nsp7-8 and pET-28a-His6-nsp7-11 (with an HRV 3C protease cleavage site between the 6x- His tag and the coding sequence) were obtained from GenScript (Piscataway, NJ).
    pET-28a-
    suggested: None
    pET-28a-His6-nsp7-11
    suggested: None
    The pGEX- 6P-1-nsp5 expression plasmid was transformed into E. coli Rosetta gami competent cells and cultured in LB media at 37 °C with 100 μg/mL ampicillin.
    pGEX-
    suggested: None
    6P-1-nsp5
    suggested: None
    Software and Algorithms
    SentencesResources
    The gene encoding SARS-CoV-2 nsp10 was cloned into the pGEX-6P-1 vector to generate an expression construct containing an N-terminal GST tag and an HRV 3C protease cleavage site (GST3CNsp10).
    SARS-CoV-2
    suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
    The gel band intensity for nsp7-11 was calculated using ImageJ software (https://imagej.nih.gov/ij/index.html) and plotted against the concentration of binders using the GraphPad Prism Version 9.3.1 (GraphPad Software, La Jolla California USA, www.graphpad.com).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Crosslink spectral matches found in Proteome Discoverer were exported and converted to sequence spectrum list format using Excel (Microsoft).
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Excel
    suggested: None
    The data was reduced using BioXTAS RAW 2.0.3 (81).
    BioXTAS
    suggested: None
    They are also provided in the SM as PyMOL sessions.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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: 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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  3. Version published to 10.1101/2022.05.27.493767v1 on bioRxiv