Pro108Ser mutation of SARS-CoV-2 3CLpro reduces the enzyme activity and ameliorates the clinical severity of COVID-19

  1. Kodai Abe
  2. Yasuaki Kabe
  3. Susumu Uchiyama
  4. Yuka W. Iwasaki
  5. Hirotsugu Ishizu
  6. Yoshifumi Uwamino
  7. Toshiki Takenouchi
  8. Shunsuke Uno
  9. Makoto Ishii
  10. Takahiro Maruno
  11. Masanori Noda
  12. Mitsuru Murata
  13. Naoki Hasegawa
  14. Hideyuki Saya
  15. Yuko Kitagawa
  16. Koichi Fukunaga
  17. Masayuki Amagai
  18. Haruhiko Siomi
  19. Makoto Suematsu
  20. Kenjiro Kosaki
  21. Keio Donner Project

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Abstract

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CL pro ) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CL pro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CL pro revealed that the Kcat/Km of the 3CL pro enzyme containing Ser108 was 58% lower than that of Pro108 3CL pro . Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CL pro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CL pro inhibitor.

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  1. Version published to 10.1038/s41598-022-05424-3
  2. Version published to 10.1101/2020.11.24.20235952v2 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.11.24.20235952: (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 point mutations including non-synonymous and synonymous mutations were annotated with ANNOVAR software and assessed with VarSifter (https://research.nhgri.nih.gov/software/VarSifter/).
    ANNOVAR
    suggested: (ANNOVAR, RRID:SCR_012821)
    The multiple amino acid sequence alignments of various β-coronaviruses were compared with Molecular Evolutionary Genetic Analysis software (MEGA, https://www.megasoftware.net/) (Supplementary Table 3).
    Molecular Evolutionary Genetic Analysis
    suggested: None
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    The functional relevance of non-synonymous mutations was predicted with a Protein Variation Effect Analyzer (PROVEAN v1.1.3, http://provean.jcvi.org/seq_submit.php), the calculations of which are not dependent on sequence conservation among animals.
    PROVEAN
    suggested: (PROVEAN, RRID:SCR_002182)
    Cloning and protein preparation of SARS-CoV-2 3CLpro: The SARS-CoV-2 3CLpro DNA fragments encoding the Wuhan strain or the strain containing Pro108Ser in non-structural polyprotein 5 (NSP5) gene were prepared using a reverse transcription kit (SuperScript III, ThermoFishher) and were amplified by PCR using primers (forward: 5’-TTTGGATCCAGTGGTTTTAGAAAAATGGCA-3’, reverse: 5’-TTTGTCGACTCATTGGAAAGTAACACCTGAGCA-3’).
    ThermoFishher
    suggested: None
    The kinetic parameters were determined with GraphPad Prism 8 software and the initial rate measurement of the substrate cleavage.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Figures of the c (s20, w) distribution were generated using the program GUSSI (version 1.3.2) (29).
    GUSSI
    suggested: (GUSSI, RRID:SCR_014962)
    To determine the dissociation constant of the monomer-dimer equilibrium (KD), the concentration dependence of the weight-average sedimentation coefficient was fitted to the monomer-dimer self-association model implemented in the program SEDPHAT (version 15.2b) (30,31)
    SEDPHAT
    suggested: (SEDPHAT, RRID:SCR_016254)
    Mass spectra were transformed using MassLynx (Waters) and acquired over the m/z range of 100–2000.
    MassLynx
    suggested: (MassLynx , RRID:SCR_014271)
    Protein structure modeling and stabilization analysis: The three-dimensional (3D) structure was visualized using PyMol v2.4.
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

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  4. Version published to 10.1101/2020.11.24.20235952v1 on medRxiv