Distinct genetic determinants and mechanisms of SARS-CoV-2 resistance to remdesivir

  1. Laura J. Stevens
  2. Andrea J. Pruijssers
  3. Hery W. Lee
  4. Calvin J. Gordon
  5. Egor P. Tchesnokov
  6. Jennifer Gribble
  7. Amelia S. George
  8. Tia M. Hughes
  9. Xiaotao Lu
  10. Jiani Li
  11. Jason K. Perry
  12. Danielle P. Porter
  13. Tomas Cihlar
  14. Timothy P. Sheahan
  15. Ralph S. Baric
  16. Matthias Götte
  17. Mark R. Denison

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Abstract

The nucleoside analog remdesivir (RDV) is an FDA-approved antiviral for the treatment of SARS- CoV-2 infections, and as such it is critical to understand potential genetic determinants and barriers to RDV resistance. In this study, SARS-CoV-2 was subjected to 13 passages in cell culture with increasing concentrations of GS-441524, the parent nucleoside of RDV. At passage 13 the RDV resistance of the lineages ranged from 2.7-to 10.4-fold increase in EC 50 . Sequence analysis of the three lineage populations identified non-synonymous mutations in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two of the three lineages encoded the S759A substitution at the RdRp Ser 759 -Asp-Asp active motif. In one lineage, the V792I substitution emerged first then combined with S759A. Introduction of the S759A and V792I substitutions at homologous nsp12 positions in viable isogenic clones of the betacoronavirus murine hepatitis virus (MHV) demonstrated their transferability across CoVs, up to 38-fold RDV resistance in combination, and a significant replication defect associated with their introduction. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a ∼10- fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, while nsp12-V792I diminished the UTP concentration needed to overcome the template-dependent inhibition associated with RDV. The in vitro selected substitutions here identified were rare or not detected in the >6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in various clinical settings.

One Sentence Summary

SARS-CoV-2 develops in vitro resistance to remdesivir by distinct and complementary mutations and mechanisms in the viral polymerase

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  1. ScreenIT

    SciScore for 10.1101/2022.01.25.477724: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    A549 cells overexpressing the human ACE2 receptor (A549-hACE2) (46) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 1% MEM Non-Essential Amino Acids Solution (
    A549
    suggested: None
    Three wells of Vero E6 cells were treated with 0.5 μM GS-441524, and three other wells were treated with 0.1% DMSO (vehicle controls), each well representing one lineage.
    Vero E6
    suggested: None
    Viral replication assays: A549-hACE2 or DBT-9 cells were seeded at 1 x 105 cells per well in 24-well plates (Corning) and allowed to reach confluence within 24 h.
    DBT-9
    suggested: None
    A549-hACE2 cells were adsorbed with MOI = 0.01 PFU/ml SARS-CoV-2 passaged population virus or plaque-isolated sub-lineages.
    A549-hACE2
    suggested: RRID:CVCL_A5KB)
    Recombinant DNA
    SentencesResources
    The pFastBac-1 (Invitrogen, Burlington, Ontario, Canada) plasmid with codon-optimized synthetic DNA sequences (GenScript, Piscataway, NJ) coding for a portion of 1ab polyproteins of SARS-CoV-2 (NCBI: QHD43415.1), containing only nsp5, nsp7, nsp8, and nsp12, was used as starting material for protein expression in insect cells (Sf9, Invitrogen).
    pFastBac-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Data and Code Availability: The bioinformatic pipeline utilized for all RNA-seq datasets is available at https://github.com/DenisonLabVU/CoVariant.git.
    bioinformatic
    suggested: (QFAB Bioinformatics, RRID:SCR_012513)
    All sequencing datasets are publicly available at the NCBI Sequence Read Archive (SRA) under BioProject PRJNA787945 (RNA-seq) and PRJNA787608 (Nanopore).
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    All fragments containing mutations were Sanger sequenced to ensure mutations were present before use in further studies (GeneWiz, South Plainfield, NJ)
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Amino acid locations were confirmed through sequence alignment using MacVector and CLC Workbench (QIAGEN)
    MacVector
    suggested: (MacVector, RRID:SCR_015700)
    The pooled library was loaded onto a quality-checked MinION flowcell with 1491 functional sequencing pores, and sequencing was performed using the MinKNOW GUI over 72 hours.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    The second module of the MutALink pipeline calls and quantifies variant allele frequencies for candidate variants using Nanopolish(52).
    MutALink
    suggested: None
    Read counts were corrected manually for duplicate counting between combinations, and the frequency of each genotype in each sample passage compared to total mapped reads was reported and visualized using the Python package, seaborn(54)
    Python
    suggested: (IPython, RRID:SCR_001658)
    Mathematical and statistical analyses: The EC50 value was calculated in GraphPad Prism 8 as the concentration at which there was a 50% decrease in viral replication relative to vehicle alone (0% inhibition).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your code and 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.
    • 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 10.1101/2022.01.25.477724 on bioRxiv