The roles of APOBEC-mediated RNA editing in SARS-CoV-2 mutations, replication and fitness

  1. Kyumin Kim
  2. Peter Calabrese
  3. Shanshan Wang
  4. Chao Qin
  5. Youliang Rao
  6. Pinghui Feng
  7. Xiaojiang S. Chen

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.

Article activity feed

  1. Version published to 10.1038/s41598-022-19067-x
  2. ScreenIT

    SciScore for 10.1101/2021.12.18.473309: (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.
    RandomizationIn this first 2-cycle PCR amplification, the forward and reverse primers were attached to barcodes consists of 15 randomized nucleotides as the Unique Identifier (UID), plus four tri-nucleotides designating four different experimental conditions: TGA for A1+A1CF; CAT for A3A; GTC for A3G; and ACG for Ctrl
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    , anti-HA mAb (HA.C5, Abcam, 1:3,000), and anti-α-tubulin mAb from mouse (GT114, GeneTex, 1:5,000) as primary antibodies.
    anti-HA
    suggested: None
    anti-α-tubulin
    suggested: None
    GT114
    suggested: None
    Cy3-labelled goat-anti-mouse mAb (PA43009, GE Healthcare, 1:3,000) was subsequently used as a secondary antibody.
    mAb
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lentivirus was produced by lentiviral vector system pLVX-TetOne-Puro (Clon-tech) in HEK293T cells.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    The Caco-2 stable cell lines were generated by transducing with the lentivirus for 24 hrs and selected with 5 µg/ml of puromycin.
    Caco-2
    suggested: None
    For SARS-CoV-2 propagation, Vero E6-hACE2 cells were used.
    Vero E6-hACE2
    suggested: None
    To assess the effect of APOBEC (A1+A1CF, A3A, and A3G) on SARS-CoV-2 RNA replication, the Caco-2-APOBEC stable cells (about 2× 105 cells) were plated in 12-well plates.
    Caco-2-APOBEC
    suggested: None
    To assess the effect of APOBEC (A1+A1CF, A3A, and A3G) on SARS-CoV-2 viral progeny production, plaque assay on Vero E6-hACD2 cells was used.
    Vero E6-hACD2
    suggested: None
    Recombinant DNA
    SentencesResources
    Lentivirus was produced by lentiviral vector system pLVX-TetOne-Puro (Clon-tech) in HEK293T cells.
    pLVX-TetOne-Puro
    suggested: RRID:Addgene_124797)
    The cells were then co-transfected with lentiviral packaging vectors, 1.0 μg of pdR8.91 (Gag-Pol-Tat-Rev, Addgene), 0.5 μg of pMD2.
    pMD2
    suggested: None
    G (VSV-G, Addgene), and 1.7 μg of the pLVX-TetOne-Puro vector encoding the APOBEC proteins, using 20 μL of X-tremeGENE 9 transfection reagent (Sigma).
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    We wrote Python scripts to analyze the sequencing data.
    Python
    suggested: (IPython, RRID:SCR_001658)

    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 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.

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

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.12.18.473309 on bioRxiv