The stem loop 2 motif is a site of vulnerability for SARS-CoV-2

  1. Valeria Lulla
  2. Michal P. Wandel
  3. Katarzyna J. Bandyra
  4. Rachel Ulferts
  5. Mary Wu
  6. Tom Dendooven
  7. Xiaofei Yang
  8. Nicole Doyle
  9. Stephanie Oerum
  10. Rupert Beale
  11. Sara M. O’Rourke
  12. Felix Randow
  13. Helena J. Maier
  14. William Scott
  15. Yiliang Ding
  16. Andrew E. Firth
  17. Kotryna Bloznelyte
  18. Ben F. Luisi

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Abstract

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stemloop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3’ UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 infection in cell culture. Our results thus permit us to suggest that the s2m element is a site of vulnerability readily targeted by ASOs, which show promise as anti-viral agents.

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  2. Rapid Reviews Infectious Diseases

    Anders Näär

    Review 2: "Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation"

    This manuscript offers a gapmer-based therapeutic strategy targeting the highly conserved s2m element present in the SARS-CoV-2 genome. While potentially informative, the study's claims require testing with wildtype SARS-CoV-2 virus and animal models to be substantiated.

  3. Rapid Reviews Infectious Diseases

    Valerio Orlando

    Review 3: "Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation"

    This manuscript offers a gapmer-based therapeutic strategy targeting the highly conserved s2m element present in the SARS-CoV-2 genome. While potentially informative, the study's claims require testing with wildtype SARS-CoV-2 virus and animal models to be substantiated.

  4. Rapid Reviews Infectious Diseases

    Hasan Cubuk

    Review 1: "Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation"

    This manuscript offers a gapmer-based therapeutic strategy targeting the highly conserved s2m element present in the SARS-CoV-2 genome. While potentially informative, the study's claims require testing with wildtype SARS-CoV-2 virus and animal models to be substantiated.

  5. ScreenIT

    SciScore for 10.1101/2020.09.18.304139: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: All cell lines tested negative for mycoplasma.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    HEK293ET, HeLa and A459 cells, as well as all stable cell lines, were grown in IMDM medium supplemented with 10% FCS at 37 °C in 5% CO2.
    HEK293ET
    suggested: RRID:CVCL_6996)
    HeLa
    suggested: None
    A459
    suggested: RRID:CVCL_A459)
    To examine the effect of gapmers on GFP expression, HeLa or A549 cells with a stably integrated GFP-s2m/s2m_scr reporter were seeded at 5 × 104 cells per well in 24-well plates.
    A549
    suggested: None
    Huh7.5.1 and HEK293T cells were transfected in triplicate with Lipofectamine 2000 reagent (Invitrogen), using the protocol in which suspended cells are added directly to the RNA complexes in 96-well plates.
    Huh7.5.1
    suggested: RRID:CVCL_E049)
    HEK293T
    suggested: None
    SARS-CoV-2 infection assay: Vero E6 cells (NIBC, UK) were grown in DMEM (containing 10 % FBS) at 37 °C and 5 % CO2 in 96 well imaging plates (Greiner 655090).
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Quantitative Gel Analysis and SHAPE Reactivity Calculation: Signal intensity of each band on the PAGE gel was detected using ImageQuant TL software and normalized to the total signal of the whole lane.
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    Motion correction, ctf estimation and particle-picking were performed in Warp (Tegunov and Cramer, 2019) and 2D/3D alignments and averaging were carried out with cryoSPARC 2.15 (Punjani et al., 2017)
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Data were analysed in FlowJo (v10.7.1).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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 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 33. 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

    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.

  6. Version published to 10.1101/2020.09.18.304139 on bioRxiv