Targeting stem-loop 1 of the SARS-CoV-2 5′ UTR to suppress viral translation and Nsp1 evasion

  1. Setu M. Vora
  2. Pietro Fontana
  3. Tianyang Mao
  4. Valerie Leger
  5. Ying Zhang
  6. Tian-Min Fu
  7. Judy Lieberman
  8. Lee Gehrke
  9. Ming Shi
  10. Longfei Wang
  11. Akiko Iwasaki
  12. Hao Wu

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Abstract

The COVID-19 pandemic and the ever-evolving variants of SARS-CoV-2 are taking a toll on human health. Despite the successful rollout of vaccines, effective therapies are still urgently needed. Our studies here showing that Nsp1 selectively blocks translation of host but not viral proteins by proper coordination of its N- and C-terminal domains to advance our understanding on SARS-CoV-2 pathogenesis. Our finding that stem-loop 1, a highly conserved sequence in the SARS-CoV-2 5′ UTR, is necessary and sufficient for bypassing Nsp1-mediated shutdown led to the design of antisense oligonucleotides targeting this sequence that make viral translation susceptible to Nsp1 shutdown, interfere with viral replication, and protect SARS-CoV-2–infected mice. This strategy of turning SARS-CoV-2’s own virulence against itself could be harnessed therapeutically.

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  1. Version published to 10.1073/pnas.2117198119
  2. ScreenIT

    SciScore for 10.1101/2021.09.09.459641: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variableCell culture: HEK293T (ATCC CRL-3216, female)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T (ATCC CRL-3216, female)
    HEK293T
    suggested: None
    , HeLa cells (ATCC CCL2, female), and, and Vero E6 (ATCC CRL1586) were purchased from the American Type Culture Collection, and Expi293 cells (female) were from ThermoFisher.
    HeLa
    suggested: None
    Vero E6
    suggested: None
    Expi293
    suggested: RRID:CVCL_D615)
    Immunofluorescence and quantitative microscopy: To visualize the expression of mScarlet and MBP-tagged SARS-CoV-2 Nsp1 (WT, CT, NT, Nsp1-linker1 and Nsp1-linker2), transfected HeLa or 293T cells were fixed with 4% formaldehyde for 10 minutes at room temperature, permeabilized with PBS + 0.25% Triton X-100, and blocked with 3% BSA.
    293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids and transfection: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.
    pDONR207
    suggested: RRID:Addgene_100600)
    pDB-His-MBP
    suggested: RRID:Addgene_123365)
    pCMV-Dest
    suggested: None
    Full-length 265 nt 5′ UTR of SARS-CoV-2 was subcloned to replace the 5’ UTR of human CMV in the pLV-mScarlet vector using a Hifi one-step kit (Gibson Assembly,
    pLV-mScarlet
    suggested: None
    Full-length, SL1 alone or ΔSL1 5′ UTR of SARS-CoV-2 were cloned into pLV-mScarlet vector or pGL3 basic vector.
    pGL3
    suggested: RRID:Addgene_48743)
    Software and Algorithms
    SentencesResources
    Image data were analysed and quantified using Image J (NIH) or MetaXpress software (Molecular Devices).
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    MetaXpress
    suggested: (MetaXpress, RRID:SCR_016654)
    All graphs were plotted and analysed with GraphPad Prism 5 software. p > 0.05 was considered statistically not significant, and the following denotations were used: ***p < 0.001, **p < 0.01 and *p < 0.05.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    One caveat is that these measurements were taken in late timepoints when viral RNAs had already started to enter the endomembrane system for packaging and would thus be shielded from the translation in the cytosol, skewing the number of elongating transcripts per copy(Finkel et al., 2021). A role for selective and direct translational block of host mRNAs is also supported by in vitro translation experiments (Mendez et al., 2021; TIDU et al., 2020). Nonetheless it is likely that both mRNA degradation and inhibition of translation are mechanistically and functionally linked and both are major contributors to SARS-CoV-2 virulence. The exact molecular basis for how Nsp1 NT coordinates with SL1 of SARS-CoV-2 5’ UTR to bypass the translation inhibition is still not yet clear. A previous study suggested that both FL and ΔCT Nsp1 directly bind the SL1 region of the CoV-2 5’ UTR by gel shift (Vankadari et al., 2020). However, this study was performed under low salt conditions, and we could not detect such interactions under physiological salt concentrations (data not shown). Thus, the molecular details of NT coordination with SL1 remain elusive, and it is likely mediated by an indirect physical interaction. Future studies will be critical for teasing out the functional interactions between Nsp1 and host proteins. Our data also demonstrates that a single cis-acting element in the SARS-CoV-2 5’UTR, the SL1 stem-loop, is both necessary and sufficient for evading Nsp1-mediated host shutdo...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 10, 14, 19, 26 and 29. 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.

    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 10.1101/2021.09.09.459641v1 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.09.18.302901: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Equal portions of the cell lysate were run on a 15% SDS-PAGE gel and blotted onto a PVDF membrane, which was subsequently probed with HRP Anti-Strep tag antibody (Abcam) and developed with an ECL substrate (Amersham Biosciences).
    Anti-Strep tag antibody ( Abcam )
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T, Expi293F and HeLa cells were purchased from the American Type Culture Collection (ATCC), and cultured in Dulbecco’s Modified Eagle medium (DMEM) or Expi293 Expression Medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) at 37 °C with 5% CO2.
    HEK293T
    suggested: None
    HeLa
    suggested: None
    Software and Algorithms
    SentencesResources
    Movies were motion-corrected and dose-weighted using MotionCor2 (Zheng et al., 2017).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    Patch contrast transfer function (CTF) estimation was performed using cryoSPARC (Punjani et al., 2017).
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Inspection, model building, and manual adjustment were carried out in Coot (Emsley and Cowtan, 2004).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Real-space refinement was performed using PHENIX (Adams et al., 2010).
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    All representations of densities and structural models were generated using Chimera, ChimeraX (Goddard et al., 2018), and Pymol (DeLano, 2002).
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Image data were analyzed and quantified using Image J (NIH) or MetaXpress software (Molecular Devices).
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    MetaXpress
    suggested: (MetaXpress, RRID:SCR_016654)
    All graphs were plotted and analyzed with GraphPad Prism 5 software. p > 0.05 was considered statistically not significant, and the following denotations were used: **p < 0.001 and *p < 0.05.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 12 and 13. 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.

  5. Version published to 10.1101/2020.09.18.302901v1 on bioRxiv