In vivo structure and dynamics of the RNA genome of SARS-Cov-2

  1. Yan Zhang
  2. Kun Huang
  3. Dejian Xie
  4. Jian You Lau
  5. Wenlong Shen
  6. Ping Li
  7. Dong Wang
  8. Zhong Zou
  9. Shu Shi
  10. Hongguang Ren
  11. Meilin Jin
  12. Grzegorz Kudla
  13. Zhihu Zhao

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Abstract

The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3’ -UTR and 5’ -UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide direct evidence of interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE, forming a “high-order pseudoknot” embedding FSE, which might help ribosome stalling at frameshift sites. More importantly, we found that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome cyclization is weakened and genome domains remain stable. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, describes dynamics of RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.

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    SciScore for 10.1101/2021.01.15.426526: (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

    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Chlorocebus sabaeus (Green monkey) VeroE6 (female, RRID:CVCL_YQ49) were purchased from American Type Culture Collection (ATCC, id: ATCC CRL-1586).
    detected: (JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Vero E6 and VeroE6/TMPRSS2 cells were cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a humidified CO2 incubator.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    VeroE6 cells were inoculated with SARS-CoV-2 strain Wuhan-Hu-1 at MOI=0.01 pfu/cell for 24 hours.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    SARS-CoV-2 virus strain Wuhan-Hu-1 was kindly provided by (Wuhan institute of viology).
    Wuhan-Hu-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Viewpoint histograms and arc plots were plotted with ggplot2 R package [43].
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    For the spatial location of particular gene loci, we used 1 point/20 balls to calculate the position of specific genes in the whole 3D simulation, and then modify the pymol results by a python script.
    pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Differential interaction identifying: Differential gene expression analysis was performed using DESeq2 [32].
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)

    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 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.
    • 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

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  2. Version published to 10.1101/2021.01.15.426526 on bioRxiv