Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites

  1. Nicole Doyle
  2. Jennifer Simpson
  3. Philippa C. Hawes
  4. Helena J. Maier

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Abstract

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

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  1. Version published to 10.3390/v13122540
  2. ScreenIT

    SciScore for 10.1101/2021.11.04.467246: (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

    Antibodies
    SentencesResources
    After washing, Alexa Fluor secondary antibody (Invitrogen) in blocking buffer was incubated on cells for 1 h, followed by washing, labeling of nuclei using 4’,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) or ToPro3 (ThermoFisher) and mounting onto glass slides with Vectashield (Vector Labs, Peterborough).
    Vectashield ( Vector Labs , Peterborough) .
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Huh7 cells (ATCC) were maintained in DMEM supplemented with 10% FBS.
    Huh7
    suggested: None
    VeroE6 cells (LGC Standards Ltd.) were maintained in DMEM supplemented with 10% FBS.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Porcine LLC-PK1 cells (ATCC CL-101 [46]) were maintained in DMEM supplemented with 10% FBS.
    LLC-PK1
    suggested: None
    Software and Algorithms
    SentencesResources
    BrU; Sigma Aldrich) and 15 μM actinomycin D (ActD; Sigma Aldrich) at 30 min prior to each timepoint.
    BrU; Sigma Aldrich
    suggested: None
    ActD; Sigma Aldrich
    suggested: None
    After washing, Alexa Fluor secondary antibody (Invitrogen) in blocking buffer was incubated on cells for 1 h, followed by washing, labeling of nuclei using 4’,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) or ToPro3 (ThermoFisher) and mounting onto glass slides with Vectashield (Vector Labs, Peterborough).
    DAPI; Sigma Aldrich
    suggested: None
    STED Images were deconvolved using Huygens Professional software 18.10 (Scientific Volume Imaging, Netherlands).
    Huygens Professional
    suggested: None
    Figures were assembled using Adobe Photoshop.
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)

    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: 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 10.1101/2021.11.04.467246v1 on bioRxiv