The SARS CoV-1 3a protein disrupts Golgi complex morphology and cargo trafficking

  1. Rex R. Gonzales
  2. Carolyn E. Machamer

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Abstract

Coronaviruses assemble by budding into the endoplasmic reticulum-Golgi intermediate compartment, but the pathway of egress from infected cells is not well understood. Efficient egress of infectious bronchitis virus (a gamma coronavirus, CoV) requires neutralization of Golgi pH by the envelope (E) protein. This results in reduced rates of cargo traffic and disrupts Golgi morphology, but it protects the spike protein from aberrant proteolysis. The severe acute respiratory syndrome (SARS) CoV-1 E protein does not disrupt the Golgi, however. We show here that in transfected cells, the ORF3a protein of SARS CoV-1 disrupts Golgi morphology, cargo trafficking and luminal pH. Unlike the infectious bronchitis virus E protein, these functions of the SARS CoV-1 3a protein appear to require its viroporin activity. Thus, neutralization of acidic compartments may be a universal feature of CoV infection, although different viral proteins and mechanisms may be used to achieve this outcome.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.19.440492: (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 AuthenticationContamination: They were routinely screened for mycoplasma contamination.
    Authentication: We determined specificity by immunoblotting and immunofluorescence using control and cells expressing SARS CoV-1 3a.

    Table 2: Resources

    Antibodies
    SentencesResources
    Commercial primary antibodies were mouse anti-GM130 and mouse anti-p230 (BD Transduction Labs), and rabbit anti-giantin (Covance Research Products)
    anti-GM130
    suggested: None
    anti-p230
    suggested: None
    anti-giantin
    suggested: None
    Fluorescent secondary antibodies were from Life Technologies: Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 568 donkey anti-mouse IgG, and Cy5 donkey anti-rabbit IgG.
    anti-mouse IgG
    suggested: None
    Cy5 donkey anti-rabbit IgG
    suggested: None
    After washing, secondary antibodies (IRDye 800 donkey anti-rabbit IgG, LiCOR) were applied for 1h at RT and after washing, blots were quantified on LiCOR Odyssey CLx system and Image Studio (LiCOR)
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Transfection: Vero cells were plated at 2×105 per 35 mm dish on coverslips for indirect immunofluorescence microscopy, or 4×105 in 6 well dishes for trafficking experiments.
    Vero
    suggested: None
    Recombinant DNA
    SentencesResources
    CoV-1 (Tor2 strain) was originally obtained from the Institute for Genomic Research and subcloned into pBluescript (Stratagene) prior to transferring to pCAGGS-MCS [17] after PCR amplification with KpnI and XhoI restriction sites.
    pBluescript
    suggested: None
    pCAGGS-MCS
    suggested: None
    The pCAGGS plasmids encoding IBV E and SARS CoV-1 E have been previously described [10,18].
    pCAGGS
    suggested: RRID:Addgene_18926)
    Plasmids pCAGGS/SARS S and pCAGGS/SARS M were previously described [20], as was the pCAGGS/VSVG plasmid [21].
    pCAGGS/SARS
    suggested: None
    pCAGGS/VSVG
    suggested: None
    The pGnT1-pHorin and TGN-pHlorin plasmids [22] were kindly provided by Dr. Yasuke Maeda (Osaka University, Japan).
    pGnT1-pHorin
    suggested: None
    TGN-pHlorin
    suggested: None
    For immunofluorescence, 0.5 ug of SARS 3a or IBV E (with or without 0.2 ug of one of the pHlorin plasmids) by adding to 100 ul OPTI-MEM (Life Technologies) with diluted XtremeGene-9 (Roche), per the manufacturer’s instructions.
    pHlorin
    suggested: None
    For the trafficking experiments, 0.5 ug of 3a or E was mixed with 0.5 ug SARS CoV S or VSV-G.
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    Images were acquired on an Axioskop microscope (Zeiss) equipped for epifluorescence using an Orca-03G CCD camera (Hamamatsu) and iVision software (BioVision Technologies).
    iVision
    suggested: (iVision, RRID:SCR_014786)
    Images (acquired with the same parameters) were analyzed by outlining the stained Golgi elements after background subtraction using the trapezoid tool in ImageJ (Fiji), and the area occupied by each marker was quantified using the measure tool in the analysis menu.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Analysis was in Prism GraphPad 8.0.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data were collected and analyzed using FACS Diva 8.0 software and Excel.
    Excel
    suggested: None

    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 limitation of our study is that we only examined cells expressing the viral proteins from cDNA. In the future, it will be important to measure the luminal pH of Golgi, endosomes and lysosomes in infected cells. Our previous results with IBV E suggest that the phenotypes in cells overexpressing the protein might be exaggerated but accurately reflect the function in infected cells [7, 11, 12]. Proteolytic processing of S in both SARS CoV-1 and -2 infected cells when the 3a protein is compromised will also need to be assessed. Importantly, specific inhibition of 3a channel function with repurposed or new drugs may provide additional treatments for SARS, MERS and COVID-19 [3, 32].

    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.

    About SciScore

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  2. Version published to 10.1101/2021.04.19.440492v1 on bioRxiv