Inhibition of Arenavirus Entry and Replication by the Cell-Intrinsic Restriction Factor ZMPSTE24 Is Enhanced by IFITM Antiviral Activity

  1. Robert J. Stott-Marshall
  2. Toshana L. Foster

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Abstract

In the absence of effective vaccines and treatments, annual outbreaks of severe human haemorrhagic fever caused by arenaviruses, such as Lassa virus, continue to pose a significant human health threat. Understanding the balance of cellular factors that inhibit or promote arenavirus infection may have important implications for the development of effective antiviral strategies. Here, we identified the cell-intrinsic zinc transmembrane metalloprotease, ZMPSTE24, as a restriction factor against arenaviruses. Notably, CRISPR-Cas9-mediated knockout of ZMPSTE24 in human alveolar epithelial A549 cells increased arenavirus glycoprotein-mediated viral entry in pseudoparticle assays and live virus infection models. As a barrier to viral entry and replication, ZMPSTE24 may act as a downstream effector of interferon-induced transmembrane protein (IFITM) antiviral function; though through a yet poorly understood mechanism. Overexpression of IFITM1, IFITM2, and IFITM3 proteins did not restrict the entry of pseudoparticles carrying arenavirus envelope glycoproteins and live virus infection. Furthermore, gain-of-function studies revealed that IFITMs augment the antiviral activity of ZMPSTE24 against arenaviruses, suggesting a cooperative effect of viral restriction. We show that ZMPSTE24 and IFITMs affect the kinetics of cellular endocytosis, suggesting that perturbation of membrane structure and stability is likely the mechanism of ZMPSTE24-mediated restriction and cooperative ZMPSTE24-IFITM antiviral activity. Collectively, our findings define the role of ZMPSTE24 host restriction activity in the early stages of arenavirus infection. Moreover, we provide insight into the importance of cellular membrane integrity for productive fusion of arenaviruses and highlight a novel avenue for therapeutic development.

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  1. Version published to 10.3389/fmicb.2022.840885
  2. ScreenIT

    SciScore for 10.1101/2021.04.12.439453: (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
    Membranes were blocked in 5% milk in PBS with 0.1% Tween®20 (PBS-T) for 30 min prior to incubation with specific primary antibodies: mouse anti-IFITM1 (Proteintech, 60074-1-Ig, 1:5000), rabbit anti-IFITM2 (Proteintech, 12769-1-AP, 1:5000), rabbit anti-IFITM3 (Proteintech, 11714-1-AP, 1:5000), rabbit anti-ZMPSTE24 antibody (Abcam ab38450, 1:1000), mouse anti-FLAG (Sigma, F1804, 1:2000), mouse anti-HA (Abcam ab18181, 1:5000)
    anti-IFITM1
    suggested: None
    60074-1-Ig
    suggested: (Proteintech Cat# 60074-1-Ig, RRID:AB_2233405)
    anti-IFITM2
    suggested: (Proteintech Cat# 12769-1-AP, RRID:AB_2122089)
    12769-1-AP
    suggested: (Proteintech Cat# 12769-1-AP, RRID:AB_2122089)
    anti-IFITM3
    suggested: (Proteintech Cat# 11714-1-AP, RRID:AB_2295684)
    11714-1-AP
    suggested: (Proteintech Cat# 11714-1-AP, RRID:AB_2295684)
    anti-ZMPSTE24
    suggested: (Abcam Cat# ab38450, RRID:AB_778983)
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    F1804
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-HA
    suggested: (Abcam Cat# ab18181, RRID:AB_444303)
    After washing membranes with PBS-T at RT, horseradish peroxidase-conjugated (HRP) horse anti-mouse IgG (CST, 7076S, 1:5000) and goat anti-rabbit IgG (CST, 7074S, 1:5000) secondary antibodies in 5% milk in PBS-T were added and membranes incubated for 1 h at RT with gentle shaking.
    anti-mouse IgG
    suggested: (Cell Signaling Technology Cat# 7076, RRID:AB_330924)
    7076S
    suggested: (Cell Signaling Technology Cat# 7076, RRID:AB_330924)
    anti-rabbit IgG
    suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)
    7074S
    suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)
    Lysed samples were centrifuged and supernatants were immunoprecipitated with 5µg/ml mouse monoclonal anti-IFITM2/3 antibody (Proteintech, 66081-1-Ig) for 1.5 h at 4°C.
    anti-IFITM2/3
    suggested: (Proteintech Cat# 66081-1-Ig, RRID:AB_11182821)
    Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT and stained overnight at 4°C with the appropriate primary antibodies (rabbit anti-ZMPSTE24, Abcam ab38450, 1:100 or mouse monoclonal anti-HA, Abcam ab18181, 1:500 or rabbit anti-EEA1, CST 2411S, 1:100 or rabbit anti-Rab9A, CST 5118T, 1:200, or rabbit anti-LAMP1, Invitrogen 14-1079-80, 1:500.
    anti-EEA1
    suggested: (SICGEN Cat# AB0005-200, RRID:AB_2333135)
    anti-Rab9A
    suggested: None
    anti-LAMP1
    suggested: (LSBio (LifeSpan Cat# LS-C84918-500, RRID:AB_1664591)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and expression constructs: Human embryonic kidney 293T (293T; ATCC), kidney epithelial Vero (Vero; ATCC), human lung adenocarcinoma epithelial (A549; ATCC) and A549 cells expressing ZMPSTE24 or the individual IFITM proteins were cultured in Dulbecco’s Modified Eagle Medium (DMEM), high glucose, GlutaMAX™ Supplement (Gibco) with 10% heat inactivated FBS (Gibco) and 200
    A549
    suggested: None
    Retroviral vectors were made by transfecting 293T cells with the pCMV-Gag-Pol murine leukaemia virus (MLV) packaging construct (kindly gifted by Professor Jonathan Ball), the pLHCX or pQXCIP packaging vector of interest and pCMV VSV-G using 1mg/ml PEI®-MAX (Polysciences).
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Passage and titration of Mopeia virus (MOPV): The UVE/MOPV/UNK/MZ/Mozambique 20410 strain of MOPV was obtained from European Virus Archive and mycoplasma-free virus stocks were propagated in Vero cells in DMEM supplemented with 2% FCS.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Flow cytometry analyses were performed using a BD FACSCanto II flow-cytometer (Becton Dickinson), collecting 10,000 events, and analysed using FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Z stacks were taken for all stained conditions and images were deconvolved with the Zeiss ZEN deconvolution software and analysed using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    For each qPCR reaction, 10ng of cDNA was used with the Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix under the following conditions: 50°C 2 mins, 95°C 2 mins then 40 cycles of 95°C 15 secs, 55°C 15 secs, 72°C 1 min
    Applied Biosystems™
    suggested: (Applied Biosystems, RRID:SCR_005039)
    Statistical analysis: All statistical analyses were carried out using GraphPad Prism v9.0.2.
    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: 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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  3. Version published to 10.1101/2021.04.12.439453v1 on bioRxiv