Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice

  1. Ashley Zani
  2. Adam D. Kenney
  3. Jeffrey Kawahara
  4. Adrian C. Eddy
  5. Xiao-Liang Wang
  6. Mahesh KC
  7. Mijia Lu
  8. Emily A. Hemann
  9. Jianrong Li
  10. Mark E. Peeples
  11. Luanne Hall-Stoodley
  12. Adriana Forero
  13. Chuanxi Cai
  14. Jianjie Ma
  15. Jacob S. Yount

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Abstract

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo , K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

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    SciScore for 10.1101/2021.12.22.473914: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Experimental procedures were approved by the OSU Institutional Biosafety Committee and OSU BSL3 Advisory
    Sex as a biological variableBoth male and female mice were used in our experiments.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Presence of virus replication in each well was assessed by visual examination of cytopathic effect along with antibody staining for N protein antigen (mouse monoclonal antibody 40143-MM08, Sino Biological) with fluorescent secondary antibody detection.
    N protein antigen
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Both viruses were plaque purified on Vero-TMPRSS2 cells (kindly provided by Dr. Shan-Lu Liu, The Ohio State University), and viral genomes from individual plaques were sequenced to identify virus lacking mutations in the furin cleavage site of the Spike protein.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Virus plaques with intact furin cleavage sites were propagated on Vero-TRMPSS2 cells.
    Vero-TRMPSS2
    suggested: None
    Viral stocks and research samples were titered via TCID50 measurements on Vero E6 cells using 10-fold serial dilutions in triplicate.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    K18-hACE2 hemizygous mice were purchased from Jackson Laboratories.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    IFITM3-KO mice were crossed with K18-hACE2 mice to obtain F1 mice possessing a K18-hACE2 allele with heterozygous IFITM3 KO.
    IFITM3-KO
    suggested: None
    F1 mice were then bred with IFITM3 KO mice to generate mice carrying hACE2 transgene and homozygous KO of IFITM3.
    IFITM3 KO
    suggested: None
    Software and Algorithms
    SentencesResources
    Quantification of these images was performed using ImageJ software and the color deconvolution method.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The principal components analysis and volcano plots were re-formatted in PRISM graphing software using values downloaded from ROSALIND.
    PRISM
    suggested: (PRISM, RRID:SCR_005375)
    The topGO R library was used to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction
    topGO
    suggested: (topGO, RRID:SCR_014798)

    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.

  2. Version published to 10.1101/2021.12.22.473914v1 on bioRxiv