LY6E impairs coronavirus fusion and confers immune control of viral disease

  1. Stephanie Pfaender
  2. Katrina B. Mar
  3. Eleftherios Michailidis
  4. Annika Kratzel
  5. Ian N. Boys
  6. Philip V’kovski
  7. Wenchun Fan
  8. Jenna N. Kelly
  9. Dagny Hirt
  10. Nadine Ebert
  11. Hanspeter Stalder
  12. Hannah Kleine-Weber
  13. Markus Hoffmann
  14. Hans-Heinrich Hoffmann
  15. Mohsan Saeed
  16. Ronald Dijkman
  17. Eike Steinmann
  18. Mary Wight-Carter
  19. Matthew B. McDougal
  20. Natasha W. Hanners
  21. Stefan Pöhlmann
  22. Tom Gallagher
  23. Daniel Todt
  24. Gert Zimmer
  25. Charles M. Rice
  26. John W. Schoggins
  27. Volker Thiel

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Abstract

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  1. Version published to 10.1038/s41564-020-0769-y
  2. ScreenIT

    SciScore for 10.1101/2020.03.05.979260: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All procedures used in this study complied with federal and institutional guidelines enforced by the UTSW Institutional Animal Care and Use Committee (IACUC).
    Randomizationnot detected.
    BlindingSlides were analyzed by an independent pathologist (UTSW Animal Resource Center) who was blinded to experimental conditions.
    Power Analysisnot detected.
    Sex as a biological variableIn vivo infection, viral titers, liver ALT, and liver histology: Six to twelve-week-old female and male mice were injected intraperitoneally with MHV-A59 diluted in PBS to the indicated titers or PBS for a mock infection control.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    24 hours post-infection, cells were dissociated using Accumax (Sigma-Aldrich), fixed in 1% PFA, permeabilized per manufacturer’s protocol (BD Cytofix/Cytoperm), and stained for nucleoprotein (1:500) and a goat anti-mouse AlexaFluor488-conjugated secondary antibody (1:2000).
    anti-mouse
    suggested: None
    IHIC and splenocytes (4 × 105) from infected or mock-infected mice were stained with a fluorescent viability dye (Ghostdye Violet 450, Tonbo Biosciences), treated with anti-CD16/CD32 (Tonbo Biosciences), stained with antibodies that recognize surface lineage markers, and then fixed in 1% PFA/PBS.
    anti-CD16/CD32
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HCoV-229E viruses were propagated on Huh-7 cells, MERS-CoV and SARS-CoV were propagated on VeroB4 cells, HCoV-OC43 was propagated on HCT-8 cells38, and MHV stocks were propagated on 17Cl1 cells.
    Huh-7
    suggested: None
    VeroB4
    suggested: CCLV Cat# CCLV-RIE 1146, RRID:CVCL_1912)
    For testing the virus panel against LY6E, 1 × 104 stable LY6E expressing or control Huh7.5 cells were seeded in a 96-well plate.
    Huh7.5
    suggested: RRID:CVCL_7927)
    Briefly, 6 × 105 293LTV cells were seeded in a 6-well plate and transfected using Lipofectamine 2000 (Invitrogen) which was complexed with DNA plasmids driving the expression of either VSV G protein (positive control), the respective CoV S proteins, or the fluorophore mCherry (negative control).
    293LTV
    suggested: RRID:CVCL_JZ09)
    The empty expression plasmid (pCAGGS-mCherry) as well as a construct encoding for VSV G served as negative and positive control, respectively. 2 × 105 stable LY6E expressing or empty control Huh7 cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with a plasmid encoding the second half of the split-luciferase protein (Rluc8155-156 DSP8-11).
    Huh7
    suggested: None
    Generation of recombinant VSV vector driving CoV S protein expression (VSV*ΔG(CoV)): Following extraction of total RNA from MERS-CoV (strain EMC) infected VeroE6 cells, the cDNA encoding the MERS-CoV S protein was generated by reverse transcription (RevertAid Premium Reverse Transkriptase, ThermoScientific).
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    For heterologous cell-cell fusion assay, BHK-21 cells were infected with VSV*ΔG(CoV S) at a MOI of 1.
    BHK-21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    Viral titers in liver and spleen were determined from frozen organs after weighing, homogenization, and plaque assay on L929 cells.
    L929
    suggested: ECACC Cat# 86032004, RRID:CVCL_4238)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Ly6etm1a ES cells were obtained from the EUCOMM consortium58 and microinjected into C57BL/6J blastocysts by the UTSW Transgenic Technology Center.
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Ly6etm1a/+ mice were crossed with FLPe-expressing mice (B6N.129S4-Gt(ROSA)26Sortm1(FLP1)Dyn/J, #16226, Jackson Laboratories) to obtain Ly6efl/+ offspring, which were bred to homozygosity.
    Ly6etm1a/+
    suggested: None
    B6N.129S4-Gt(ROSA)26Sortm1 ( FLP1)Dyn/J
    suggested: None
    Ly6efl/+
    suggested: None
    Conditional Ly6efl/fl mice were bred to Vav1-iCre transgenic mice (B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J, #008610, Jackson Laboratories) to obtain Ly6eΔHSC/+ (Ly6efl/+; Vav1-iCre) offspring.
    Ly6efl/fl
    suggested: None
    Vav1-iCre
    suggested: None
    B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J
    suggested: RRID:IMSR_JAX:008610)
    Ly6eΔHSC/+ mice were bred to obtain Ly6eΔHSC (Ly6efl/fl; Vav1-iCre) offspring, which harbor a deletion of exon 3 and 4 in hematopoietic stem cells (HSC).
    Ly6eΔHSC/+
    suggested: None
    Ly6efl/fl; Vav1-iCre
    suggested: None
    Software and Algorithms
    SentencesResources
    Data was analyzed with FlowJo Software (Treestar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Differences in data were tested for significance using GraphPad Prism v8.3.1 for Windows (GraphPad).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The RNAseq data discussed in this publication have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (GSE146074).
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 33. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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

    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 10.1101/2020.03.05.979260v1 on bioRxiv