IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition

  1. Caterina Prelli Bozzo
  2. Rayhane Nchioua
  3. Meta Volcic
  4. Jana Krüger
  5. Sandra Heller
  6. Christina M. Stürzel
  7. Dorota Kmiec
  8. Carina Conzelmann
  9. Janis Müller
  10. Fabian Zech
  11. Desiree Schütz
  12. Lennart Koepke
  13. Elisabeth Braun
  14. Rüdiger Groß
  15. Lukas Wettstein
  16. Tatjana Weil
  17. Johanna Weiß
  18. Daniel Sauter
  19. Jan Münch
  20. Federica Diofano
  21. Christine Goffinet
  22. Alberto Catanese
  23. Michael Schön
  24. Tobias Böckers
  25. Steffen Stenger
  26. Kei Sato
  27. Steffen Just
  28. Alexander Kleger
  29. Konstantin M.J. Sparrer
  30. Frank Kirchhoff

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Abstract

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

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  1. Version published to 10.1101/2020.08.18.255935v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.08.18.255935: (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
    Inibition by IFITM antibodies and peptides: Calu-3 cells were seeded in 48-well format (peptides assays), or in 24-well format (antibodies assay), 24h later cells were treated with increasing concentrations (20 and 80µg/ml) of IFITMs derived peptides (human IFITM2 long: EEQEVAMLGVPHNPAPPMSTVIH, human IFITM2 short: QEVAMLGVPHNAPPMST-VIH, mouse IFITM2 long: EEYGVTELGEPSNSAVVRTTVIN, human IFITM3 long: EEHEVAVLGAPHNPAPPTSTVIH, scrambled IFITM2: EGESGVTTATVEVVIERNN-LPY) or blocking antibodies (15 and 30 µg/ml) (α-ACE2 AK (AC18Z), Santa Cruz Biotechnology sc-73668; α-IFITM1
    α-ACE2 AK
    suggested: None
    Gut organoids were treated with increasing concentrations (15 and 30 µg/ml) of IFITMs derived peptides (mouse IFITM2 antibody blocking peptide Santa Cruz sc-373676 P) and blocking antibodies (α-ACE2 AK (AC18Z), Santa Cruz Biotechnology sc-73668, α-IFITM1/2/3 (F-12) Santa Cruz Biotechnology sc-374026) as indicated.
    F-12
    suggested: (Santa Cruz Biotechnology Cat# sc-374026, RRID:AB_10916884)
    Afterwards cells were washed three times with PBS and fixed with 100µl of Reagent A (FIX & PERM Fixation and Permeabilization Kit, Nordic MUbio) for 30 minutes at room temperature, washed three time with PBS and stained with primary antibody (α-IFITM1 Cell Signaling 13126 S, α-IFITM2 Cell Signaling 13530S, α-IFITM3 Proteintech 11714-1-AP, α-IFITM1/2/3 (F-12) Santa Cruz Biotechnology sc-374026,) diluted 1:20 in PBS or in Reagent B (FIX & PERM Fixation and Permeabilization Kit Nordic MUbio) for 1 h at 4°C.
    α-IFITM1/2/3
    suggested: None
    Cells were washed three times with PBS and stained with secondary antibody (Goat Anti-Rabbit IgG H&L (PE), ab72465,
    Anti-Rabbit IgG
    suggested: (Abcam Cat# ab72465, RRID:AB_1269142)
    Sections were permeabilized with 0.5 % Triton-X for 30 min at RT and stained over night with primary antibodies (rabbit anti-IFITM1 Cell Signaling 13126 S, 1:500 or rabbit anti-IFITM2 Cell Signaling #13530S, 1:500 or rabbit anti-IFITM3 Cell Signaling #59212S, 1:250 or anti-SARS-CoV-2 N 1:500 or anti-E-Cadherin 1:500) diluted in antibody diluent (Zytomed) in a wet chamber at 4°C.
    anti-IFITM2 Cell Signaling #13530S
    suggested: None
    anti-IFITM3
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    anti-E-Cadherin
    suggested: None
    Proteins were stained using primary antibodies against IFITM1 (α-IFITM1, Cell Signaling #13126 S, 1:1000,), IFITM2 (α-IFITM2 Cell Signaling #13530S, 1:1000), IFITM3 (α-IFITM3 Cell Signaling #59212S, 1:1000) SARS Spike CoV-2 (SARS-CoV-1/-2 (COVID-19) spike antibody [1A9], GTX-GTX632604, 1:1000), VSV-M
    α-IFITM1 , Cell Signaling #13126 S
    suggested: None
    α-IFITM2 Cell Signaling #13530S
    suggested: None
    α-IFITM3 Cell Signaling #59212S
    suggested: None
    GTX-GTX632604
    suggested: None
    Mouse Monoclonal Anti-VSV-M Absolute antibody, ABAAb01404-21.0, 1:1000), actin (Anti-beta Actin antibody Abcam, ab8227, 1:5000 Abcam,), ACE2 (Rabbit policclonal anti-ACE2 Abcam, ab166755, 1:1000) and Infrared Dye labelled secondary antibodies (LI-COR IRDye).
    Anti-VSV-M
    suggested: (Kerafast Cat# EB0011, RRID:AB_2734773)
    ABAAb01404-21.0 , 1:1000) , actin
    suggested: None
    Anti-beta Actin
    suggested: (Abcam Cat# ab8227, RRID:AB_2305186)
    ab8227
    suggested: None
    anti-ACE2
    suggested: None
    ab166755
    suggested: None
    For staining following antibodies were used: IFITM1 (α-IFITM1 Cell Signaling 13126 S)
    IFITM1
    suggested: (Cell Signaling Technology Cat# 13126, RRID:AB_2798126)
    α-IFITM1
    suggested: None
    , IFITM2 (α-IFITM2 Abcam 236735), IFITM3 (α-IFITM3 Cell Signaling 59212S), SARS Spike CoV-2 (SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9], GTX-GTX632604)
    IFITM2
    suggested: None
    α-IFITM2
    suggested: None
    α-IFITM3
    suggested: None
    , blotted onto polyvinylidene difluoride (PVDF) membrane, blocked in 5% milk and probed with rabbit anti-V5 (Cell Signaling #13202), mouse anti-FLAG (Sigma #F1804) and rat anti-GAPDH (Biolegend #607902) antibodies, followed by goat anti-mouse, anti-rabbit and anti-rat secondary fluorescent antibodies (LI-COR).
    anti-V5
    suggested: None
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-GAPDH
    suggested: (BioLegend Cat# 607902, RRID:AB_2734503)
    anti-mouse,
    suggested: None
    anti-rabbit
    suggested: None
    anti-rat
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Human embryonic kidney 293T cells (HEK293T; ATCC) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml).
    293T
    suggested: None
    Caco-2 (human epithelial colorectal adenocarcinoma) cells were maintained in DMEM containing 10% FCS, glutamine (2 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml), NEAA supplement (Non-essential amino acids (1 mM)), sodium pyruvate (1 mM).
    Caco-2
    suggested: None
    Calu-3 (human epithelial lung adenocarcinoma) cells were cultured in Minimum Essential Medium Eagle (MEM) supplemented with 10% FCS (during viral infection) or 20% (during all other times), penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mM), and NEAA supplement (1 mM).
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Vero cells (ATCC, CCL-81) cells were maintained in DMEM containing 2.5% FCS, glutamine (2 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml), NEAA supplement (Non-essential amino acids (1 mM)), sodium pyruvate (1 mM).
    Vero
    suggested: None
    Human hESC cultivation and gut organoids differentiation: Human embryonic stem cell (hESC) line HUES8 (Harvard University) was used with permission from the Robert Koch Institute according to the “Approval according to the stem cell law” AZ 3.04.02/0084.
    HUES8
    suggested: RRID:CVCL_B207)
    Target cell assay: HEK293T cells were transiently transfected using PEI38 with pLV-EF1a-human ACE2-IRES-puro and pCG-IFITM1-IRES_eGFP or pCG-IFITM2-IRES_eGFP or pCG-IFITM3-IRES_BFP.
    HEK293T
    suggested: None
    SARS-CoV-2 virus stock production: BetaCoV/Netherlands/01/NL/2020 or BetaCoV/ France/IDF0372/2020 was propagated on Vero E6 infected at an MOI of 0.003 in serum-free medium containing 1 μg/ml trypsin as previously described16.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    2 h post-treatment, cells were infected with SARS-CoV-2 with an MOI of 0.05.
    SARS-CoV-2
    suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
    Images were acquired on a Zeiss LSM 710 and processed using ImageJ (Fiji).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Statistics: Statistical analyses were performed using GraphPad PRISM 8 (GraphPad Software).
    GraphPad PRISM
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    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.

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  3. Version published to 10.1101/2020.08.18.255935v1 on bioRxiv