Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

  1. Guoli Shi
  2. Abhilash I. Chiramel
  3. Tiansheng Li
  4. Kin Kui Lai
  5. Adam D. Kenney
  6. Ashley Zani
  7. Adrian Eddy
  8. Saliha Majdoul
  9. Lizhi Zhang
  10. Tirhas Dempsey
  11. Paul A. Beare
  12. Swagata Kar
  13. Jonathan W. Yewdell
  14. Sonja M. Best
  15. Jacob S. Yount
  16. Alex A. Compton

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Abstract

SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Significance

Rapamycin is an immunosuppressant used in humans to treat cancer, autoimmunity, and other disease states. Here, we show that rapamycin and related compounds promote the first step of the SARS-CoV-2 infection cycle—entry into cells—by disarming cell-intrinsic immune defenses. We outline the molecular basis for this effect by identifying a rapamycin derivative that is inactive, laying the foundation for improved mTOR inhibitors that do not suppress intrinsic immunity. We find that rapamycin analogs that promote SARS-CoV-2 entry are those that activate TFEB, a transcription factor that triggers the degradation of antiviral membrane proteins inside of cells. Finally, rapamycin administration to rodents prior to SARS-CoV-2 challenge results in enhanced viral disease, revealing that its use in humans may increase susceptibility to infection.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.15.440067: (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 with Odyssey Blocking Buffer in PBS (Li-COR) and incubated with the following primary antibodies diluted in Odyssey Antibody Diluent (Li-COR): anti-IFITM1 (60074-1-Ig; Proteintech), anti-IFITM2 (66137-1-Ig; Proteintech), anti-IFITM3 (EPR5242, ab109429; Abcam), anti-actin (C4, sc-47778; Santa Cruz Biotechnology), anti-hACE2 (ab15348; Abcam), and anti-CoV Spike (PA1-41165; Thermo Fisher).
    anti-IFITM1
    suggested: None
    anti-IFITM2
    suggested: None
    anti-IFITM3
    suggested: (Abcam Cat# ab109429, RRID:AB_10865792)
    EPR5242
    suggested: None
    anti-actin
    suggested: (Santa Cruz Biotechnology Cat# sc-47778, RRID:AB_626632)
    C4
    suggested: (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189)
    anti-hACE2
    suggested: None
    anti-CoV
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HeLa-ACE2, HeLa-ACE2-TMPRSS2, HeLa-DPP4, and A549-ACE2 cell lines were produced by transducing cells with lentivirus packaging pWPI encoding ACE2, DPP4, or TMPRSS2 and selecting with blasticidin.
    HeLa-ACE2-TMPRSS2
    suggested: None
    A549-ACE2
    suggested: None
    HeLa IFITM1/2/3 Knockout (C5-9) cells were purchased from ATCC (CRL-3452).
    HeLa IFITM1/2/3
    suggested: None
    In brief, HEK293T cells were transfected with 2 μg pcDNA3.1 and CoV, CoV-2, or MERS Spike using Lipofectamine2000 (Thermo Fisher).
    HEK293T
    suggested: None
    Supernatants were harvested 24 hours later and titers were determined on plaque assays performed in Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Briefly, six million 293T cells were seeded in a T75 flask.
    293T
    suggested: None
    50 ng p25 equivalent of virus was added to HeLa-ACE2 cells for 2 hours.
    HeLa-ACE2
    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: 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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04461340RecruitingEfficacy and Safety of Sirolimus in COVID-19 Infection
    NCT04341675RecruitingSirolimus Treatment in Hospitalized Patients With COVID-19 P…
    NCT04371640WithdrawnSirolimus in COVID-19 Phase 1

    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

    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 10.1101/2021.04.15.440067 on bioRxiv