Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2
Guoli Shi, Abhilash I. Chiramel, Tiansheng Li, Kin Kui Lai, Adam D. Kenney, Ashley Zani, Adrian C. Eddy, Saliha Majdoul, Lizhi Zhang, Tirhas Dempsey, Paul A. Beare, Swagata Kar, Jonathan W. Yewdell, Sonja M. Best, Jacob S. Yount, Alex A. Compton
Guoli Shi, Abhilash I. Chiramel, Tiansheng Li, Kin Kui Lai, Adam D. Kenney, Ashley Zani, Adrian C. Eddy, Saliha Majdoul, Lizhi Zhang, Tirhas Dempsey, Paul A. Beare, Swagata Kar, Jonathan W. Yewdell, Sonja M. Best, Jacob S. Yount, Alex A. Compton
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Research Article

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

Abstract

Severe acute respiratory syndrome coronavirus 2 (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 an increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increased susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. We identified 1 rapalog (ridaforolimus) that was less potent in this regard and demonstrated that rapalogs promote spike-mediated entry into cells, by triggering the degradation of the antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increased virus entry inhibited mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitated its nuclear translocation and triggered 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.

Authors

Guoli Shi, Abhilash I. Chiramel, Tiansheng Li, Kin Kui Lai, Adam D. Kenney, Ashley Zani, Adrian C. Eddy, Saliha Majdoul, Lizhi Zhang, Tirhas Dempsey, Paul A. Beare, Swagata Kar, Jonathan W. Yewdell, Sonja M. Best, Jacob S. Yount, Alex A. Compton

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Figure 3

Rapalogs promote SARS-CoV-2 infection in HeLa-ACE2 cells.

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Rapalogs promote SARS-CoV-2 infection in HeLa-ACE2 cells. (A) HeLa-ACE2 ...
(A) HeLa-ACE2 cells were treated with varying concentrations of everolimus or DMSO for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID50 of supernatants recovered 24 hours after infection. TCID50 (PFU/mL) values are shown. (B) HeLa-ACE2 cells were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID50 per milliliter of supernatants recovered 24 hours after infection. TCID50 per milliliter values were normalized to 100 in the DMSO condition. (C) HeLa-ACE2 cells were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 72 hours after infection. Luciferase units were normalized to 100 in the DMSO condition. (D) HeLa-ACE2 cells from C were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti-IFITM2, anti-IFITM1, anti-IFITM3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. (E) IFITM3 levels from D were normalized to actin levels and summarized from 5 independent experiments. (F) HeLa-ACE2 cells were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours, and cells were fixed, stained with DAPI and anti–IFITM2/-3, and imaged by confocal immunofluorescence microscopy. Images represent stacks of 5 Z-slices, and 1 representative image is shown per condition. Original magnification, ×63. Means and the standard error were calculated from 3–6 experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA versus DMSO.