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

Abstract

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of 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 infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Fig. 1. Opposing effects of IFITM proteins on SARS-CoV-2 infection.

a Quantification of VSV(luc)ΔG*SARS-CoV-2-S entry by measuring luciferase activity in HEK293T cells transiently expressing the indicated IFITM proteins. Bars represent the mean of three independent experiments (±SEM), exact p values are provided in supplementary data 1. b Calu-3 cells treated with non-targeting (CTRL) or IFITM1, 2, or 3 siRNAs or a combination of the three and infected with VSV(luc)ΔG*SARS-CoV-2-S particles. Bars represent the mean of three independent experiments (±SEM). c Quantification of viral N RNA levels by qRT-PCR 48 h post-infection with SARS-CoV-2 (MOI 0.05) in the supernatant of HEK293T cells transiently expressing ACE2 alone or together with the indicated IFITM proteins. Bars represent the mean of three independent experiments (±SEM), exact p values are provided in the supplementary data 1. d Quantification of viral N RNA levels by qRT-PCR in the supernatant of Calu-3 cells, collected 48 h post-infection with SARS-CoV-2 (MOI 0.05). Cells were transfected with control (CTRL) or IFITM1, 2, and/or 3 targeting siRNA or a combination of the three and either treated with IFN-β or left untreated as indicated. Bars represent the mean of four independent experiments (±SEM). ***p < 0.0001. e Infectious SARS-CoV-2 particles in the supernatant of (d) as assessed by TCID50 assay. Bars represent the mean of three independent experiments (±SEM), exact p values are provided in Supplementary Data 1. f Infectious SARS-CoV-2 particles in the supernatant of (d) as assessed by plaque-forming unit assay. g Quantification of (f). Bars represent the mean of three independent experiments (±SEM). siRNA CTRL vs. siRNA IFITM1 p = **0.0012, siRNA CTRL vs. siRNA IFITM2 p = ****<0.0001, siRNA CTRL vs. siRNA IFITM3 p = *0.0197, siRNA CTRL vs. siRNA IFITM1–3 p = **0.0020). h, i Correlation of infectious viral particle analysis (h, TCID50; i, plaques) of (e, g) with qPCR data from (d) values showed calculated as a Pearson correlation (h: r = 0.82, p < 0.0001, i: r = 0.83, p = 0.0002). a, c, d, g Unpaired t test with Welch’s correction. b, e Unpaired t tests. p Values are indicated as *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2. Role of IFITMs in SARS-CoV-2 replication in SAEC.

a Expression of IFITM1, IFITM2, and IFITM3 in SAEC after stimulation with IFN-α2 (500 U/ml, 72 h), IFN-β (500 U/ml, 72 h), or IFN-γ (200 U/ml, 72 h). Immunoblots of whole-cell lysates were stained with α-IFITM1, α-IFITM2, α-IFITM3, and α-GAPDH. The experiment was performed twice to similar results. b, c Quantification of SARS-CoV-2N RNA in the supernatants of SAEC that were left untreated or treated with IFN-β (b) or IFN-γ (c) 48 h post-infection with SARS-CoV-2 (MOI 2.5). Shown are viral RNA levels relative to those obtained upon treatment with the control siRNA (100%). Bars represent the means of three independent experiments (±SEM). (b, untreated panel) siRNA CTRL vs. siRNA IFITM1 p = **0.007; (b, IFN-β treated panel) siRNA CTRL vs. siRNA IFITM1/2/3 p = ***0.0001; (c, untreated panel) siRNA CTRL vs. siRNA IFITM1 p = ** 0.0092. d Quantification of intracellular viral N RNA levels in Calu-3 cells 6 h (left panel) and 24 h (middle panel) post-infection with SARS-CoV-2 (MOI 0.05). (right panel) Quantification of viral N RNA levels in the supernatant 24 h post-infection. Values were normalized to GAPDH and calculated relative to the control (set to 100%). Cells were transiently transfected with siRNA either control (CTRL) or targeting IFITM1, 2, 3, or a combination of the three as indicated. Bars represent the mean of three independent experiments, measured in duplicates (±SEM), exact p values are provided in Supplementary Data 1. d Unpaired t test with Welch’s correction. b, c Unpaired t tests. p Values are indicated as *p < 0.05; **p < 0.01; ***p < 0.001 or were not significant (p > 0.05).

Fig. 3. IFITM2 promotes SARS-CoV-2 entry and interacts with the Spike protein.

a Quantification and exemplary images of a proximity ligation assay (PLA) between the SARS-CoV-2 Spike and IFITM proteins in Calu-3 cells infected with SARS-CoV-2 for 2 h at 4 °C. DAPI (blue), nuclei. PLA signal (yellow), proximity between S/IFITMs. Bars represent the mean of three independent experiments, (300 cells, ±SEM), two-sided Wilcoxon matched-pairs test, ****p < 0.0001. Scale bar, 20 μm. b Quantification and exemplary images of a PLA in SAEC using a similar setup as (a). DAPI (blue), nuclei. PLA signal (yellow), proximity between S/IFITMs. Scale bar, 20 µm. Bars represent the mean of two independent experiments (80 cells, ±SEM). c Relative interaction between SARS-CoV-2 Spike and human IFITM proteins measured by MaMTH protein-protein interaction assay in cotransfected HEK293T B0166 Gaussia luciferase reporter cells. Bars represent the mean of triplicate transfections performed in two independent experiments. d Co-Immunoprecipitation (Co-IP) of IFITM proteins by the Spike protein in HEK293T cells overexpressing SARS-CoV-2 S and IFITM1, IFITM2, or IFITM3, as indicated. Twenty-four-hours post-transfection, cells were harvested. The Co-IP was repeated twice to similar results.

Fig. 4. Impact of IFITMs on the ACE2-SARS-CoV-2 S proximity.

a Quantification and exemplary images of a PLA between SARS-CoV-2 Spike and ACE2 in Calu-3 depleted of IFITM1, IFITM2, or IFITM3 and infected with genuine SARS-CoV-2. Bars represent the mean of four independent experiments (100 cells ±SEM), two-sided Wilcoxon matched-pairs test, ****p < 0.0001. b Quantification and exemplary images of a PLA between Spike and ACE2 in Calu-3 cells depleted of IFITM2 and infected with SARS-CoV-2 virus on ice for 2 h and then incubated for 15 min at 37 °C. Bars represent the mean of four independent experiments (300 cells, ±SEM), two-sided Wilcoxon matched-pairs test, ****p < 0.0001. c Quantification and exemplary images of a PLA assay between Spike and RAB5A as in (c). Bars represent the mean of four independent experiments (400 cells, ±SEM), two-sided Wilcoxon matched-pairs test, ****p < 0.0001. DAPI (blue), nuclei. PLA signal (yellow). Scale bar, 20 µm. d Summary of the quantification shown in panels (b) (Spike-ACE2) and (c) (Spike-RAB5α) proximity upon SARS-CoV-2 infection. Individual data points and statistics are provided in panels (b) and (c). e Exemplary images of the colocalization (white) of PLA foci (red) indicating SARS-CoV-2 Spike and IFITM2 with early (EEA1, green, upper panel) or late endosomal markers (Rab7a, green, lower panel) in Calu-3 cells. Cells were infected with SARS-CoV-2 for 2 h at 4 °C or 2 h at 4 °C and 15 min at 37 °C as indicated. DAPI (blue), nuclei. f Quantification of the percentage of colocalization between the PLA signal and the indicated endosomal markers in 225 × 225 µm images. Bars represent the mean of four individual images (±SEM), unpaired t test, **p = 0.0031. g Quantification of the combined PLA (IFITM2/Spike) in (e). Bars represent the mean of 120 cells (±SEM) over two independent experiments. The experiment was replicated twice to similar results. Two-sided Wilcoxon matched-pairs test, ****p < 0.0001. a–c, e Scale bars, 20 μm.

Fig. 5. IFITM blocking antibodies and IFITM derived peptides target the N-terminal domain.

a Alignment of the amino acid sequence of human IFITM1, 2, and 3. Binding sites of IFITM blocking antibodies are indicated and the region of origin of the IFITM derived peptides highlighted. b Quantification of viral N RNA levels in the supernatant of Calu-3 cells treated with α-ACE2, α-IFITM1, α-IFITM2, α-IFITM3, and α-IFITM1-3 antibodies (7.5, 15, or 30 µg/ml) 1 h before infection (SARS-CoV-2, MOI 0.05), collected 48 h post-infection. Bars represent the mean of two (30 µg/ml α-IFITM2) or three (all other concentrations) independent experiments each measured in technical duplicates (±SEM), unpaired t-test. All p values were calculated compared to CTRL (ACE2, 15 µg/ml: 0.0011, ACE2, 30 µg/ml: <0.00001; IFITM2, 30 µg/ml: <0.00001; IFITM1-3, 15 µg/ml: 0.004; IFITM1-3, 30 µg/ml: <0.00001). c Quantification of viral N RNA levels in the supernatant of Calu-3 cells treated with IFITM-derived peptides (20 or 80 µg/ml) as indicated for 1 h before infection (MOI 0.05), collected 48 h post-infection. Bars represent the mean of three (hIFITM2 long and short peptide) or four (CTRL, hIFITM3 and scrambled) independent experiments each measured in technical duplicates (±SEM), unpaired t test. All p values were calculated compared to CTRL (hIFITM2 long, 20 µg/ml: 0.005; hIFITM2 long, 80 µg/ml: <0.0001; hIFITM2 short, 20 µg/ml: 0.0009; hIFITM2 short, 80 µg/ml: 0.0002; hIFITM3, 20 µg/ml: 0.0031). p Values are indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 or were not significant (p > 0.05).

Fig. 6. Blocking antibodies and IFITM-derived peptides treatment decrease SARS-CoV-2 infection in gut organoids and cardiomyocytes.

a Immunohistochemistry of gut organoids treated with blocking antibodies and a mouse IFITM2 (mIFITM2) derived peptide and infected with SARS-CoV-2 (MOI 0.15). Organoids were stained with α-SARS-CoV-2 N (red), E-Cadherin (green), and DAPI (blue). Scale bar, 100 µm (left panel). Quantification of SARS-CoV-2 N fluorescence, normalized to DAPI (right panel). Bars represent the mean of CTRL:9, α-ACE2 15 µg/ml:8, α-ACE2 30 µg/ml:2, α-IFITM1-3 15 µg/ml:5, α-IFITM1-3 30 µg/ml:6, mIFITM2 peptide 15 µg/ml:6, or mIFITM2 peptide 30 µg/ml:2 individual images over one experiment (±SEM), unpaired t-test with Welch’s correction All p values were calculated compared to CTRL (α-ACE2 15 µg/ml: 0.0104, α-ACE2 30 µg/ml: 0.0093, α-IFITM1-3 30 µg/ml: 0.0002, mIFITM2 peptide 15 µg/ml: 0.0295, mIFITM2 peptide 30 µg/ml: 0.005). b Quantification of viral N RNA levels in the supernatant of SARS-CoV-2 infected cardiomyocytes (increasing MOIs as indicated) at indicated time points. Lines represent a single experiment measured in duplicates. c Immunoblot of IFITM1, IFITM2, and IFITM3 in cardiomyocytes infected with SARS-CoV-2. Whole-cell lysates were stained with α-IFITM1, α-IFITM2, α-IFITM3, and α-GAPDH. n = 1. d Quantification of viral N RNA levels in the supernatant of SARS-CoV-2 infected cardiomyocytes (MOI 0.1) treated with IFITM-derived peptides, collected at indicated time points post-infection. Bars represent the mean of two independent experiments each measured in technical duplicates. Non-infected controls were below the quantification level (<1000). Exact p values are provided in Supplementary Data 1. p Values are indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 or were not significant (p > 0.05).