Fig 2: DRAM1 Interacts with LAT1 and SLC1A5 Amino Acid Transporters to Stimulate mTOR Activation(A) Saos2 TetOn-DRAM1 cells treated with or without doxycycline for 24 h before incubation in full DMEM or EBSS for 3 h. Cells were harvested and lysed prior to immunoprecipitation of DRAM1 using anti-Myc-tag antibody. Immunoblotting from total protein extracts (INPUT) or elutions (IP DRAM1) was undertaken to detect SLC7A5 (LAT1), SLC1A5, DRAM1 (Myc tag), and ERK2 (loading control). A representative result from at least 3 independent experiments is shown.(B) Saos2 TetOn-DRAM1 cells expressing the indicated short hairpin RNA (shRNA) constructs were grown in the presence or absence of doxycycline for 24 h. Myc-tagged proteins were immunoprecipitated and samples analyzed by western blotting to detect DRAM-1 (Myc tag), SLC7A5, SLC1A5, or SLC3A2. ERK2 was used as a loading control.(C) Quantifications of SLC7A5 and SLC1A5 bound to DRAM1 from 3 independent experiments. Data are mean ± SEM. *p < 0.05.(D–F) Experiments were performed using Dram1flox/flox MEF expressing Cre recombinase (-/-) or a control vector (fl/fl).(D) Cells were starved in EBSS with, where indicated, 2 mM glutamine for 3 h before incubation in EBSS containing 0.8 mM leucine for 20 min.(E) Cells were starved in EBSS containing glutamine for 3 h before incubation for 20 min in EBSS containing 0.8 mM leucine, 10 mM D-phenylalanine, and 25 µg/mL cycloheximide as indicated.(F) Cells were starved for 3 h in EBSS containing glutamine alone or supplemented with L-?-glutamyl-p-nitroanilide (?-GPNA) before treatment with EBSS containing 0.8 mM leucine alone or supplemented with 100 nM rapamycin for 20 min.(D–F) mTOR activation was detected by measuring phospho-S6 kinase levels by western blotting. Total S6 kinase and ERK2 levels were used as loading controls.See also Figure S3.

Fig 3: SLC1A5 is required for efficient entry of pandemic SARS-CoV-2 D614G into host cells. (A) Confocal microscopy images of Huh-7.5 cells. Control (Sh-pLKo) and SLC1A5 downmodulated (Sh#-SLC1A5) cells were stained with anti-hACE2 (red), anti-SLC1A5 (green) and 647-labeled SARS-CoV-2-RBD (white). Scale bars are indicated. (B) Graph representing the number of hACE2, SLC1A5 and SARS-CoV-2-RBD triple positive spots/cell, Means ± SEM are indicated. (Two-tailed t-test p-values: **, <0.01). (C) Pearson correlation between hACE2 and SARS-CoV-2-RBD Means ± SEM are indicated. (p-values were calculated using two tailed t tests: ***, <0.001). (D) Graph representing the number of hACE2/SLC1A5 double positive spots/cell in the presence or absence of SARS-CoV-2-RBD. Means ± SEM are indicated. (p-values were calculated using two-way ANOVA ***, <0.001). For A-D a total of n = 8 cells/Sh-pLKo and n = 7 cells/Sh#2-SLC1A5 were analyzed over two independent experiments. (E) SARS-CoV-2-RBD quantitative binding to cells by ELISA assay. Data are expressed as percentages referred to Sh-pLKo signals and represent means ± SD of three independent experiments. (p-values were calculated using two tailed t-tests: *, <0.05; **, <0.01; ***, <0.001). (F) Scheme describing our experimental approach. Cells expressing either physiological or reduced levels of SLC1A5 were transduced with Spike-DEL-LUC pseudoparticles and assessed for viral entry. (G) Spike-DEL-LUC pseudoparticle assay. Samples prepared as in (F) were analyzed for luciferase signal intensity. Huh-7.5 cells were either infected with the indicated pseudoparticles concentrations or left untreated (NT). Histograms represents means ± SEM of three independent experiments. (Two tailed t-test p-values: *, <0.05; **, <0.01; ***, <0.001). (H) SARS-CoV-2-RBD binding assay in Huh-7.5 cells treated with V-9302. Data are expressed as percentages referred to the signals of control cells and represent means ± SD of three independent experiments. (Two tailed t-test p-value ***, <0.001). (I) Spike-DEL-LUC pseudoparticles entry assay on Huh-7.5 cells treated with increasing concentrations of V-9302. Not Treated cells (NT) values were set at 100%. Data are expressed as means ± SEM (n = 3). (Two tailed t-test p-value ***, <0.001). (J) Western blot analysis on total (T), cytosolic (C) and membrane (M) fractions of Huh-7.5 cells treated in vitro with either 15 µM V-9302 or DMSO as a control. GAPDH: cytosolic control; hACE2: membrane control. (K) SARS-CoV-2 viral entry assay in Huh-7.5 cells with SLC1A5 downmodulation. Cells transduced with either control plasmid Sh-pLKo or Sh#2-SLC1A5, were infected with 0.1 MOI of SARS-CoV2 viral particles. Histograms represent the relative expression levels. Means ± SD (n = 4) are shown. (Two tailed t-test p-value: ****, <0.0001).

Fig 4: SLC1A5 partly colocalizes with SARS-CoV-2-RBD in human lung tissues. (A) Representative images of normal lung tissues derived from patients listed in Fig. 2A. SLC1A5 was detected with rabbit-anti-SLC1A5 primary antibodies and Alexa-Fluor 568-labeled anti-rabbit secondary antibodies (red). 647-labeled SARS-CoV-2-RBD was used (green). Colocalized signals are shown in yellow. Nuclei were stained with DAPI. Scale bars are indicated. (B) Quantification of SLC1A5 positive cells per mm2 of tissue. Each dot represents the number of positive cells/Field of View (n = 15 FOVs/sample). Means ± SEM are indicated. (C) Quantification of SARS-CoV-2-RBD positive cells per mm2 of tissue. Each dot represents the number of positive cells/Field of View (n = 15 FOVs/samples). Means ± SEM are indicated. (D) Quantification of SLC1A5 and SARS-CoV-2-RBD double positive cells in each analyzed lung tissue sample. Graphs represent the number of SARS-CoV-2-RBD and hACE2 double positive cells/mm2 of tissue. Each dot represents the number of double positive cells/Field of View (n = 15 FOVs/sample). Means ± SEM are indicated. (E) Pearson correlation index between SLC1A5 and SARS-CoV-2-RBD represented by Violin plots. Medians are shown in green (n = 15 analyzed pixels/FOVs/samples). (F-G) Representative images of one selected cell from the human lung tissue of individual #1 acquired with Spinning Disk video-confocal structure illumination (VCS-SIM) (F) and STED (G) microscopy. hACE2 and SLC1A5 proteins are shown in red and blue, respectively. 647-labelled-SARS-CoV-2-RBD is shown in green. Nuclei were stained with DAPI. Scale bars are indicated. All boxes represent the spatial interaction between hACE2, SLC1A5 and SARS-CoV-2-RBD proteins by 2D rendering on a single Z-plan at higher magnification. (H) Pearson correlation index represented by Violin plots. Medians are shown in green (n = 22 analyzed triple positive spots). (Kruskal–Wallis test, p-value <0.001, ***, p-value <0.0001, ****).

Fig 5: SLC1A5 interacts with hACE2/SARS-CoV-2-RBD in human cell lines. (A) Total number of proteins detected by mass spectrometry, upon hACE2 pulldown with or without SARS-CoV-2-RBD. (B) GO enrichment analysis of proteins specific of the hACE2 + SARS-CoV-2-RBD pulldown. (C) List of membrane proteins specific for the hACE2 + SARS-CoV-2-RBD pulldown. (D) Top 10 interactors of hACE2 in the presence of SARS-CoV-2-RBD. The 90 proteins specific of the hACE2 + SARS-CoV-2-RBD pulldown were crossed with previously published SARS-CoV-2 interactomes and each protein was given a score (refer to the “Methods” section for details). (E) SLC1A5 was immunoprecipitated from HEK-hACE2 cells, previously incubated with recombinant SARS-CoV-2-RBD protein, using anti-SLC1A5 antibodies. The blot is representative of three independent experiments. (F) hACE2 and SLC1A5 colocalization in HEK cells. Confocal microscopy images obtained from hACE2-HEK cells stained with anti-hACE2 (red) and anti-SLC1A5 (green) antibodies. Nuclei were stained with DAPI. Scale bars are indicated. (G) hACE2, SLC1A5 and SARS-CoV-2-RBD colocalization in HEK cells. Top, hACE2 (red) and SLC1A5 (green) proteins were stained as in (F). 647-labelled-SARS-CoV-2-RBD is shown in white. Nuclei were stained with DAPI. Scale bars are indicated. Bottom, Pearson correlation index between hACE2 and SARS-CoV-2-RBD (n = 28 co-labeled spots) and between hACE2 and SLC1A5 either in the presence or absence of SARS-CoV-2-RBD (n = 100 co-labeled spots/sample). (Kruskal–Wallis test, p-value <0.001, ***). (H) Representative confocal image of Huh-7.5 cells, incubated in live with 647-labeled SARS-CoV-2-RBD protein. Immunofluorescence staining was performed as described in (G); (a-e) images of single and multiple spots at lower magnification. Scale bars are indicated. (f–g) Images showing the colocalization signals between hACE2 and SAR-CoV-2-RBD (grey). (1–3) boxes representing the spatial interaction between hACE2, SLC1A5 and SARS-CoV-2-RBD proteins by 2D reconstruction on a single Z-plan. Scale bars are indicated.