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. 2023 Jan 27;12:e79946. doi: 10.7554/eLife.79946

Figure 3. Metabolic intervention of SARS-CoV-2 shows the antiviral effect of PPARα activation.

(A) Left: Schematic depicting potential drug interactions with the metabolic landscape of SARS-CoV-2 infection. Right: Schematic of the relationship between PPARα and fatty acid oxidation in our model. (B) Microscopic analysis of lipid accumulation in lung cells infected by SARS-CoV-2 (USA-WA1/2020) at MOI 2 exposed to different drugs for 96 hr compared to DMSO-treated (vehicle) and mock-infected controls. Cells treated with PPARα agonist fenofibrate showed a significant decrease in phospholipid content (n=3, p<0.001). (C) Lactate over glucose ratio of SARS-CoV-2 infected primary lung cells treated with various drugs. Fenofibrate significantly reduced the lactate-to-glucose ratio by 60% (n=3; p<0.01) normalizing the metabolic shift induced by infection. (D) Quantification of SARS-CoV-2 viral RNA over treatment with a physiological concentration of various drugs or DMSO (vehicle). Treatment with 20 µM fenofibrate (Cmax) reduced SARS-CoV-2 viral load by 2-logs (n=3; p<0.001). Treatment with 10 µM cloperastine reduced viral load by 2.5–3-fold (n=3; p<0.05). (E) Cell number post-treatment was unaffected by all drugs tested. (n=3). (F) Microscopic analysis of lipid accumulation in lung cells infected by SARS-CoV-2 (hCoV-19/Israel/CVL-45526-NGS/2020) and B.1.617.2 variant of concern (hCoV-19/Israel/CVL-12806/2021) exposed to structurally different PPARα agonists for 5 days compared to DMSO-treated cells (vehicle). Cells treated with any PPARα agonists showed a significant decrease in phospholipid content in both viruses (n=6, p<0.001). (G) Quantification of SARS-CoV-2 viral RNA over treatment with a physiological concentration of various PPARα agonists or DMSO (vehicle). Treatment with 20 µM fenofibrate, 50 µM bezafibrate, or 1 µM WY-14643 reduced SARS-CoV-2 viral load by 3–5-logs (n=6; p<0.001). Treatment with 50 µM conjugated (9Z,11E)-linoleic acid and 50 µM oleic acid reduced viral load by 2.5-logs (n=6; p<0.01 in alpha variant). (H) Microscopic analysis of lipid accumulation in lung cells infected by SARS-CoV-2 and B.1.617.2 variant of concern (delta) exposed to PPARα agonist fenofibrate with 4 µM of lipid catabolism inhibitor, etomoxir (ETO) for 5 days compared to DMSO-treated (vehicle). Cells treated with fenofibrate showed a significant decrease in phospholipid content in both viruses (n=6, p<0.001). Phospholipid decrease was reversed by the addition of etomoxir. (I) Quantification of SARS-CoV-2 viral RNA exposed to the PPARα agonist fenofibrate with or without 4 µM of lipid catabolism inhibitor, etomoxir, or DMSO (vehicle). Treatment with 20 µM fenofibrate reduced SARS-CoV-2 viral load by 4–5-logs (n=6; p<0.001). Fenofibrate antiviral effect was reversed by the addition of etomoxir. (J) Microscopic analysis of lipid accumulation in PPARα or NT CRISPR-knockout lung cells (methods) infected by SARS-CoV-2 and B.1.617.2 variant of concern (delta) exposed to PPARα agonist fenofibrate with 4 µM of lipid catabolism inhibitor, etomoxir compared to DMSO-treated (vehicle). PPARα or NT CRISPR-knockout cells treated with fenofibrate did not show a decrease in phospholipid content in either virus and was unaffected by etomoxir (n=6). (K) Quantification of SARS-CoV-2 viral RNA after treatment with the PPARα agonist fenofibrate with or without 4 µM of lipid catabolism inhibitor, etomoxir, or DMSO (vehicle). Genetic inhibition of PPARα causes cells to be refractory to fenofibrate treatment and the addition of etomoxir (n=6). * p<0.05, ** p<0.01, *** p<0.001 in a two-sided heteroscedastic student’s t-test against control. Bar = 30 µm. Error bars indicate S.E.M.

Figure 3—source data 1. Raw measurements, mean, standard error, and student t-test values were used to create the display items in Figure 3.

Figure 3.

Figure 3—figure supplement 1. Metabolic regulators in SARS-CoV-2 infection in vitro.

Figure 3—figure supplement 1.

(A) Schematic depicting the metabolic landscape of SARS-CoV-2 infection. Potential drugs (white boxes) and their therapeutic targets are marked on the chart. (B) Table summarizing FDA-approved drugs that interfere with SARS-CoV-2-induced metabolic alterations. (C) Microscopic analysis of lipid accumulation and cell number in alpha variant (USA-WA1/2020) infected bronchial epithelial cells at MOI 2 after treatment with different metabolic regulators, compared to mock-infected bronchial epithelial cells. Bar = 30 µm. (D) Quantification of SARS-CoV-2 viral RNA 24 hours post-infection, prior to treatment. Analysis shows no difference in viral quantities (n = 3).
Figure 3—figure supplement 2. PPARα agonism anti-viral mechanism is ligand-wide and fatty oxidation dependent in SARS-CoV-2 infection in vitro.

Figure 3—figure supplement 2.

(A) Microscopic analysis of lipid accumulation and cell number in alpha variant (hCoV-19/Israel/CVL-45526-NGS/2020) infected bronchial epithelial cells at TCID100 after treatment with different PPARα agonists (n=6, p<0.05). (B) Quantification of alpha variant SARS-CoV-2 viral RNA before treatment with a physiological concentration of various PPARα agonists or DMSO (vehicle; n=6). (C) Microscopic analysis of lipid accumulation and cell number in delta variant (hCoV-19/Israel/CVL-12806/2021) infected bronchial epithelial cells at TCID50 after treatment with different PPARα agonists (n=6, p<0.001). (D) Quantification of delta variant SARS-CoV-2 viral RNA before treatment with a physiological concentration of various PPARα agonists or DMSO (vehicle; n=6). (E–F) Microscopic analysis of lipid accumulation and cell number in (E) alpha variant (hCoV-19/Israel/CVL-45526-NGS/2020) infected bronchial epithelial cells at TCID50 after 5 days of treatment with 20 µM fenofibrate or 20 µM fenofibrate and 4 µM CPT1α inhibitor etomoxir (n=6, p<0.01). (F) delta variant (hCoV-19/Israel/CVL-12806/2021) infected bronchial epithelial cells at TCID50 after treatment with 20 µM fenofibrate or 20 µM fenofibrate and 4 µM CPT1α inhibitor etomoxir. (G) Quantification of alpha and delta variant SARS-CoV-2 viral RNA before treatment with 20 µM fenofibrate or 20 µM fenofibrate and 4 µM CPT1α inhibitor etomoxir (n=6). * p<0.05, ** p<0.01, *** p<0.001. Bar = 50 µm. Error bars indicate S.E.M.
Figure 3—figure supplement 3. PPARα is required for fenofibrate rescue and etomoxir reversal in SARS-CoV-2 infection in vitro.

Figure 3—figure supplement 3.

(A) Gene expression of PPARα and CPT1α by qRT-PCR in bronchial epithelial cells or PPARα CRISPR-KO bronchial epithelial cells. Analysis shows a significant decrease in PPARα expression and its target gene CPT1α (n=6, p<0.001). (B) Western blot analysis of PPARα protein in bronchial epithelial cells or PPARα CRISPR-KO bronchial epithelial cells. Microscopic analysis of lipid accumulation and viability in (C) alpha variant (hCoV-19/Israel/CVL-45526-NGS/2020) infected PPARα K/O bronchial epithelial cells at TCID50 after 5 days of treatment with 20 µM fenofibrate or 20 µM fenofibrate and 4 µM CPT1α inhibitor etomoxir. (D) Delta (hCoV-19/Israel/CVL-12806/2021) infected PPARα K/O bronchial epithelial cells at TCID50 after 5 days of treatment with 20 µM fenofibrate or 20 µM fenofibrate and 4 µM CPT1α inhibitor etomoxir (n=6). (E) Quantification of alpha and delta variant SARS-CoV-2 viral RNA in infected PPARα K/O bronchial before treatment with 20 µM fenofibrate or 20 µM fenofibrate and 4 µM CPT1α inhibitor etomoxir (n=6). * p<0.05, ** p<0.01, *** p<0.001. Bar = 50 µm. Error bars indicate S.E.M.
Figure 3—figure supplement 3—source data 1. Complete raw and unedited blots assembly used to determine PPARα expression.
(1) original and (2) inverted files of the full raw unedited alpha tubulin blot. (3) original and (4) inverted files of the full raw unedited PPAR alpha blot. (5) The uncropped blots with the relevant bands are clearly labeled.