SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition

Abstract

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Fig. 1. SARS-CoV-2 infection alters TCA cycle metabolism.

a Glutamine entry into the TCA cycle is reduced in SARS-CoV-2 infected cells. Mass isotopomer analysis of TCA cycle metabolites in mock or SARS-CoV-2 infected Vero kidney epithelial cells after 24 h incubation with U-¹³C-glutamine. Each isotopomer is a different color, which is defined in the figure. P values compare the total fraction labeled between mock and SARS-CoV-2 infected cells (n = 3 biologically independent samples). b The oxidative TCA cycle is decreased in SARS-CoV-2 infected (red bars) Vero cells after 24 h infection compared with mock infected (gray bars) Vero cells (n = 3 biologically independent samples). c The reductive TCA cycle is maintained in SARS-CoV-2 infected (red bars) Vero cells compared with mock infected (gray bars) Vero cells after 24 h infection (n = 3 biologically independent samples). d Levels of αKGC members OGDH and DLD are reduced in SARS-CoV-2 infected Vero cells (representative of two biological independent samples). e Glucose-derived carbon entry into the TCA cycle is increased in SARS-CoV-2 infected cells. Mass isotopomer analysis of TCA cycle metabolites in mock or SARS-CoV-2 infected Vero kidney epithelial cells after 24 h incubation with U-¹³C-glucose. Each isotopomer is a different color, which is defined in the figure. P values compare the total fraction labeled between mock and SARS-CoV-2 infected cells (n = 3 biologically independent samples). f Glucose-derived carbon entry into the TCA cycle using PC is increased in SARS-CoV-2 infected cells. Fraction of labeled metabolites in mock (gray bars) or SARS-CoV-2 infected (red bars) Vero cells after 24 h incubation with 3-¹³C-glucose (n = 3 biologically independent samples). g Levels of PDH complex members are decreased in SARS-CoV-2 infected Vero cells (representative of two biological independent samples). h Schematic of the changes in TCA cycle metabolism in SARS-CoV-2 infected cells. Glutamine entry is reduced (blue arrow) and pyruvate entry using PC is increased (red arrow). Unless indicated, data are the mean ± s.e.m. and P values were obtained by two-way ANOVA with Sidak’s multiple comparison test.

Fig. 2. SARS-CoV-2 infection increases mTORC1 activity.

a mTORC1 activity is increased in SARS-CoV-2 infected Vero kidney epithelial cells. Protein was extracted from Vero cells infected with SARS-CoV-2 for 24 h and immunoblotted for the indicated proteins (representative of two biological independent samples). b mTORC1 activity is increased in ALI mucociliary cultures derived from two different donors. Protein was extracted from ALI cultures infected with SARS-CoV-2 for 72 h and immunoblotted for the indicated proteins (n = 2 biological independent samples). c pAKT and pERK are increased in SARS-CoV-2 infected NHBE ALI cultures (representative of two biological independent samples). d mTORC1 activity is increased in COVID-19 patient lungs. IHC staining of p4EBP1 and the macrophage marker CD68 on lungs from COVID-19 and non-COVID-19 patients. Images are representative of two out of five COVID-19 patient lungs and nine out of nine non-COVID-19 lungs. Red arrows indicate cells positive for p4EBP1. Scale bar 50 µM.

Fig. 3. mTORC1 inhibitors reduce SARS-CoV-2 replication.

a mTORC1 inhibitors reduce SARS-CoV-2 levels in Vero kidney epithelial cells. Immunofluorescence of cells infected with SARS-CoV-2 and treated with 25 nM of the indicated non-FDA approved mTORC1 inhibitors for 72 h. Cells were stained for dsRNA (red) to show SARS-CoV-2 levels. DAPI (blue) stained the nuclei of the Vero cells (n = 3 biologically independent samples). b The FDA-approved mTORC1 inhibitor rapamycin reduces SARS-CoV-2 levels in NHBE ALI mucociliary cultures. Immunofluorescence of cells infected with SARS-CoV-2 and treated with 1 µM rapamycin for 48 h. Cells were stained with an anti-SARS coronavirus antibody (green) to show SARS-CoV-2 levels. DAPI (blue) stained the nuclei of the mucociliary cells (n = 3 biologically independent samples). c FDA-approved mTORC1 inhibitors reduce SARS-CoV-2 protein expression. NHBE ALI cultures were infected with SARS-CoV-2 and treated with 1 µM rapamycin. Protein was extracted after 48 h and immunoblotted with an anti-SARS coronavirus antibody to determine SARS-CoV-2 protein expression. Multiple exposures of SARS-CoV-2 antibody are shown (representative of three biological independent samples). d FDA-approved mTORC1 inhibitors reduce SARS-CoV-2 gene expression. NHBE ALI cultures were infected with SARS-CoV-2 (red bar) and treated with 1 µM rapamycin (gray bar). RNA was extracted after 48 h and SARS-CoV-2 N gene expression quantified relative to RPLP0 using qPCR. Data are normalized to SARS-CoV-2 solvent cells (n = 3 biologically independent samples. Data are mean ± s.e.m. and P values were obtained by one-way ANOVA with Tukey’s multiple comparison’s test). Scale bar 30 µM.