Summary of Study ST001709

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001094. The data can be accessed directly via it's Project DOI: 10.21228/M89394 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST001709
Study Title	SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition
Study Summary	Viruses hijack host cell metabolism to acquire the building blocks required for viral replication. Understanding how SARS-CoV-2 alters host cell metabolism could lead to potential treatments for COVID-19, the disease caused by SARS-CoV-2 infection. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface 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 host cell oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes in host cell metabolism, we show that SARS-CoV-2 increases activity of mTORC1, a master regulator of anabolic metabolism, in cell lines and patient lung stem cell-derived airway epithelial cells. We also show evidence of mTORC1 activation in COVID-19 patient lung tissue. Notably, mTORC1 inhibitors reduce viral replication in kidney epithelial cells and patient-derived lung stem cell cultures. This suggests that targeting mTORC1 could be a useful antiviral strategy for SARS-CoV-2 and treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.
Institute	University of California, Los Angeles
Department	Biomedical Sciences
Laboratory	Heather Christofk
Last Name	Matulionis
First Name	Nedas
Address	615 Charles E Young Dr S, BSRB 354-05
Email	nmatulionis@mednet.ucla.edu
Phone	310-206-0163
Submit Date	2021-02-19
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2021-02-24
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001094
Project DOI:	doi: 10.21228/M89394
Project Title:	SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition
Project Summary:	Viruses hijack host cell metabolism to acquire the building blocks required for viral replication. Understanding how SARS-CoV-2 alters host cell metabolism could lead to potential treatments for COVID-19, the disease caused by SARS-CoV-2 infection. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface 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 host cell oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes in host cell metabolism, we show that SARS-CoV-2 increases activity of mTORC1, a master regulator of anabolic metabolism, in cell lines and patient lung stem cell-derived airway epithelial cells. We also show evidence of mTORC1 activation in COVID-19 patient lung tissue. Notably, mTORC1 inhibitors reduce viral replication in kidney epithelial cells and patient-derived lung stem cell cultures. This suggests that targeting mTORC1 could be a useful antiviral strategy for SARS-CoV-2 and treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.
Institute:	University of California, Los Angeles
Department:	Biomedical Sciences
Laboratory:	Heather Christofk
Last Name:	Matulionis
First Name:	Nedas
Address:	615 Charles E Young Drive South Los Angeles, CA 90095
Email:	nmatulionis@mednet.ucla.edu
Phone:	310-206-0163

Subject:

Subject ID:	SU001786
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Cell_type	Sample_type
SA159041	Sample_08	HEK293T_ACE2	Mock
SA159042	Sample_09	HEK293T_ACE2	Mock
SA159043	Sample_07	HEK293T_ACE2	Mock
SA159044	Sample_02	HEK293T_ACE2	Mock
SA159045	Sample_03	HEK293T_ACE2	Mock
SA159046	Sample_01	HEK293T_ACE2	Mock
SA159047	Sample_10	HEK293T_ACE2	SARS-CoV-2
SA159048	Sample_11	HEK293T_ACE2	SARS-CoV-2
SA159049	Sample_12	HEK293T_ACE2	SARS-CoV-2
SA159050	Sample_04	HEK293T_ACE2	SARS-CoV-2
SA159051	Sample_06	HEK293T_ACE2	SARS-CoV-2
SA159052	Sample_05	HEK293T_ACE2	SARS-CoV-2
SA159053	Sample_25	NHBE-ALI	Mock
SA159054	Sample_26	NHBE-ALI	Mock
SA159055	Sample_27	NHBE-ALI	Mock
SA159056	Sample_29	NHBE-ALI	SARS-CoV-2
SA159057	Sample_28	NHBE-ALI	SARS-CoV-2
SA159058	Sample_30	NHBE-ALI	SARS-CoV-2
SA159059	Sample_33	Vero	Mock
SA159060	Sample_31	Vero	Mock
SA159061	Sample_39	Vero	Mock
SA159062	Sample_32	Vero	Mock
SA159063	Sample_15	Vero	Mock
SA159064	Sample_14	Vero	Mock
SA159065	Sample_13	Vero	Mock
SA159066	Sample_37	Vero	Mock
SA159067	Sample_38	Vero	Mock
SA159068	Sample_20	Vero	Mock
SA159069	Sample_19	Vero	Mock
SA159070	Sample_21	Vero	Mock
SA159071	Sample_41	Vero	SARS-CoV-2
SA159072	Sample_42	Vero	SARS-CoV-2
SA159073	Sample_36	Vero	SARS-CoV-2
SA159074	Sample_40	Vero	SARS-CoV-2
SA159075	Sample_22	Vero	SARS-CoV-2
SA159076	Sample_17	Vero	SARS-CoV-2
SA159077	Sample_16	Vero	SARS-CoV-2
SA159078	Sample_18	Vero	SARS-CoV-2
SA159079	Sample_23	Vero	SARS-CoV-2
SA159080	Sample_34	Vero	SARS-CoV-2
SA159081	Sample_24	Vero	SARS-CoV-2
SA159082	Sample_35	Vero	SARS-CoV-2

Showing results 1 to 42 of 42

Showing results 1 to 42 of 42

Collection:

Collection ID:	CO001779
Collection Summary:	Please refer to the Treatment and SamplePrep sections.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR001799
Treatment Summary:	Cells were infected with SARS-CoV-2 for 2 hr, at which point the virus was removed and the media replaced with DMEM containing 10 mM U-13C-glucose (Cambridge Isotopes), 10 mM 3-13C-glucose or 4 mM U-13C-glutamine (Cambridge Isotopes). ALI culture media was serum free, and Vero and HEK293T-ACE2 media contained 10% dialyzed FBS.

Sample Preparation:

Sampleprep ID:	SP001792
Sampleprep Summary:	To extract metabolites, we washed the cells with ice-cold 150 mM ammonium acetate, pH 7.3, and then added 500 uL 80% methanol and incubated for 20 minutes at -80°C. Cells were then scraped off the plate, vortexed and centrifuged for 10 minutes at maximum speed. We dried 400 uL of the supernatant under vacuum and stored the dried metabolites at -80°C. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile(ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 min at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials.

Sampleprep ID:

SP001792

Sampleprep Summary:

To extract metabolites, we washed the cells with ice-cold 150 mM ammonium acetate, pH 7.3, and then added 500 uL 80% methanol and incubated for 20 minutes at -80°C. Cells were then scraped off the plate, vortexed and centrifuged for 10 minutes at maximum speed. We dried 400 uL of the supernatant under vacuum and stored the dried metabolites at -80°C. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile(ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 min at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials.

Combined analysis:

Analysis ID	AN002783	AN002784
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	SeQuant ZIC-HILIC (150 x 2.1mm,5um)	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Peak Area	Peak Area

Chromatography:

Chromatography ID:	CH002059
Chromatography Summary:	Samples were run on a Vanquish (Thermo Scientific) UHPLC system with mobile phase A (20 mM ammonium carbonate, pH 9.7) and mobile phase B (100% Acetonitrile) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min.
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Temperature:	35°C
Flow Gradient:	100% Acetonitrile
Flow Rate:	150 µL/min
Internal Standard:	10 nM Trifluoromethanesulfonate (extraction buffer)
Solvent A:	100% water; 20 mM ammonium carbonate, pH 9.7
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS002579
Analysis ID:	AN002783
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	POSITIVE

MS ID:	MS002580
Analysis ID:	AN002784
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	NEGATIVE