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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13338/m_MTBLS13338_LC-MS_alternating_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13338/a_MTBLS13338_LC-MS_alternating_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13338/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13338/s_MTBLS13338.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13338We used Riken, HMDB and MassBank as reference spectral librariesMetaboLightsPublicLiquid Chromatography MS - alternating - reverse phaseFor both ionization modes, LC separation was performed using an Elute UHPLC system (Bruker Daltonics, Germany) with mobile phase A (water) and mobile phase B (acetonitrile, ACN), both containing 0.1% FA. The LC gradient consisted of a 30-minute runtime, including column washing, as follows: T0–T2: 99% A / 1% B, T2–T17: 1% A / 99% B, T17–T20: maintained at 99% B / 1% A, T20–T20.10: flow increased to 0.35 mL/min, transitioning to 1% B / 99% A, T20.10–T28.30: maintained at 1% B / 99% A, T28.30–T30: flow returned to 0.25 mL/min until the end. The autosampler was maintained at 4 °C. Samples were injected using the ul Pickup mode with a 5 µL injection volume for both polarities. Separation was performed on a T-ReX Elute M-column Kit (Bruker Daltonics, Germany), which includes an Intensity Solo HPLC Column C18 (100 × 2 mm, 2 μm) coupled to a VanGuard pre-column, thermostated at 35 °C.SARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I Inhibition.IMIBICÃngela Peralbo-MolinaA-549 cellmass spectrometry assaySamples were transferred to 2 mL tubes and subjected to magnetic separation. Subsequently, 200 µL of methanol (MeOH) was added, and the samples were agitated for 10 minutes using a BeatBox. Following incubation, the magnets were removed, and the samples were centrifuged at 10 °C for 10 minutes. The supernatants were transferred to new Eppendorf tubes and centrifuged again at 10 °C for 6 minutes. Finally, the supernatants were transferred to vials containing inserts. Quality control (QC) was performed using 35 µL per sample. Pellets were retained and stored for potential downstream analyses.Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS13338Transito García-García. Department of Genetics, Immunogenomics and Molecular Pathogenesis Group, UIC Zoonoses and Emergent Diseases ENZOEM, University of Córdoba, Córdoba, Spain. Departamento de Genética, Campus Universitario de Rabanales, Universidad de Córdoba, Spain. b52gagat@uco.es. +34957218730.Transito García-García. Department of Genetics, Immunogenomics and Molecular Pathogenesis Group, UIC Zoonoses and Emergent Diseases ENZOEM, University of Córdoba, Córdoba, Spain.. Departamento de Genética, Campus Universitario de Rabanales, Universidad de Córdoba, Spain. b52gagat@uco.es. +34957218730.Initial data evaluation was carried out using DataAnalysis 6.1 software (Bruker Daltonics). Untargeted data processing was performed in MetaboScape 8.0.2 (Bruker Daltonics) using the T-ReX algorithm for mass calibration, peak picking, time alignment, and within-batch correction.All samples were analyzed in triplicate and randomized, with pooled QC samples injected every 10 samples to allow for normalization. Feature merging was conducted to combine signals detected in both polarities. Missing values were addressed using the T-ReX automatic region complete extraction, which re-extracts features below the detection threshold in a targeted manner. Only features present in all QC samples were retained.QC dilution series (100%, 80%, 60%, 40%, 20%, 10%) were used to filter features via a custom R script. For each metabolite, a pseudo-calibration curve was constructed from the QC dilution series, and only metabolites with a coefficient of determination (R²) > 0.65 were retained for downstream analysis, ensuring that only reliable biological signals were considered.SampleRun referenceangela.peralbo@imibic.orgCell culture, lentivirus production and transduction A549 pulmonary epithelial cells (ATCC CRM-CCL-185; RRID: CVCL_0023) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 41966029) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco, 1027016) and 1% Penicillin-Streptomycin (100U/ml) (Gibco, 15070063). THP1 (ATCC CRM-TIB-202; RRID: CVCL_0006) were cultured in Roswell Park Memorial Institute (RPMI) (Gibco, 11875093) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco, 1027016) and 1% Penicillin-Streptomycin (100U/ml) (Gibco, 15070063). ORF7a accessory protein coding sequences were cloned into pLVX-EF1α-IRES-Puro Cloning and Expression Lentivector (Clontech, Takara, 631253) to generate pseudotyped lentiviral particles encoding ORF7a protein (Wuhan-Hu-1 isolate). Cells were transduced by incubating them with lentivirus at a MOI of 10 for 24 h followed by 2 µg/ml puromycin treatment to start the selection of successfully transduced cells. Differentiation of THP1 into macrophage-like cells (THP1-M0) was induced by treating 80 ng/ml PMA phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P8139) for 24 hours. After PMA exposure, the cells were washed with PBS and incubated with fresh medium for an additional 24–72 hours prior to experiments to allow complete differentiation. All cells were cultured at 37°C in a 5% CO2, 90% humidity atmosphere. MetabolomicsMetabolomicsMitochondriaProteomicsORF7a protein, SARS-CoV-2MetabolomicsMitochondriaProteomicsORF7a protein, SARS-CoV-2MS acquisition: 4D PASEF4D PASEF experiments were conducted using a TIMS-TOF Pro instrument (Bruker Daltonics, Germany) in both positive and negative ion modes. Instrument parameters were as follows:End plate offset: 500 V; capillary voltage: 3500 VDry gas flow: 8 L/min at 240 °CNebulizer gas: 2.5 barPeak detection threshold: 100 countsScan mode: PASEF, mass range 20–1300 Da for MS and MS²Acquisition cycle: 0.1 s, mobility range 0.45–1.45 Vs/cm² (positive) and 0.45–1.30 Vs/cm² (negative)Collision energy: 10 eVTIMS and mass calibration were performed weekly using the Agilent ESI LC-MS tuning mix. Online recalibration was performed immediately after each sample using a mixture of Agilent ESI LC-MS tune mix and 10 mM sodium formate (1:1), injected via a syringe pump. Segment switching during the 30-minute LC-MS runtime was achieved using a conventional 6-port divert valve.Gluconic acidOroxindinglu-gluLPS 20:42-Naphthalenesulfonic acid2'-Deoxyguanosine 5'-monophosphateElaidic acidGuanosine 5'-monophosphateNAD⁺Uridine 5'-monophosphateLauryldiethanolamineLPE 20:23b-Hydroxy-5-cholenoic acidOxidized glutathioneLPE 17:1Glucose 1-phosphateUridineLPE 18:1alanine4-(4-hydroxyphenyl)sulfonylphenolAdenosine 5'-monophosphatephytosphingosineLPE 16:1GlutathionefalseSARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I InhibitionSARS-CoV-2 reprograms host metabolism to promote viral replication and evade immune responses. While infection is known to impair mitochondrial function and enhance glycolysis, the role of viral accessory proteins in these alterations remains unclear. Here, we investigate the metabolic impact of the accessory protein ORF7a. Lentiviral expression of ORF7a in human lung epithelial (A549) and monocytic (THP1) cells, coupled with integrated transcriptomic, proteomic, and metabolomic analyses, revealed profound dysregulation of glucose and lipid metabolism. ORF7a impaired oxidative phosphorylation, reducing basal and maximal respiration, inducing mitochondrial depolarization, and increasing reactive oxygen species. Mechanistically, ORF7a upregulated pyruvate dehydrogenase kinase 4 (PDK4), promoting pyruvate dehydrogenase (PDH) complex phosphorylation and suppressing pyruvate oxidation. However, pharmacological PDK4 inhibition did not restore respiration. High-resolution respirometry in frozen samples revealed impaired complex I function, while Blue Native-PAGE demonstrated defective respiratory supercomplex assembly. By linking enzymatic inhibition with structural destabilization, our study uncovers a functional vulnerability of the mitochondrial respiratory chain to viral manipulation. These findings establish ORF7a as a key modulator of host metabolic reprogramming and highlight mitochondrial pathways as potential therapeutic targets in COVID-19.2026-07-092025-11-18MTBLS13338MTBLC16449MTBLC5390MTBLC29042MTBLC16192MTBLC17345MTBLC16695MTBLC16856MTBLC17858MTBLC16704MTBLC229635MTBLC145247MTBLC64575MTBLC183648MTBLC131692MTBLC229266MTBLC27997MTBLC46961MTBLC89234MTBLC44229MTBLC57540MTBLC33198MTBLC16027MTBLC34372MTBLC61282CHEBI:16449CHEBI:5390CHEBI:29042CHEBI:16192CHEBI:17345CHEBI:16695CHEBI:16856CHEBI:17858CHEBI:16704CHEBI:229635CHEBI:145247CHEBI:64575CHEBI:183648CHEBI:131692CHEBI:229266CHEBI:27997CHEBI:46961CHEBI:89234CHEBI:44229CHEBI:57540CHEBI:33198CHEBI:16027CHEBI:34372CHEBI:61282