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dstone.ucsf.edu"],"submitter":["Robyn Kaake"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Sars-cov-2","orf8","pyroptosis","ace2","il-1β","macrophage","Sars-cov-2; orf8; macrophage; ace2; pyroptosis; il-1β; brodalumab","brodalumab"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD069220"],"tissue":["Primary Cell","Blood","Macrophage"],"sample_protocol":["All experiments were performed in triplicate. Primary human macrophages were generated from peripheral blood mononuclear cells (PBMCs) by positive selection of CD14+ monocytes after Ficoll density separation, followed by 6 days of differentiation in serum-free medium supplemented with macrophage colony-stimulating factor (M-CSF, 50 ng/mL). M1 macrophages were obtained by stimulating for 2 days with lipopolysaccharide (LPS, 10 ng/mL) and interferon-γ (IFN-γ, 50 ng/mL), and M2 macrophages by stimulating for 2 days with interleukin-4 (IL-4, 10 ng/mL). For each condition, 1×E6 cells were plated per well (12-well plate), treated with SARS-CoV-2 ORF8 protein (1 µg/mL), and harvested at 12 h and 48 h. Cells were washed with PBS and lysed in the RIPA buffer, and lysates were stored at −80 °C. Frozen cell pellets were resuspended in 100μL of urea lysis buffer (8M urea, 150mM NaCl2, 100mM Tris) on ice, sonicated for 30 seconds, prior to one round of freeze thaw. For each lysate, 50μg was reduced with 2mM TCEP (room temperature (RT), 30 min), and alkylated with 10mM iodoacetamide (dark, RT, 30 min). Iodoacetamide was quenched with 10mM DTT (dark, RT, 30 min). Buffer exchange and protein digestion were performed on a KingFisher Flex unit using a protein aggregation approach, plates setup as follows: 1) 96-well comb; 2) lysate/bead mixtures; 3-5) 95% acetonitrile wash; 6-7) 70% ethanol wash; and 8) digestion elution buffer. Prior to automated run, the lysate/bead mixtures were prepared by mixing together 20µL MagReSyn Amine beads with 100μL of lysate and 280µL of 100% acetonitrile. On beads protein aggregation was induced by 4 cycles of mixing (1 min), pause (5 min), and collection (24 min total time). Beads were transferred to plate 3, released and washed by 5 cycles of mixing (repeated in plates 4-5). Beads were transferred to plate 6, released and washed by 5 cycles of mixing (repeated in plate 7). Finally, protein bound beads were released into plate 8 with freshly prepared digestion elution buffer (150 µL of 50 mM ammonium bicarbonate (pH 7.8) with 0.5μg Lys-C and 0.5μg trypsin) and proteolytic digestion proceeded overnight at 37 °C with agitation at 800 RPM. The resulting peptides were filtered (0.45 µm membranes), acidified with 5μL formic acid, and dried by vacuum centrifugation. Dried samples were resuspended in 25μL 0.1% formic acid (FA) in water, and 1μL of peptide was injected on a Orbitrap Exploris 480 mass spectrometer coupled to a Vanquish Neo liquid chromatography instrument (Thermo Fisher Scientific). Peptides were separated on a 150μm x 15cm PepSep column (Bruker, 1.5μm beads) over a 90min gradient (flow rate of 600 nL/min). Buffer A consisted of 0.1% FA in water, and buffer B was 0.1% FA in 80% acetonitrile. Spectra were continuously acquired in a data-dependent acquisition (DDA) mode: one full scan was acquired in the Orbitrap (350-1250 m/z at 120,000 resolution with a normalized AGC target of 300%), followed by as many MS/MS scans as could be acquired on the most abundant ions in 2 seconds in the Orbitrap (1500 resolution; Normalized collision energy type; HCD collision energy of 28%; normalized AGC target of 200%, maximum injection time set to Auto, and isolation window of 1.6 m/z). Singly and unassigned charge states were rejected and dynamic exclusion was enabled after n=1 time, with an exclusion duration of 20 seconds (tolerance of ±10 ppm)."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Raw MS files were analyzed using Fragpipe (version 21.1). MS/MS spectra were searched against the human proteome (UniProt reviewed, downloaded 09 May 2024; concatenated with equal sized reversed database and common contaminants). Peptide ion intensities from the output of Fragpipe were summarized to protein intensities using the R Bioconductor package MSstats (version 4.8.3). The functions dataProcess and groupComparison were applied with default settings, except for setting \"MBimpute = FALSE\" in dataProcess. Proteins with an adjusted p-value ≤ 0.05 and an absolute log₂ fold change > 1 were considered significant."],"omics_type":["Proteomics"],"labhead":["Robyn Midori Kaake"],"instrument_platform":[""],"labhead_affiliation":["Univeristy of California San Francisco (UCSF), Department of Bioengineering and Therapeutic Sciences, USA, San Francisco"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["Not available"],"submitter_mail":["robyn.kaake@gladstone.ucsf.edu"],"submitter_affiliation":["Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA"],"submitter_country":["United States"],"additional_accession":[]},"is_claimable":false,"name":"Secreted ORF8 reprograms macrophages to enhance SARS-CoV-2 infection of lung epithelial cells","description":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets the respiratory epithelium, yet severe disease features diffuse lung injury and hyperinflammatory syndromes driven by dysregulated immune activation. Emerging evidence indicates that resident and infiltrating immune cells in the lung can encounter the virus early in infection and, under specific conditions, become infected. This process amplifies local inflammation and facilitates viral propagation in the lower airways and distal lung regions, where angiotensin converting enzyme 2 (ACE2) expression is limited. However, how epithelial and immune cell compartments interact to produce the hallmark pulmonary pathology of SARS-CoV-2 infection remains unresolved. Here we show that secreted ORF8, a SARS-CoV-2 accessory protein, drives inflammatory lung pathology by increasing macrophage permissively to infection, triggering pyroptosis, and amplifying viral replication in alveolar epithelial cells. Co-culture of macrophages with human alveolar type II (AT2) cells overrides ORF8’s previously reported inhibition of AT2 infection, restoring productive viral replication. In vivo, IL-17RA blockade with brodalumab counteracts ORF8 activity, lowering viral burden and attenuating pulmonary inflammation and fibrosis. These findings reveal a paracrine role for ORF8 in reprogramming macrophages, thereby establishing a feedforward proviral circuit that accelerates lung pathology in COVID-19 and are particularly relevant given the recurrent emergence of SARS-CoV-2 variants with either intact or deleted ORF8 since the beginning of the pandemic.","dates":{"publication":"2026-08-07","submission":"2025-10-09"},"accession":"PXD069220","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:1538","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:10804","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211",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