Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/FileSetup.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_3.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_17.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_21.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_18.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_19.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_Input_6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075408/221117_RL_Apex_3.pdResultprimaryOK200bagarcia@wustl.eduORLANDO SCUDEROData-dependent acquisitionMass SpectrometryBottom-up proteomicsRnase l-dependent bodies (rlbs)Stress granules (sgs)Dna virusesAdenovirus 5 (ad5)https://www.ebi.ac.uk/pride/archive/projects/PXD075408Cell CultureThe peptide mixture was separated using a Dionex Ultimate 3000 high-performance liquid chromatography (HPLC) system (Thermo Scientific) equipped with a two-column setup, consisting of a reversed-phase trap column (Acclaim PepMap100 C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm, Thermo Scientific) and a reversed-phase analytical column (35 cm, 75 μm i.d., 360 μm o.d., packed with Pur C18AQ, 2.4 μm; Dr. Maisch). Loading buffer was 0.1% trifluoroacetic acid (Merck Millipore) in water. Buffer A was 0.1% formic acid, and Buffer B was 80% acetonitrile (ACN) + 0.1% formic acid. The HPLC was coupled online with a Q-Exactive-HF mass spectrometer (Thermo Scientific). Peptides were eluted using a 140 min ACN gradient (90 minute 5%–25% ACN gradient, followed by a 30 minute 25%–45% ACN gradient, a 10 minute 45%-95% gradient, with a final 10-minute isocratic step at 5% ACN) at a flow rate of 300 nl/min for IP and input samples. The MS instrument was controlled by Xcalibur software (Thermo Fisher Scientific). Samples were batch-randomized to account for instrument variation. The data dependent acquisition (DDA) MS method was designed with the MS1 having a window of 400–1000 m/z, AGC target of 1e6 and maximum inject time (MIT) of 75 ms with the MS2 having automated windows, AGC target of 100% and MIT of 75 ms. Fragmentation was performed with high-energy collisional dissociation (HCD) using normalized collision energies (NCE) of 27%. The selection for ions were charges 2–8, minimum peak intensity of 1e4, and a 3 s maximum cycle time.PrideThe raw mass spectrometer files were processed for protein identification using the Proteome Discoverer (v2.4, Thermo Scientific) and the Sequest HT algorithm with a peptide mass tolerance of 10 ppm, fragment m/z tolerance of 0.02 Da, and a false discovery rate (FDR) of 1% for proteins and peptides. Quantification was performed using a label-free approach using the “Precursor ions quantifier” node, and peptide abundances were rolled up into protein abundance using the summed abundance algorithm using only unique or razor peptides. All peak lists were searched against the UniProtKB/Swiss‐Prot database of Human sequences (9606; downloaded November 2022) using the parameters as follows: enzyme, trypsin; maximum missed cleavages, 2; fixed modification, carbamidomethylation (C); variable modifications, oxidation (M), protein N‐terminus acetylation. All subsequent protein-level analysis of streptavidin-enriched APEX2-labeled proteins (APEX) and total lysate control (Input) abundance quantification data was performed using custom R scripts. Proteins were filtered to include only those identified by at least 1 unique peptide and with peptide q-value < 0.01. APEX and Input abundance quantification values within each sample were transformed to log2 values and normalized by the sample median to account for technical variation in the abundances across samples. APEX and Input abundance means for each protein, in each condition, were obtained by calculating the arithmetic average of the abundance quantification values across the three biological replicates of each condition. Biological replicates for which abundance quantification values were not obtained were removed from the calculation of the mean. Relative protein APEX and Input abundances within each condition were obtained by calculating the statistical z-scores based on the average and standard deviation within the average abundance quantifications of the respective condition. APEX abundances were normalized by the respective input abundances for each replicate of all infections and treatments. Log2 fold changes of APEX abundances for compared conditions were obtained by comparing the average normalized quantification of the respective conditions. Log2 fold changes were imputed for cases in which the protein was identified in 3/3 replicates in one condition and 0/3 replicates in the compared condition. In these cases, the log2 fold change was defined as the minimum or maximum of the fold changes within the respective condition. Statistical p-values associated with the APEX abundance log2 fold changes were calculated using unpaired, two-sided, student’s t-tests comparing the three replicate normalized quantifications of each compared condition. P-values were calculated only for comparisons in which each condition had quantifications in at least 2 of 3 biological replicates. When comparing APEX abundances across conditions, statistically enriched proteins were defined as those proteins that were identified in 3/3 biological replicates in one condition and 0/3 replicates in the compared condition, or identified in 3/3 biological replicates in one condition and exhibiting a log2 fold change > 1 and p-value < 0.05 versus the compared condition.ProteomicsBenjamin A. GarciaPARTIALDepartment of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, Missouri, USAHomo Sapiens (human)10.1371/JOURNAL.PPAT.1014452scuderoo@chop.eduChildren's Hospital of PhiladelphiaUnited StatesfalseDifferential assembly of RNP granules via activation of distinct dsRNA sensors by adenovirus mutantsRecognition of dsRNA triggers antiviral defense mediated by PKR and OAS3/RNase L pathways through translational arrest and RNA decay. This is accompanied by assembly of distinct cytoplasmic ribonucleoprotein (RNP) condensates termed stress granules (SGs) and RNase L-dependent bodies (RLBs). Here we show that adenovirus infection differentially modulates dsRNA sensors and RNP granule assembly. Infection with splicing-defective ∆E4 mutant leads to dsRNA accumulation and activation of both PKR and OAS3/RNase L, promoting formation of RLB-like granules. In contrast, mutants lacking virus-associated (VA) RNAs trigger PKR activation and assembly of SGs despite absence of detectable dsRNA. Proteomic analysis revealed distinct protein compositions of canonical SGs and RLBs, which were reflected in virus-induced granules. While ∆VA-induced granules were PKR-dependent, ∆E4 mutants induced RLB-like granules independently of PKR and RNase L. In these cells, granule assembly coincided with translational arrest independent of eIF2α phosphorylation, indicating additional pathways linking nuclear dsRNA sensing to translational control and RNP granule assembly during viral infection. These findings provide novel insights into how distinct dsRNA sensors modulate translation and RNP condensates in response to stress.2026-07-102026-03-09PXD075408NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:9778NEWT:150475NEWT:9417NEWT:347515NEWT:1216979NEWT:307972NEWT:32046NEWT:544496NEWT:5180NEWT:256737NEWT:2042546NEWT:115104NEWT:1081927NEWT:67825NEWT:43179NEWT:13076NEWT:1249668NEWT:376741NEWT:317NEWT:55153NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:415540NEWT:4081NEWT:876138NEWT:554NEWT:98334NEWT:426428NEWT:237561NEWT:6928NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:29204NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:3885NEWT:746360NEWT:6239NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:3641NEWT:8782NEWT:1263854NEWT:1000589NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:1283NEWT:931281NEWT:1000561NEWT:7029NEWT:1283300NEWT:6183NEWT:334747NEWT:61235NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NCBITaxon:6191NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:185431NEWT:83332NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:44685NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:1123869NEWT:7370NEWT:75058NEWT:83906NEWT:607699NEWT:6282NEWT:208964NEWT:1134506NEWT:575584NEWT:296543NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:1182590NEWT:8726NEWT:6669NEWT:10090NEWT:935293NEWT:64152NEWT:749200NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:100816NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:632957NEWT:373995NEWT:5689NEWT:645463NEWT:544404NEWT:3702NEWT:129249NEWT:9925NEWT:8839NEWT:4232NEWT:990119NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:421932NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:128161NEWT:106592NEWT:1117957NEWT:9913NEWT:1432138NEWT:10312NEWT:1424507NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:180066NEWT:67767NEWT:46835NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:10306NEWT:1911079NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:3635NEWT:5811NEWT:235443NEWT:1480154NEWT:1274414NEWT:59202NEWT:9975NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:884019NEWT:169963NEWT:36329NEWT:1147787NEWT:72407NEWT:9606NEWT:367830NEWT:157295NEWT:178616NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:1450511NEWT:360094NEWT:470NEWT:84023NEWT:9838NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:33952NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:9940NEWT:327160NEWT:573NEWT:9031NEWT:1274420NEWT:70910000-0002-6625-9387