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One 15 cm plate was used for each sample and three replicates of each condition were prepared, including uninfected controls. Cells were harvested at 24 hpi and pelleted by centrifugation (1100 rpm for 5 min.). For 3xFlag-tagged samples, cells were resuspended in 150 µL SDS lysis buffer pre-heated to 95°C (1% SDS, 50 mM Tris/HCl pH 8.0, 150 mM NaCl). Samples were heated at 95°C for 10 min. 500 µL of RIPA w/o SDS (50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1.1% Triton X-100, 0.55% Sodium deoxycholate) was added to each sample, followed by sonication (30-50% power for 3 x 1 sec.) until samples were no longer viscous. 850 µL RIPA w/o SDS was added to each sample, debris were pelleted by centrifugation (13.2K rpm for 10 min. at 4°C), and supernatants transferred to fresh tubes. 40 µL of Anti-FLAG M2 Magnetic Beads (Sigma, #M8823) were pre-washed 3x with RIPA (50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS) and then added to each sample. Lysates were incubated with beads overnight at 4°C with rotation. Beads were then washed 3x with RIPA, and then 3x with RIPA w/o detergent (50 mM Tris/Hcl pH 8.0, 150 mM NaCl). Bound proteins were eluted in 30 µL elution buffer containing 50 mM Tris/HCL pH 8.0, 150 mM NaCl, 0.05% RapiGest SF Surfactant (Waters) and 100 µg/mL 3xFLAG peptide (MDYKDHDGDYKDHDIDYKDDDDK, Elim Biopharm) for 30 min. at room temperature on a shaker. 6His-tagged samples were prepared as described.4 Enrichment of Flag and His-tagged proteins were confirmed by WB of input, eluates, and flowthrough. Eluates were used below for both total protein analysis and identification of SUMO-conjugated peptides. LC-MS/MS Immunoprecipitated samples were diluted in buffer (2M urea, 10 mM NH4HCO3, and 2 mM DTT) and incubated at 60°C for 30 min. Iodoacetamide was added to 2 mM and samples were incubated at room temperature in the dark for 45 min. 160 ng trypsin (Gold Mass spectrometry Grade, Promega) was added and samples incubated overnight at 37 °C. Samples were desalted with C18 spin columns (Nest Group, product #HUMS18R), vacuum centrifuged to dryness, and resuspended in 0.1% formic acid for MS analysis. All samples were analyzed on an Orbitrap Eclipse MS system equipped with an Easy nLC 1200 ultra-high pressure liquid chromatography system interfaced via a Nanospray Flex nanoelectrospray source (Thermo Fisher). Samples were injected onto a fritted fused silica capillary (30 cm × 75 μm inner diameter with 15 μm tip, CoAnn Technologies) packed with ReprosilPur C18-AQ 1.9 μm particles (Dr. Maisch GmbH). Buffer A consisted of 0.1% formic acid in H2O, and buffer B consisted of 0.1% formic acid in 80% acetonitrile. Peptides were separated by an organic gradient from 5% to 35% mobile buffer B over 120 min, followed by an increase to 100% B over 10 min at a flow rate of 300 nL/min. Analytical columns were equilibrated with 3 μL buffer A."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["To build a spectral library, samples from each set of biological replicates were pooled and acquired in data-dependent manner. Data-dependent acquisition (DDA) was performed by acquiring a full scan over a m/z range of 375-1025 in the Orbitrap at 120,000 resolving power (@ 200 m/z) with a normalized AGC target of 100%, an RF lens setting of 30%, and an instrument-controlled ion injection time. Dynamic exclusion was set to 30 seconds, with a 10 p.p.m. exclusion width setting. Peptides with charge states 2-6 were selected for MS/MS interrogation using higher energy collisional dissociation (HCD) with a normalized HCD collision energy of 28%, with 3 seconds of MS/MS scans per cycle. Data-independent analysis (DIA) was performed on all individual samples. A full scan was collected at 60,000 resolving power over a scan range of 390-1010 m/z, an instrument controlled AGC target, an RF lens setting of 30%, and an instrument controlled maximum injection time, followed by DIA scans using 8 m/z isolation windows over 400-1000 m/z at a normalized HCD collision energy of 28%. The Spectronaut algorithm was used to build spectral libraries from DDA data, identify peptides/proteins, and extract intensity information from DIA data. DDA data were searched against the Homo sapiens reference proteome sequences in the UniProt database (one protein sequence per gene, downloaded on October 10, 2019). False discovery rates were estimated using a decoy database strategy. All data were filtered to achieve a false discovery rate of 0.01 for peptide-spectrum matches, peptide identifications, and protein identifications. An independent mass spectrometry experiment was also performed in collaboration with the lab of Simone Sidoli. Digested peptides were desalted using a 96-well plate filter (Orochem) packed with 1 mg of Oasis HLB C-18 resin (Waters). Briefly, the samples were resuspended in 100 µl of 0.1% TFA and loaded onto the HLB resin, which was previously equilibrated using 100 µl of the same buffer. After washing with 100 µl of 0.1% TFA, the samples were eluted with a buffer containing 70 µl of 60% acetonitrile and 0.1% TFA and then dried in a vacuum centrifuge. Samples were resuspended in 10 µl of 0.1% TFA and loaded onto a Dionex RSLC Ultimate 300 (Thermo Scientific), coupled online with an Orbitrap Fusion Lumos (Thermo Scientific). Chromatographic separation was performed with a two-column system, consisting of a C-18 trap cartridge (300 µm ID, 5 mm length) and a picofrit analytical column (75 µm ID, 25 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 µm resin. Peptides were separated using a 90 min gradient from 4-30% buffer B (buffer A: 0.1% formic acid, buffer B: 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 nl/min. The mass spectrometer was set to acquire spectra in a data-dependent acquisition (DDA) mode. Briefly, the full MS scan was set to 300-1200 m/z in the orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5x10e5. MS/MS was performed in the ion trap using the top speed mode (2 secs), an AGC target of 1x10e4 and an HCD collision energy of 35. Proteome raw files were searched using Proteome Discoverer software (v2.5, Thermo Scientific) using SEQUEST search engine and the SwissProt human + HIV database. Trypsin was specified as the digestive enzyme with up to 2 missed cleavages allowed. Mass tolerance was set to 10 pm for precursor ions and 0.2 Da for product ions. Peptide and protein false discovery rate was set to 1%. MS data analysis Protein intensities were log2 transformed, and log2 fold changes in SUMO substrates between the HIV-1 infected versus uninfected samples were calculated as the difference between the average log2 abundance of the triplicate HIV-1-infected samples and uninfected samples (Supplemental Table 1 tabs C-F, Supplemental Table 2)."],"omics_type":["Proteomics"],"labhead":["Simone Sidoli"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, United States"],"species":["Homo Sapiens (human)"],"publication":["Not available"],"submitter_mail":["simone.sidoli@gmail.com"],"submitter_affiliation":["Albert Einstein College of Medicine"],"submitter_country":["United States"],"additional_accession":[]},"is_claimable":false,"name":"HIV-1 infection induces Vif-mediated SUMOylation of host RNA splicing factors important for proper viral RNA splicing","description":"HIV-1 exploits host cell post-translation modifications (PTMs) to facilitate production of infectious particles. These modifications include SUMOylation, a dynamically regulated PTM involving covalent attachment of small ubiquitin-like modifiers (SUMOs) to lysine (K) residues of target proteins. SUMOylation modulates the activity of thousands of proteins and multiple fundamental host cellular processes, including pathways hijacked by HIV-1 to promote infection and spread. The SUMOylation of several proteins during HIV-1 infection has been characterized. However, the broad effects of HIV-1 infection on the SUMOylation of the host cell proteome is largely unknown. To date, SUMOylation has not been explored by large-scale proteomics in the context of HIV infection, where many SUMO-regulated host dependency factors remain to be identified. In this study, we performed a proteome-wide, mass spectrometry (MS)-based screen to identify proteins that are SUMOylated during HIV-1 infection. Here, and in immunoprecipitation assays, infection with HIV-1 led to the widespread increased SUMOylation of the heterogeneous nuclear ribonucleoprotein (HNRNP) A/B proteins, a protein family central to the regulation of alternative splicing. Intriguingly, this phenotype was found to be driven by expression of the HIV-1 Viral Infectivity Factor (Vif), suggesting a novel function for this protein aside from APOBEC3G degradation. We selected HNRNPA2B1 (A2/B1) and HNRNPA3 for further study, where depletion of these proteins led to the altered splicing of HIV-1 viral RNAs and dramatically reduced HIV-1 infectivity. Considering the enrichment of SUMOylation sites within the RNA-binding domains of the HNRNPA/B family, our data suggest a novel mechanism involving HIV-1-induced, Vif-mediated SUMOylation of host RNA splicing factors as a means to regulate HIV-1 alternative splicing. Broadly, our findings suggest that infection with HIV-1 alters the SUMOylation of many unexplored host cellular proteins, and provides a significant proteomic resource for their future mechanistic study.","dates":{"publication":"2026-04-28","submission":"2026-03-12"},"accession":"PXD075554","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:241368","NEWT:2","NEWT:157546","NEWT:190802","NEWT:35554","NEWT:150475","NEWT:9417","NEWT:347515","NEWT:1216979","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1081927","NEWT:67825","NEWT:13076","NEWT:1249668","NEWT:317","NEWT:1736309","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:4081","NEWT:876138","NEWT:554","NEWT:98334","NEWT:237561","NEWT:10036","NEWT:7574","NEWT:1351","NEWT:7215","NEWT:272563","NEWT:507601","NCBITaxon:79857","NCBITaxon:6157","NEWT:95648","NEWT:746360","NEWT:6239","NEWT:1589","NEWT:470150","NEWT:135622","NEWT:216257","NEWT:6915","NEWT:9986","NEWT:101510","NEWT:4054","NEWT:3880","NEWT:8782","NEWT:1000589","NEWT:1902","NEWT:85962","NEWT:160488","NEWT:28104","NEWT:317447","NEWT:7955","NCBITaxon:2","NEWT:985076","NEWT:7959","NEWT:2261","NEWT:4565","NEWT:1264690","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:931281","NEWT:7029","NEWT:1283300","NEWT:334747","NEWT:61235","NCBITaxon:79824","NCBITaxon:4563","NEWT:5755","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:436486","NEWT:6287","NEWT:2242","NEWT:300641","NEWT:4784","NEWT:727","NEWT:9796","NEWT:725","NEWT:360106","NEWT:260707","NEWT:287","NEWT:10117","NEWT:10239","NCBITaxon:6191","NEWT:10116","NEWT:1280","NEWT:1836","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:44685","NEWT:2697049","NEWT:1148","NEWT:11676","NEWT:55571","NEWT:100226","NCBITaxon:6073","NEWT:4530","NEWT:4896","NEWT:6279","NEWT:1123869","NEWT:7370","NEWT:83906","NEWT:6282","NEWT:1134506","NEWT:575584","NEWT:1773","NEWT:38783","NEWT:8727","NEWT:1895","NEWT:1182590","NEWT:8726","NEWT:10090","NEWT:935293","NEWT:749200","NEWT:4120","NEWT:5693","NEWT:8724","NEWT:51511","NEWT:92867","NEWT:8723","NEWT:990346","NEWT:5334","NEWT:145953","NEWT:257309","NEWT:230741","NEWT:284812","NCBITaxon:10359","NCBITaxon:1313","NEWT:43330","NEWT:242619","NEWT:44544","NEWT:632957","NEWT:373995","NEWT:544404","NEWT:3702","NEWT:129249","NEWT:8839","NEWT:4232","NEWT:990119","NEWT:2758385","NEWT:4113","NEWT:837","NEWT:11298","NEWT:171101","NEWT:196627","NEWT:408172","NEWT:5691","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:627025","NEWT:400772","NEWT:1097677","NEWT:3708","NEWT:106592","NEWT:1117957","NEWT:9913","NEWT:1432138","NEWT:10312","NEWT:4100","NEWT:1076","NEWT:6763","NEWT:803","NEWT:8030","NEWT:29722","NEWT:380394","NEWT:1692259","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:180066","NEWT:67767","NEWT:46835","NEWT:135588","NEWT:1843183","NEWT:95486","NEWT:58002","NEWT:9103","NEWT:4577","NEWT:5664","NEWT:2157","NEWT:146479","NEWT:10306","NEWT:1911079","NEWT:8022","NEWT:145943","NEWT:3635","NEWT:5811","NEWT:1480154","NEWT:235443","NEWT:1274414","NEWT:59202","NEWT:3197","NEWT:9615","NEWT:10299","NEWT:860688","NEWT:884019","NEWT:169963","NEWT:36329","NEWT:1147787","NEWT:72407","NEWT:9606","NEWT:367830","NEWT:157295","NEWT:410289","NEWT:373153","NEWT:915099","NEWT:74940","NEWT:1450511","NEWT:470","NEWT:84023","NEWT:9838","NCBITaxon:9615","NEWT:1193501","NEWT:3055","NEWT:6326","NEWT:6689","NEWT:2762","NEWT:5476","NEWT:1174673","NEWT:562","NEWT:33952","NEWT:1274432","NEWT:1274426","NEWT:1423","NEWT:4932","NEWT:70448","NEWT:9825","NEWT:1274423","NEWT:3603","NEWT:698936","NEWT:2759","NEWT:3847","NEWT:39946","NEWT:9823","NEWT:9940","NEWT:573","NEWT:9031","NEWT:1274420","NEWT:7091"],"ORCID":["0000-0001-9073-6641"]}}