Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/macroH2A_KO_pure.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_01.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_02.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_01.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_03.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_02.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_03.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_01.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_01.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_01.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_01.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_02.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_02.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_macroH2A_KO_PNGaseF_03.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/012324_pure_PNGaseF_03.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD061284/UniProt_HomoSapiens_HCMV_Towne_2024_01_24.fastaprimaryOK200kentsisresearchgroup@gmail.comAlex KentsisData-dependent acquisitionMass SpectrometryBottom-up proteomicsViral Infectious DiseaseNot availableHistone ptmsChromatinMacroh2a1Human cytomegalovirus (hcmv)https://www.ebi.ac.uk/pride/archive/projects/PXD061284Cell CultureFibroblastPreparation of purified viral particles for mass spectrometry analysis. All chemicals used for preparation of mass spectrometry samples were of at least sequencing grade and purchased from Sigma-Aldrich (St Louis, MO), unless otherwise stated. The sucrose cushion purified HCMV viral particles were lysed in 1% sodium dodecyl sulfate (SDS), 50 mM triethylammonium bicarbonate (TEAB) buffer, pH 8.5 by tip-probe sonication on ice. Lysates were centrifuged at 14,000xg for 15min at 4°C, and the supernatant was used for further analysis. The 1% SDS detergent and lipids were removed from samples prior mass spectrometry analysis by precipitation using chloroform (CHCl3)-methanol (MeOH) precipitation [PMID: 6731838]. The protein pellet from CHCl3-MeOH precipitation was resuspended in 6 M urea and 2 M thiourea in 50 mM ammonium bicarbonate, pH 8.0. Protein concentration was determined using Bradford assay kit (ThermoScientific) according to the manufacturers’ instructions. Protein reduction was carried out using 10mM DTT for 1 hour at room temperature followed by alkylation with 30 mM iodoacetamide in the dark at room temperature for 40 min. After alkylation, proteins were digested first with endopeptidase Lys-C (Wako, mass spectrometry grade) for 3 hours, after which the solution was diluted 10 times with 20 mM ammonium bicarbonate, pH 8.0. Subsequently, samples were digested with trypsin (Promega) at an enzyme to substrate ratio of approximately 1:50 for 12 hours at room temperature. The samples were acidified with 5% formic acid (FA) to a pH ≤ 3 and desalted using Poros Oligo R3 RP columns (PerSeptive Biosystems) packed in a P200 stage tip with C18 3M plug (3M Bioanalytical Technologies). Purified peptide samples were dried by lyophilization and stored at −20°C until further analysis. Enzymatic deglycosylation. Tryptic peptides were resuspended in 100µl of 20 mM ammonium bicarbonate, pH = 7.5 before adding 500 U PNGase F (NewEngland Biolabs, Ipswich, MA) for 12 h at 37°C. The reaction was stopped by acidification using 1% final concentration of formic acid and the peptides desalted as described above. Liquid Chromatography-Tandem Mass Spectrometry Analysis. Samples were dissolved in buffer A (0.1% formic acid; FA) and analyzed using an LC-MS/MS system consisting of an Ekspert NanoLC 425 chromatograph (Eksigent, Redwood City, CA, USA) coupled to an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA). The samples were separated on a 30 cm fused silica capillary analytical column (75 µm ID). All columns were homemade and packed with ReproSil-Pur C18 AQ 3 μm reversed-phase material (Dr. Maisch, Ammerbuch-Entringen, Germany). The peptides were eluted using 120 min gradients from buffer B (100% Acetonitrile (ACN), 0.1% FA) and introduced into the MS instrument via nano-electrospray. A full MS scan in the mass area of 300–1200 Da was performed in the Orbitrap with a resolution of 120,000 FWHM (200 m/z), an AGC target value of 1x106, and a maximum injection time of 100 ms. For each full scan, the settings for the higher energy collision dissociation (HCD) were as follows: AGC target value of 5×104, maximum injection time of 22 ms, isolation window of 1.6 m/z, normalized collision energy of 30, and a dynamic exclusion window of 60 s.PrideRaw MS files were analyzed by PEAKS Studio (v11.5) software platform (Bioinformatics Solutions Inc). MS/MS spectra were searched against the UniProt-human and HCMV (Towne) database (Version January 2024, 20,585 entries). For all searches, MS1 and HCD MS2 mass tolerances were set to 10 ppm and 0.02 Da, respectively. Cysteine carbamidomethylation was set as fixed, while methionine oxidation and acetylation (protein N-term) were set as variable modifications, with a maximum of two variable modifications per peptide. Only peptides containing 6–45 residues and up to 2 missed trypsin cleavages were considered. Each protein quantification value was log2 transformed and subsequently normalized by the HCMV MCP (UL86) protein abundance within each run. Cellular Component association analysis was performed using the STRING database (v12.0) with a false discovery rate (FDR) <1%. Each GO term was ranked based on the STRING strength score (represents the confidence level of functional associations between proteins or gene ontology (GO) terms, derived from integrated evidence sources such as experimental data, text mining, co-expression, and curated databases, with higher scores indicating stronger enrichment or interaction reliability) [PMID: 33237311].ProteomicsAlex Kentsis, MD, PhDTow Center for Developmental Oncology, Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Departments of Pediatrics, Pharmacology, and Physiology & Biophysics, Weill Cornell Medical College, Cornell University, New York, NY, USA.COMPLETECytomegalovirusHomo Sapiens (human)40781068 Kelnhofer-Millevolte LE, Smith JR, Nguyen DH, Wilson LS, Lewis HC, Arnold EA, Brinkley MR, Shin K, Ahn JH, Kim ET, Kulej K, Geballe AP, Ramachandran S, Avgousti DC. Human cytomegalovirus induces neuronal gene expression through IE1 for viral maturation. Nat Commun. 2025 16(1):7316 10.1038/s41467-025-61915-7kentsisresearchgroup@gmail.comMemorial Sloan Kettering Cancer CenterUnited States10.6019/PXD061284Viral invasion of the host cell causes some of the most dramatic changes in biology. Human cytomegalovirus (HCMV) extensively remodels host cells, altering nuclear shape and generating a cytoplasmic viral-induced assembly compartment (vIAC). How these striking morphology changes occur in the context of host gene regulation is still emerging. Histone variant macroH2A1 is both important for maintaining nuclear integrity and functions to promote herpes simplex virus infection. Therefore, we hypothesized it may also function in cytomegalovirus infection. Here, we discovered that macroH2A1 is necessary for HCMV-induced cellular reorganization and formation of infectious progeny. Using RNA-seq in infected cells, we find that while all viral genes are highly expressed in the absence of macroH2A1, many HCMV-induced host genes are not. Remarkably, hundreds of these HCMV-induced macroH2A1-dependent host genes are associated with a neuronal signature. Further, we find that HCMV immediate early protein, IE1, is both necessary and sufficient to induce these neuronal genes, providing a mechanism of activation. Together, our findings demonstrate that HCMV hijacks a dormant neuronal secretory pathway through chromatin manipulation for efficient virion maturation.Human cytomegalovirus induces neuronal gene expression through IE1 for viral maturation.Kelnhofer-Millevolte Laurel E LE, Smith Julian R JR, Nguyen Daniel H DH, Wilson Lea S LS, Lewis Hannah C HC, Arnold Edward A EA, Brinkley Mia R MR, Shin Kihye K, Ahn Jin-Hyun JH, Kim Eui Tae ET, Kulej Katarzyna K, Geballe Adam P AP, Ramachandran Srinivas S, Avgousti Daphne C DCfalseHuman cytomegalovirus induces neuronal gene expression through IE1 for viral maturationViral invasion of the host cell causes some of the most dramatic changes in biology. Human cytomegalovirus (HCMV) extensively remodels host cells, altering nuclear shape and generating a cytoplasmic viral-induced assembly compartment (vIAC). How these striking morphology changes occur in the context of host gene regulation is still emerging. Here, we discovered that histone variant macroH2A1 is necessary for formation of infectious progeny and HCMV-induced reorganization of the host cell. Using RNA-seq we found that while all viral genes were highly expressed in the absence of macroH2A1, many HCMV-induced host genes were not. Remarkably, hundreds of these HCMV-induced macroH2A1-dependent host genes are associated with neuronal synapse formation and vesicle trafficking. Further, we found that HCMV immediate early protein, IE1, is sufficient to induce these neuronal genes, providing a mechanism of activation. Together, our findings demonstrate that HCMV hijacks a dormant neuronal secretory pathway through chromatin manipulation for efficient virion maturation.2026-02-092025-02-27PXD061284NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:71647NEWT:2NEWT:157546NEWT:474186NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:1496NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:6938NEWT:1129NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:44139NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NEWT:2164133NCBITaxon:183674NEWT:1316931NEWT:224308NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:511145NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:431241NEWT:931281NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NEWT:145481NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:246196NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:161934NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:5507NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:296543NEWT:316435NEWT:42157NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:1034304NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:1077286NEWT:943129NEWT:243277NEWT:990119NEWT:7067NEWT:2850NEWT:1111708NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:930945NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:1194599NEWT:272844NEWT:54571NEWT:9598NEWT:8030NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:84588NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:537457NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:644223NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:1679NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NCBITaxon:138NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:571NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:40479NEWT:317NEWT:9646NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:273068NEWT:313625NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:434271NEWT:747679NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:38018NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITa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