Landscape of Epstein-Barr virus gene expression and perturbations in cancer

Landscape of Epstein-Barr virus gene expression and perturbations in cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Landscape of Epstein-Barr virus gene expression and perturbations in cancer Ka-Wei Tang, Yarong Tian, Guojiang Xie, Alan Bäckerholm, Isak Holmqvist, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3911441/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Epstein-Barr virus (EBV) is the causative agent for multiple neoplastic diseases of epithelial and lymphocytic origin1-3. The heterogeneity of the viral elements expressed and the mechanisms by which these coding and non-coding genes maintain cancer cell properties in vivo remain elusive4,5. Here we conducted a multi-modal transcriptomic analysis of EBV-associated neoplasms and identified that the ubiquitously expressed RPMS1 non-coding RNAs support cancer cell properties by disruption of the interferon response. Our map of EBV expression shows a variable, but pervasive expression of BNLF2 discerned from the overlapping LMP1 RNA in bulk sequencing data. Using long-read single-molecule sequencing, we identified three new viral elements within the RPMS1 gene. Furthermore, single-cell sequencing datasets allowed for the separation of cancer cells and healthy cells from the same tissue biopsy and the characterization of a microenvironment containing interferon gamma excreted by EBV-stimulated T-lymphocytes. In comparison with healthy epithelium, EBV-transformed cancer cells exhibited increased proliferation and inhibited immune response induced by the RPMS1-encoded microRNAs. Our atlas of EBV expression shows that the EBV-transformed cancer cells express high levels of non-coding RNAs originating from RPMS1 and that the oncogenic properties are maintained by RPMS1 microRNAs. Through bioinformatic disentanglement of single cells from cancer tissues we identified a positive feedback loop where EBV-activated immune cells stimulate cancer cells to proliferate, which in turn undergo viral reactivation and trigger an immune response. Biological sciences/Cancer/Tumour virus infections Biological sciences/Cancer/Cancer genomics Biological sciences/Computational biology and bioinformatics/Computational models Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataTable1Listofdatasets.xlsx Extended Data Table 1 ExtendedDataTable2PolyAcontainingreads.xlsx Extended Data Table 2 ExtendedDataTable3Housekeepinggenestpmvalues.xlsx Extended Data Table 3 ExtendedDataTable4EBVgenestpmvalues.xlsx Extended Data Table 4 ExtendedDataTable5ListofcellnumbersinscRNASeq.xlsx Extended Data Table 5 ExtendedDataTable6CpmvaluesofEBVgenesinscRNASeqasbulk.xlsx Extended Data Table 6 ExtendedDataTable7DifferentialgeneexpressionforNPC.xlsx Extended Data Table 7 ExtendedDataTable8Primersandoligonucleotides.xlsx Extended Data Table 8 ExtendeddataFigure1EBVRNA.pdf Extended data Figure 1 ExtendeddataFigure2RNApeakanalysis.pdf Extended data Figure 2 ExtendeddataFigure3RPMS1copynumber.pdf Extended data Figure 3 ExtendedDataFigure4EBVexpression.pdf Extended Data Figure 4 ExtendedDataFigure5BulkGSEA.pdf Extended Data Figure 5 ExtendedDataFigure6Stromalcellcomposition.pdf Extended Data Figure 6 ExtendedDataFigure7Singlecellexpressionmerged.pdf Extended Data Figure 7 ExtendedDataFigure8SinglecellGSEA.pdf Extended Data Figure 8 ExtendedDataFigure9EBVmicroRNAexpression.pdf Extended Data Figure 9 ExtendedDataFigure10Expressionofcytokinesandifnpathway.pdf Extended Data Figure 10 SupplementaryInformation.pdf Supplementary Information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3911441","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":270262203,"identity":"115ed0fe-0cdc-415f-8563-3e44503a7906","order_by":0,"name":"Ka-Wei Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACCRCRAGYyHyBJiwGQZEsgQQsDWAuPAXFaJNvPGD54wPBH3px/zefXPAx1cgS1SPPkGBsAHWa4c8bbbdY8DIeNCWqRY8jdJgHUwrjhxtltxjwMBxIbCGrhf7v9B1CL/YYbZ54BtdTVE9QiLZG7DRRiiRvO9zA/5mFgTiDoMMkZ7z9LJBgYJ2+4wWbGOMfgsCFBWyTOpyV+/FEhZ7vh/OHHH95U1MkTtAUCQDEikcAmwUBk1EAB/wHmDyRpGAWjYBSMghEDALXxOr5/yXehAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1189-5092","institution":"University of Gothenburg","correspondingAuthor":true,"prefix":"","firstName":"Ka-Wei","middleName":"","lastName":"Tang","suffix":""},{"id":270262204,"identity":"fa0d9104-1aac-4715-a152-804b5a03b448","order_by":1,"name":"Yarong Tian","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Yarong","middleName":"","lastName":"Tian","suffix":""},{"id":270262205,"identity":"49c3cec7-8c55-472a-a137-4ce5a8dbf32b","order_by":2,"name":"Guojiang Xie","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Guojiang","middleName":"","lastName":"Xie","suffix":""},{"id":270262206,"identity":"9ab2c5cc-2ccf-406e-9f52-47095fc72011","order_by":3,"name":"Alan Bäckerholm","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Alan","middleName":"","lastName":"Bäckerholm","suffix":""},{"id":270262207,"identity":"23b2b777-cf1d-48ae-a1c4-8726c076fa0b","order_by":4,"name":"Isak Holmqvist","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Isak","middleName":"","lastName":"Holmqvist","suffix":""},{"id":270262208,"identity":"4328efc5-a286-49d4-8aaa-3d9ed6f9cefc","order_by":5,"name":"Diana Vracar","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Diana","middleName":"","lastName":"Vracar","suffix":""},{"id":270262209,"identity":"09596718-0171-4a08-8305-ab5fe8b800db","order_by":6,"name":"Jianqiong Lin","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Jianqiong","middleName":"","lastName":"Lin","suffix":""},{"id":270262210,"identity":"35245f5c-13f0-4bc4-a1f3-eceb3ba5272c","order_by":7,"name":"Jonas Carlsten","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Jonas","middleName":"","lastName":"Carlsten","suffix":""},{"id":270262211,"identity":"8cc5e105-fe7b-48f9-85d4-575dd0fadb29","order_by":8,"name":"Sanna Abrahamsson","email":"","orcid":"","institution":"University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Sanna","middleName":"","lastName":"Abrahamsson","suffix":""},{"id":270262212,"identity":"d23d4632-1d84-4ecb-99dd-e24f2dd9c6c1","order_by":9,"name":"Zhentao Liu","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Zhentao","middleName":"","lastName":"Liu","suffix":""},{"id":270262213,"identity":"a7a664c3-eb81-447f-9f0a-78aa7a1a57b5","order_by":10,"name":"Yufei Huang","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Yufei","middleName":"","lastName":"Huang","suffix":""},{"id":270262214,"identity":"938f9c69-96d9-420b-9f36-13cb5f7927f0","order_by":11,"name":"Kathy Shair","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Kathy","middleName":"","lastName":"Shair","suffix":""}],"badges":[],"createdAt":"2024-01-30 18:30:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3911441/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3911441/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50531689,"identity":"ab706726-8ca4-4894-b80d-b2e9dd2f07f2","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195728,"visible":true,"origin":"","legend":"\u003cp\u003eDetection and characterization of EBV gene expression \u0026nbsp;a, RNA-Seq data from four types of neoplasms and two types of EBV cell lines were analyzed \u0026nbsp;for their EBV-content. The viral RNA from datasets containing more than 10 ppm EBV RNA \u0026nbsp;were plotted against the EBV genome. Further sub-analyses were conducted on the EBV positive tumors (box). b, Fraction of datasets with high (\u0026gt;10 ppm, red) or low (2-10 ppm, grey) \u0026nbsp;EBV content. Numbers in parentheses indicate the number of patients for each category and \u0026nbsp;percentages represent the fraction of datasets with high EBV content. c, Average coverage of \u0026nbsp;EBV RNA in the four types of neoplasms. Numbers on the x-axis correspond to the EBV \u0026nbsp;genome position. Alignment to the BamHI digestion map is shown in the bottom. d, \u0026nbsp;Magnification of the RNA reads within the BamHI-A/I region. Rightward RPMS1 exons (in \u0026nbsp;roman numerals) and leftward genes depicted in the bottom. Unique regions without overlap \u0026nbsp;with other genes are shown in dotted boxes. e, RNA coverage of transcription start site of \u0026nbsp;RPMS1 in NPC1. (F) Splice-junction reads detected within the BamHI-A/I region in NPC1. \u0026nbsp;Lines between the constitutive exons of RPMS1 are shown in bold. g, poly-A containing reads \u0026nbsp;detected at the 3’-end of RPMS1 in NPC1. Reference sequence and poly-A signal shown in the \u0026nbsp;bottom. ppm, parts per million reads; NPC, nasopharyngeal carcinoma; GAC, gastric \u0026nbsp;adenocarcinoma; eBL, endemic Burkitt’s lymphoma; sBL, sporadic Burkitt’s lymphoma; ECL, \u0026nbsp;EBV associated tumor derived cell line; LCL, lymphoblastic cell line.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/aff4cbebb468f6ff324213f5.png"},{"id":50532008,"identity":"369a4ab2-3c31-4daa-be2f-79d59f6b1030","added_by":"auto","created_at":"2024-02-02 03:01:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74901,"visible":true,"origin":"","legend":"\u003cp\u003eEBV gene expression in bulk RNA sequencing data \u0026nbsp;a-f, Heatmap depicting tpm-values of four gene regions RPMS1/BAREs, LMP1/BNLF2, \u0026nbsp;LMP2A/B and EBNA1 in NPC, GAC, eBL, sBL, ECL and LCL. Three datasets containing \u0026nbsp;additional EBV genes expressed at more than 5 tpm are marked with an asterisk. These datasets contained EBV genes indicative of lytic replication within the neoplasm. g-i, tpm-distribution \u0026nbsp;of genes with overlapping 3’. The fused RPMS1/BAREs 3’ end, compared with the unique \u0026nbsp;regions for RPMS1, BARE1, BARE2 and BARE3, the fused LMP1/BNLF2 3’ end, compared \u0026nbsp;with the unique regions for LMP1 and the fused LMP2A/2B 3’ end, compared with the unique \u0026nbsp;regions for LMP2A, LMP2B and BNRF1 in NPC, GAC, eBL and sBL. j, Generalized pathway \u0026nbsp;perturbations in principal components for respective cancer type. Principal components \u0026nbsp;correlating with highest EBV-status separation are marked with bold. tpm, transcripts per \u0026nbsp;million reads; NPC, nasopharyngeal carcinoma; GAC, gastric adenocarcinoma; eBL, endemic \u0026nbsp;Burkitt’s lymphoma; sBL, sporadic Burkitt’s lymphoma; PC, principal component.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/4083b103f79d14a3ef3001c6.png"},{"id":50531691,"identity":"b5e68ff2-ef04-42f7-98ea-5b3396171962","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228794,"visible":true,"origin":"","legend":"\u003cp\u003eNasopharyngeal carcinoma single-cell RNA sequencing datasets \u0026nbsp;a, Proportion of epithelial and stromal cells in the four studies. Striped portion of the bar shows \u0026nbsp;the fraction of EBV-expressing cells in each category. b, Classification of each sample \u0026nbsp;according to their origin and EBV-status according to EBER in situ hybridization, antibody \u0026nbsp;detection or UMI in the single-cell data. Samples showing concordant results from two analyses \u0026nbsp;are shown in green, discordant in purple and unknown in grey. c, EBV expression in datasets \u0026nbsp;containing more than 100 epithelial cells (green). The proportion of epithelial cells from each \u0026nbsp;tumor expressing fused EBV gene is shown in the respective column (orange). Genes expressed \u0026nbsp;over 2 cpm were included. d, Inferred chromosomal RNA expression throughout the genome \u0026nbsp;in T-cells (upper panel) and epithelial cells (lower panel), position on x-axis correspond to \u0026nbsp;position in respective chromosome. Epithelial cells divided by unsupervised hierarchical \u0026nbsp;clustering. Areas in red depicts inferred gains and blue loss of chromosomal segment. e-g, \u0026nbsp;Epithelial cells extracted from NPC1 were reclustered in UMAP. Cancer cells classified \u0026nbsp;according to EBV expression (blue) showed a lower sensitivity compared to cancer (red) and \u0026nbsp;healthy cell classification based on inference of chromosomal copy number variation. NPC, nasopharyngeal carcinoma; EBER, Epstein–Barr virus–encoded small RNAs; UMI, unique \u0026nbsp;molecular identifier; cpm, counts per million reads; UMAP, uniform manifold approximation \u0026nbsp;and projection.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/67eb81c35675da95037e3967.png"},{"id":50531698,"identity":"dc5fcc0d-d9a2-481e-84df-f98c78e6bcbd","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":922204,"visible":true,"origin":"","legend":"\u003cp\u003eEBV-induced host perturbations \u0026nbsp;a, Changes in biological pathways between cancer cells and healthy cells from the same \u0026nbsp;patients. Hallmarks enriched in all four EBV-positive NPCs studies are listed. The same \u0026nbsp;pathways for EBV-negative tumors and healthy controls in which basal cells were compared to \u0026nbsp;differentiated cells in non-tumor samples are shown alongside. Absence of bar indicates no \u0026nbsp;significant differences. Induced changes in the EBV-negative nasopharyngeal carcinoma cell \u0026nbsp;line HK1 transfected with RPMS1 miR-BARTs (circles) and Namalwa cells treated with \u0026nbsp;doxycycline (triangles) to upregulate RPMS1 gene are shown in the right column. b, Genes \u0026nbsp;perturbed in the same direction in multiple tumors. Enriched genes are marked with green \u0026nbsp;(upregulated) and magenta (downregulated). The x-axis shows the negative log10 of the false \u0026nbsp;discovery rate q-value (FDRq) for pathways in respective category. c, Immune response genes \u0026nbsp;downregulated in tumor cells categorized according to pathway. Genes in italics are also part \u0026nbsp;of the NF-κB pathway. d, Depiction of factors involved in viral perturbations in NPC epithelial \u0026nbsp;cells. MITSP, mitotic spindle; UVD, UV responsed down; IFNA, interferon alpha response; \u0026nbsp;IFNG, interferon gamma response; OXPH, oxidative phosphorylation; IFN, interferon.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/16300b17e1241ace2b54cdf2.png"},{"id":50659181,"identity":"816a3877-ee0e-4179-ae2f-4c88f1ac2c53","added_by":"auto","created_at":"2024-02-05 11:16:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":761246,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1_covered_db821ea9-65a8-4ab3-a745-8c348d33fba8.pdf"},{"id":50531687,"identity":"ba800bd4-af2c-4420-8ee8-fae9ddff8073","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":91764,"visible":true,"origin":"","legend":"Extended Data Table 1","description":"","filename":"ExtendedDataTable1Listofdatasets.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/cad8887efd31e021f6ad03ea.xlsx"},{"id":50531696,"identity":"aeaf1f79-ca68-41f2-830c-e68187030947","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":839031,"visible":true,"origin":"","legend":"Extended Data Table 2","description":"","filename":"ExtendedDataTable2PolyAcontainingreads.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/79694accc6418411df68d3bc.xlsx"},{"id":50531688,"identity":"10baac13-5468-40ea-8760-3f3a8f4016ba","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":26956,"visible":true,"origin":"","legend":"Extended Data Table 3","description":"","filename":"ExtendedDataTable3Housekeepinggenestpmvalues.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/55431adc10057ff0efe35980.xlsx"},{"id":50532009,"identity":"d0510747-2cbd-4c61-847f-e44087058cd5","added_by":"auto","created_at":"2024-02-02 03:01:31","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":200586,"visible":true,"origin":"","legend":"Extended Data Table 4","description":"","filename":"ExtendedDataTable4EBVgenestpmvalues.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/e308930d1f8be53cbe3e1477.xlsx"},{"id":50532007,"identity":"4788133f-c898-4a26-aba1-42509f7454b4","added_by":"auto","created_at":"2024-02-02 03:01:31","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":23863,"visible":true,"origin":"","legend":"Extended Data Table 5","description":"","filename":"ExtendedDataTable5ListofcellnumbersinscRNASeq.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/386908f7731bfaad0367b443.xlsx"},{"id":50531695,"identity":"cedbcbff-ef43-45bc-a15c-35503739321e","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":23124,"visible":true,"origin":"","legend":"Extended Data Table 6","description":"","filename":"ExtendedDataTable6CpmvaluesofEBVgenesinscRNASeqasbulk.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/9db21490ec557ce3ec53bbd7.xlsx"},{"id":50532010,"identity":"234a41f9-d8ab-4ce6-9a80-6a32a0f966ee","added_by":"auto","created_at":"2024-02-02 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02:53:31","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":10706797,"visible":true,"origin":"","legend":"\u003cp\u003eExtended data Figure 3\u003c/p\u003e","description":"","filename":"ExtendeddataFigure3RPMS1copynumber.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/c60041b758a95f212eeeada3.pdf"},{"id":50531703,"identity":"5963fbc0-8af4-49c2-ad2a-851616042360","added_by":"auto","created_at":"2024-02-02 02:53:31","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":776580,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Figure 4\u003c/p\u003e","description":"","filename":"ExtendedDataFigure4EBVexpression.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/2ba955b6c1486b230e892c94.pdf"},{"id":50531701,"identity":"d3cf733b-9e1e-4bef-9314-9f29f914c919","added_by":"auto","created_at":"2024-02-02 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02:53:32","extension":"pdf","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":46574483,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3911441/v1/daa2265c897cf7cd3148aa92.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Landscape of Epstein-Barr virus gene expression and perturbations in cancer","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3911441/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3911441/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Epstein-Barr virus (EBV) is the causative agent for multiple neoplastic diseases of epithelial and lymphocytic origin1-3. The heterogeneity of the viral elements expressed and the mechanisms by which these coding and non-coding genes maintain cancer cell properties in vivo remain elusive4,5. Here we conducted a multi-modal transcriptomic analysis of EBV-associated neoplasms and identified that the ubiquitously expressed RPMS1 non-coding RNAs support cancer cell properties by disruption of the interferon response. Our map of EBV expression shows a variable, but pervasive expression of BNLF2 discerned from the overlapping LMP1 RNA in bulk sequencing data. Using long-read single-molecule sequencing, we identified three new viral elements within the RPMS1 gene. Furthermore, single-cell sequencing datasets allowed for the separation of cancer cells and healthy cells from the same tissue biopsy and the characterization of a microenvironment containing interferon gamma excreted by EBV-stimulated T-lymphocytes. In comparison with healthy epithelium, EBV-transformed cancer cells exhibited increased proliferation and inhibited immune response induced by the RPMS1-encoded microRNAs. Our atlas of EBV expression shows that the EBV-transformed cancer cells express high levels of non-coding RNAs originating from RPMS1 and that the oncogenic properties are maintained by RPMS1 microRNAs. Through bioinformatic disentanglement of single cells from cancer tissues we identified a positive feedback loop where EBV-activated immune cells stimulate cancer cells to proliferate, which in turn undergo viral reactivation and trigger an immune response.","manuscriptTitle":"Landscape of Epstein-Barr virus gene expression and perturbations in cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-02 02:53:26","doi":"10.21203/rs.3.rs-3911441/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90051b9a-629e-46a4-b5bc-0da79b0621f1","owner":[],"postedDate":"February 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28485563,"name":"Biological sciences/Cancer/Tumour virus infections"},{"id":28485564,"name":"Biological sciences/Cancer/Cancer genomics"},{"id":28485565,"name":"Biological sciences/Computational biology and bioinformatics/Computational models"}],"tags":[],"updatedAt":"2024-02-05T14:33:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-02 02:53:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3911441","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3911441","identity":"rs-3911441","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}