Epstein–Barr virus (EBV) infection is highly prevalent in humans and is implicated in various diseases, including cancer1,2. Chronic active EBV infection (CAEBV) is an intractable disease classified as a lymphoproliferative disorder in the 2016 World Health Organization lymphoma classification1,2. CAEBV is characterized by EBV-infected T/natural killer (NK) cells and recurrent/persistent infectious mononucleosis-like symptoms3. Here, we show that CAEBV originates from an EBV-infected lymphoid progenitor that acquires DDX3X and other mutations, causing clonal evolution comprising multiple cell lineages. Conspicuously, the EBV genome in CAEBV patients harboured frequent intragenic deletions (27/77) that were also common in various EBV-associated neoplastic disorders (28/61), including extranodal NK/T-cell lymphoma and EBV-positive diffuse large B-cell lymphoma, but were not detected in infectious mononucleosis or post-transplant lymphoproliferative disorders (0/47), which suggests a unique role of these mutations in neoplastic proliferation of EBV-infected cells. These deletions frequently affected BamHI A rightward transcript microRNA clusters (31 cases) and several genes that are essential for producing viral particles (20 cases). The deletions observed in our study are thought to reactivate the lytic cycle by upregulating the expression of two immediate early genes, BZLF1 and BRLF14,5,6,7, while averting viral production and subsequent cell lysis. In fact, the deletion of one of the essential genes, BALF5, resulted in upregulation of the lytic cycle and the promotion of lymphomagenesis in a xenograft model. Our findings highlight a pathogenic link between intragenic EBV deletions and EBV-associated neoplastic proliferations.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Custom codes used for the simulation of EBV deletions are available from GitHub repository (https://github.com/yusukeokuno/ebvsimulation) with no restriction on access.
Sequence data has been deposited at the European Genome-phenome Archive (EGA), which is hosted by the EBI and the CRG, under accession number EGAS00001003159. Further information about EGA can be found on https://ega-archive.org and ref. 49. We also registered assembled sequences of the EBV genomes in DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/) under BioProject accession number PRJDB7503. The sequences can be individually via getentry (http://getentry.ddbj.nig.ac.jp/) using the accession numbers AP019012–AP019188.
Cohen, J. I., Kimura, H., Nakamura, S., Ko, Y. H. & Jaffe, E. S. Epstein–Barr virus-associated lymphoproliferative disease in non-immunocompromised hosts: a status report and summary of an international meeting, 8-9 September 2008. Ann. Oncol. 20, 1472–1482 (2009).
Swerdlow, S. H. et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 127, 2375–2390 (2016).
Quintanilla-Martinez, L., Ko, Y., Kimura, H. & Jaffe, E. in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues Revised 4th edn (Swerdlow, S. H. et al.) 355–362 (IARC, Lyon, 2017).
Iizasa, H. et al. Editing of Epstein–Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency. J. Biol. Chem. 285, 33358–33370 (2010).
Lin, X. et al. The Epstein–Barr virus BART miRNA cluster of the M81 strain modulates multiple functions in primary B cells. PLoS Pathog. 11, e1005344 (2015).
Qiu, J. & Thorley-Lawson, D. A. EBV microRNA BART 18-5p targets MAP3K2 to facilitate persistence in vivo by inhibiting viral replication in B cells. Proc. Natl Acad. Sci. USA 111, 11157–11162 (2014).
Jung, Y. J., Choi, H., Kim, H. & Lee, S. K. MicroRNA miR-BART20-5p stabilizes Epstein–Barr virus latency by directly targeting BZLF1 and BRLF1. J. Virol. 88, 9027–9037 (2014).
Longnecker, R., Kieff, E. & Cohen, J. I. in Fields Virology 6th edn, Vol. 1 (eds Knipe, D. M. et al.) 1898–1959 (Lippincott Williams & Willkins, Philadelphia, 2013).
Kimura, H. et al. EBV-associated T/NK-cell lymphoproliferative diseases in nonimmunocompromised hosts: prospective analysis of 108 cases. Blood 119, 673–686 (2012).
Ohga, S. et al. Clonal origin of Epstein–Barr virus (EBV)-infected T/NK-cell subpopulations in EBV-positive T/NK-cell lymphoproliferative disorders of childhood. J. Clin. Virol. 51, 31–37 (2011).
Ruiz-Perez, V. L. & Goodship, J. A. Ellis–van Creveld syndrome and Weyers acrodental dysostosis are caused by cilia-mediated diminished response to hedgehog ligands. Am. J. Med. Genet. C 151C, 341–351 (2009).
Jones, D. T. et al. Dissecting the genomic complexity underlying medulloblastoma. Nature 488, 100–105 (2012).
Jiang, L. et al. Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma. Nat. Genet. 47, 1061–1066 (2015).
Ichigi, Y. et al. Generation of cells with morphological and antigenic properties of microglia from cloned EBV-transformed lymphoid progenitor cells derived from human fetal liver. Cell. Immunol. 149, 193–207 (1993).
Paterson, R. L. et al. Model of Epstein–Barr virus infection of human thymocytes: expression of viral genome and impact on cellular receptor expression in the T-lymphoblastic cell line, HPB-ALL. Blood 85, 456–464 (1995).
Depledge, D. P. et al. Specific capture and whole-genome sequencing of viruses from clinical samples. PLoS ONE 6, e27805 (2011).
Lee, N., Moss, W. N., Yario, T. A. & Steitz, J. A. EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell 160, 607–618 (2015).
Palser, A. L. et al. Genome diversity of Epstein–Barr virus from multiple tumor types and normal infection. J. Virol. 89, 5222–5237 (2015).
Klinke, O., Feederle, R. & Delecluse, H. J. Genetics of Epstein–Barr virus microRNAs. Semin. Cancer Biol. 26, 52–59 (2014).
Narita, Y. et al. Pin1 interacts with the Epstein–Barr virus DNA polymerase catalytic subunit and regulates viral DNA replication. J. Virol. 87, 2120–2127 (2013).
Djavadian, R., Chiu, Y. F. & Johannsen, E. An Epstein–Barr virus-encoded protein complex requires an origin of lytic replication in cis to mediate late gene transcription. PLoS Pathog. 12, e1005718 (2016).
Qiu, J., Smith, P., Leahy, L. & Thorley-Lawson, D. A. The Epstein–Barr virus encoded BART miRNAs potentiate tumor growth in vivo. PLoS Pathog. 11, e1004561 (2015).
Yamamoto, T. et al. Epstein–Barr virus reactivation is induced, but abortive, in cutaneous lesions of systemic hydroa vacciniforme and hypersensitivity to mosquito bites. J. Dermatol. Sci. 82, 153–159 (2016).
Cohen, M. et al. Epstein–Barr virus lytic cycle involvement in diffuse large B cell lymphoma. Hematol. Oncol. 36, 98–103 (2018).
Ma, S. D. et al. A new model of Epstein–Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas. J. Virol. 85, 165–177 (2011).
Wu, C. C. et al. Epstein–Barr virus DNase (BGLF5) induces genomic instability in human epithelial cells. Nucleic Acids Res. 38, 1932–1949 (2010).
Chiu, S. H. et al. Epstein–Barr virus BALF3 mediates genomic instability and progressive malignancy in nasopharyngeal carcinoma. Oncotarget 5, 8583–8601 (2014).
Zuo, J. et al. Epstein–Barr virus evades CD4+ T cell responses in lytic cycle through BZLF1-mediated downregulation of CD74 and the cooperation of vBcl-2. PLoS Pathog. 7, e1002455 (2011).
Xu, Z. G. et al. The latency pattern of Epstein–Barr virus infection and viral IL-10 expression in cutaneous natural killer/T-cell lymphomas. Br. J. Cancer 84, 920–925 (2001).
Ma, S. D. et al. An Epstein–Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model. J. Virol. 86, 7976–7987 (2012).
Okano, M. et al. Proposed guidelines for diagnosing chronic active Epstein–Barr virus infection. Am. J. Hematol. 80, 64–69 (2005).
Koboldt, D. C. et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 22, 568–576 (2012).
Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).
Richards, S. et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–424 (2015).
Muramatsu, H. et al. Clinical utility of next-generation sequencing for inherited bone marrow failure syndromes. Genet. Med. 19, 796–802 (2017).
Kent, W. J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).
Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Feng, J. et al. GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data. Bioinformatics 28, 2782–2788 (2012).
Djavadian, R., Hayes, M. & Johannsen, E. CAGE-seq analysis of Epstein–Barr virus lytic gene transcription: 3 kinetic classes from 2 mechanisms. PLoS Pathog. 4, e1007114 (2018).
Watanabe, T. et al. The Epstein–Barr virus BDLF4 gene is required for efficient expression of viral late lytic genes. J. Virol. 89, 10120–10124 (2015).
Linnerbauer, S. et al. Virus and autoantigen-specific CD+ T cells are key effectors in a SCID mouse model of EBV-associated post-transplant lymphoproliferative disorders. PLoS Pathog. 10, e1004068 (2014).
Xiang, Z. et al. Targeted activation of human Vɣ9Vδ2-T cells controls Epstein–Barr virus-induced B cell lymphoproliferative disease. Cancer Cell 26, 565–576 (2014).
Kimura, H. et al. Quantitative analysis of Epstein–Barr virus load by using a real-time PCR assay. J. Clin. Microbiol. 37, 132–136 (1999).
Ando, S. et al. Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein–Barr virus-associated T and natural killer cell lymphoma cells. Oncotarget 7, 76793–76805 (2016).
Katsumura, K. R., Maruo, S., Wu, Y., Kanda, T. & Takada, K. Quantitative evaluation of the role of Epstein–Barr virus immediate-early protein BZLF1 in B-cell transformation. J. Gen. Virol. 90, 2331–2341 (2009).
Shimizu, N., Yoshiyama, H. & Takada, K. Clonal propagation of Epstein–Barr virus (EBV) recombinants in EBV-negative Akata cells. J. Virol. 70, 7260–7263 (1996).
Kanda, Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 48, 452–458 (2013).
Lappalainen, I. et al. The European Genome-phenome Archive of human data consented for biomedical research. Nat. Genet. 47, 692–695 (2015).
The authors would like to thank all of the subjects and families for participating in this study. The authors would like to thank N. Yoshida, M. Okada, H. Moriuchi, K. Yamamoto, S. Kamimura, Y. Horikoshi and T. Kinoshita for providing samples. The authors would also like to thank Y. Miura, Y. Imanishi, F. Ando, S. Kumagai, T. Kunogi, H. Matsuda, H.M.A. Masud and H. Namizaki for their valuable assistance. The authors acknowledge M. Nakatochi for valuable comments. The authors acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine for technical support of cell sorting and next-generation sequencing. The authors acknowledge the Human Genome Center, the Institute of Medical Science, the University of Tokyo (http://sc.hgc.jp/shirokane.html) for providing super-computing resources. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to H.K. (grant nos 25293109 and 17H04081), grants from Japan Agency for Medical Research and Development to H.K. (grant no. JP17ek0109286) and T.M. (grant no. JP17fm0208016), a grant from the Hori Sciences and Arts Foundation and from the Ministry of Health, Labor, and Welfare of Japan to H.K. (grant no. H29-Nanchi-016).
S.Kojima was supported by a grant from Sanofi K.K. All other authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Okuno, Y., Murata, T., Sato, Y. et al. Defective Epstein–Barr virus in chronic active infection and haematological malignancy. Nat Microbiol 4, 404–413 (2019). https://doi.org/10.1038/s41564-018-0334-0
Virology Journal (2020)
Nature Reviews Microbiology (2019)