Epstein–Barr virus ncRNA from a nasopharyngeal carcinoma induces an inflammatory response that promotes virus production

Abstract

The Epstein–Barr virus M81 strain, isolated from a nasopharyngeal carcinoma, induces potent spontaneous virus production in infected B cells. We found that the M81 non-coding Epstein–Barr-encoded RNA EBER2, which carries polymorphisms that are mainly restricted to viruses found in endemic nasopharyngeal carcinomas, markedly stimulated this process. M81 EBER2 increased CXCL8 expression, and this chemokine enhanced spontaneous lytic replication levels in M81-infected B cells. Both events resulted from the endocytosis of extracellular vesicles containing EBER2 that were generated by neighbouring M81-infected B cells, thereby generating a paracrine loop. These effects were strictly dependent on a functional Toll-like receptor 7 (TLR7), a sensor of single-stranded RNA located in the endosome of these cells. These unique properties of M81 EBER2 could be ascribed to its unusually high expression level and to the ability of its single-stranded region to activate TLR7; both of these properties were dependent on M81-specific polymorphisms. Thus, M81 induced chronic inflammation in its target cells and this resulted in increased virus production. These observations provide a mechanistic molecular link between M81 virus replication—a central viral function and a cancer risk factor—and the production of a chemokine involved in inflammation and carcinogenesis.

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Fig. 1: The M81 EBERs potentiate spontaneous lytic replication in infected cells.
Fig. 2: The M81 EBERs control lytic replication in vivo.
Fig. 3: EBER expression levels vary after infection with different EBV strains.
Fig. 4: M81 EBERs modulate lytic replication by amplifying the expression of CXCL8.
Fig. 5: Incubation of LCLs with EV from EBER-positive cells increase CXCL8 expression and activate lytic replication.
Fig. 6: EBERs activate TLR7.

Data availability

The data that support the findings of this study are available within the article and its Supplementary Information files, or from the corresponding author upon request. The raw microarray data that support the findings of this study have been deposited at ArrayExpress, with accession no. E-MTAB-8102.

References

  1. 1.

    Rickinson, A. B. & Kieff, E. in Fields Virology 5th edn (eds Knipe, D. M. & Howley, P. M.) 2655–2700 (Lippincott: Williams & Wilkins, 2007).

  2. 2.

    Tsai, M. H. et al. Spontaneous lytic replication and epitheliotropism define an Epstein–Barr virus strain found in carcinomas. Cell Rep. 5, 458–470 (2013).

    CAS  PubMed  Google Scholar 

  3. 3.

    Young, L. S., Yap, L. F. & Murray, P. G. Epstein–Barr virus: more than 50 years old and still providing surprises. Nat. Rev. Cancer 16, 789–802 (2016).

    CAS  PubMed  Google Scholar 

  4. 4.

    Kieff, E. & Rickinson, A. B. in Fields Virology 5th edn (eds Knipe, D. M. & Howley, P. M.) 2603–2654 (Lippincott: Williams & Wilkins, 2007).

  5. 5.

    Klinke, O., Feederle, R. & Delecluse, H. J. Genetics of Epstein–Barr virus microRNAs. Semin. Cancer Biol. 26, 52–59 (2014).

    CAS  PubMed  Google Scholar 

  6. 6.

    Lerner, M. R., Andrews, N. C., Miller, G. & Steitz, J. A. Two small RNAs encoded by Epstein–Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc. Natl Acad. Sci. USA 78, 805–809 (1981).

    CAS  PubMed  Google Scholar 

  7. 7.

    Shen, Z. C. et al. High prevalence of the EBER variant EB-8m in endemic nasopharyngeal carcinomas. PLoS ONE 10, e0121420 (2015).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Correia, S. et al. Natural variation of Epstein–Barr virus genes, proteins, and primary microRNA. J. Virol. 91, e00375-17 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Palser, A. L. et al. Genome diversity of Epstein–Barr virus from multiple tumor types and normal infection. J. Virol. 89, 5222–5237 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Hui, K. F. et al. High risk Epstein–Barr virus variants characterized by distinct polymorphisms in the EBER locus are strongly associated with nasopharyngeal carcinoma. Int. J. Cancer 144, 3031–3042 (2019).

    CAS  PubMed  Google Scholar 

  11. 11.

    Correia, S. et al. Sequence variation of Epstein–Barr virus: viral types, geography, codon usage, and diseases. J. Virol. 92, e01132-18 (2018).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Pimienta, G. et al. Proteomics and transcriptomics of BJAB cells expressing the Epstein–Barr virus noncoding RNAs EBER1 and EBER2. PLoS ONE 10, e0124638 (2015).

    Google Scholar 

  13. 13.

    Komano, J., Maruo, S., Kurozumi, K., Oda, T. & Takada, K. Oncogenic role of Epstein–Barr virus-encoded RNAs in Burkitt’s lymphoma cell line Akata. J. Virol. 73, 9827–9831 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Tsai, M. H. et al. The biological properties of different Epstein–Barr virus strains explain their association with various types of cancers. Oncotarget 8, 10238–10254 (2017).

    PubMed  Google Scholar 

  15. 15.

    Howe, J. G. & Shu, M. D. Epstein–Barr virus small RNA (EBER) genes: unique transcription units that combine RNA polymerase II and III promoter elements. Cell 57, 825–834 (1989).

    CAS  PubMed  Google Scholar 

  16. 16.

    Samanta, M., Iwakiri, D. & Takada, K. Epstein–Barr virus-encoded small RNA induces IL-10 through RIG-I-mediated IRF-3 signaling. Oncogene 27, 4150–4160 (2008).

    CAS  PubMed  Google Scholar 

  17. 17.

    Yang, L., Aozasa, K., Oshimi, K. & Takada, K. Epstein–Barr virus (EBV)-encoded RNA promotes growth of EBV-infected T cells through interleukin-9 induction. Cancer Res. 64, 5332–5337 (2004).

    CAS  PubMed  Google Scholar 

  18. 18.

    Iwakiri, D., Sheen, T. S., Chen, J. Y., Huang, D. P. & Takada, K. Epstein–Barr virus-encoded small RNA induces insulin-like growth factor 1 and supports growth of nasopharyngeal carcinoma-derived cell lines. Oncogene 24, 1767–1773 (2005).

    CAS  PubMed  Google Scholar 

  19. 19.

    Howe, J. G. & Steitz, J. A. Localization of Epstein–Barr virus-encoded small RNAs by in situ hybridization. Proc. Natl Acad Sci. USA 83, 9006–9010 (1986).

    CAS  PubMed  Google Scholar 

  20. 20.

    Ahmed, W., Philip, P. S., Tariq, S. & Khan, G. Epstein–Barr virus-encoded small RNAs (EBERs) are present in fractions related to exosomes released by EBV-transformed cells. PLoS ONE 9, e99163 (2014).

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Ahmed, W., Tariq, S. & Khan, G. Tracking EBV-encoded RNAs (EBERs) from the nucleus to the excreted exosomes of B-lymphocytes. Sci. Rep. 8, 15438 (2018).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Mulcahy, L. A., Pink, R. C. & Carter, D. R. Routes and mechanisms of extracellular vesicle uptake. J. Extracell. Vesicles 3, 24641 (2014).

    Google Scholar 

  23. 23.

    Thery, C., Amigorena, S., Raposos, G. & Claton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 30, 3.22.1–3.22.29 (2006).

    Google Scholar 

  24. 24.

    Flanagan, J., Middeldorp, J. & Sculley, T. Localization of the Epstein–Barr virus protein LMP 1 to exosomes. J. Gen. Virol. 84, 1871–1879 (2003).

    CAS  PubMed  Google Scholar 

  25. 25.

    Iwakiri, D. et al. Epstein–Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from Toll-like receptor 3. J. Exp. Med. 206, 2091–2099 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Hanten, J. A. et al. Comparison of human B cell activation by TLR7 and TLR9 agonists. BMC Immunol. 9, 39 (2008).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Bencun, M. et al. Translational profiling of B cells infected with the Epstein–Barr virus reveals 5′ leader ribosome recruitment through upstream open reading frames. Nucleic Acids Res. 46, 2802–2819 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Barbalat, R., Ewald, S. E., Mouchess, M. L. & Barton, G. M. Nucleic acid recognition by the innate immune system. Annu. Rev. Immunol. 29, 185–214 (2011).

    CAS  PubMed  Google Scholar 

  29. 29.

    Lin, W., Yip, Y. L., Jia, L., Deng, W. & Zheng, H. Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma. Nat. Commun. 9, 4663 (2018).

    PubMed  PubMed Central  Google Scholar 

  30. 30.

    Tsang, C. M. et al. Epstein–Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization. Int. J. Cancer 127, 1570–1583 (2010).

    CAS  PubMed  Google Scholar 

  31. 31.

    Swaminathan, S., Tomkinson, B. & Kieff, E. Recombinant Epstein–Barr virus with small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro. Proc. Natl Acad. Sci. USA 88, 1546–1550 (1991).

    CAS  PubMed  Google Scholar 

  32. 32.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Gregorovic, G. et al. Epstein–Barr viruses (EBVs) deficient in EBV-encoded RNAs have higher levels of latent membrane protein 2 RNA expression in lymphoblastoid cell lines and efficiently establish persistent infections in humanized mice. J. Virol. 89, 11711–11714 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Martin, H. J., Lee, J. M., Walls, D. & Hayward, S. D. Manipulation of the toll-like receptor 7 signaling pathway by Epstein–Barr virus. J. Virol. 81, 9748–9758 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Heil, F. et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526–1529 (2004).

    CAS  PubMed  Google Scholar 

  36. 36.

    Forsbach, A. et al. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J. Immunol. 180, 3729–3738 (2008).

    CAS  PubMed  Google Scholar 

  37. 37.

    Baglio, S. R. et al. Sensing of latent EBV infection through exosomal transfer of 5′pppRNA. Proc. Natl Acad. Sci. USA 113, E587–E596 (2016).

    CAS  PubMed  Google Scholar 

  38. 38.

    Waugh, D. J. & Wilson, C. The interleukin-8 pathway in cancer. Clin. Cancer Res. 14, 6735–6741 (2008).

    CAS  PubMed  Google Scholar 

  39. 39.

    Thorley-Lawson, D. A. EBV persistence—introducing the virus. Curr. Top. Microbiol. Immunol. 390, 151–209 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Chien, Y. C. et al. Serologic markers of Epstein–Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. New Engl. J. Med. 345, 1877–1882 (2001).

    CAS  PubMed  Google Scholar 

  41. 41.

    Shumilov, A. et al. Epstein–Barr virus particles induce centrosome amplification and chromosomal instability. Nat. Commun. 8, 14257 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Tischer, B. K., Smith, G. A. & Osterrieder, N. En passant mutagenesis: a two step markerless red recombination system. Methods Mol. Biol. 634, 421–430 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Feederle, R., Bartlett, E. J. & Delecluse, H. J. Epstein–Barr virus genetics: talking about the BAC generation. Herpesviridae 1, 6 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    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).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Feederle, R. et al. Epstein–Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection. Int. J. Cancer 121, 588–594 (2007).

    CAS  PubMed  Google Scholar 

  46. 46.

    Feederle, R. et al. The Epstein–Barr virus lytic program is controlled by the co-operative functions of two transactivators. Embo J. 19, 3080–3089 (2000).

    CAS  Google Scholar 

  47. 47.

    Feederle, R. et al. Epstein–Barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes. J. Virol. 80, 9435–9443 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Hessing, M., van Schijndel, H. B., van Grunsven, W. M., Wolf, H. & Middeldorp, J. M. Purification and quantification of recombinant Epstein–Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells. J. Chromatogr. 599, 267–272 (1992).

    CAS  PubMed  Google Scholar 

  49. 49.

    Greijer, A. E. et al. Quantitative multi-target RNA profiling in Epstein–Barr virus infected tumor cells. J. Virol. Methods 241, 24–33 (2017).

    CAS  PubMed  Google Scholar 

  50. 50.

    Feederle, R. et al. A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog. 7, e1001294 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Van Deun, J. et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat. Methods 14, 228–232 (2017).

    PubMed  Google Scholar 

  52. 52.

    Thery, C. & Witwer, K. W. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Fitzgerald, W. & Freeman, M. L. A system of cytokines encapsulated in extracellular vesicles. Sci. Rep. 8, 8973 (2018).

    PubMed  PubMed Central  Google Scholar 

  54. 54.

    Barrat, F. J. et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J. Exp. Med. 202, 1131–1139 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the staff of the animal facility at DKFZ for their assistance with the animal experiments; the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Whole-Genome Expression Beadchips and related services; and J. M. Middeldorp (VU -University Medical Center, Amsterdam, Netherlands), for the OT6 antibody. This study was supported by German Cancer Research Center (F100), Institut National de la Santé et de la Recherche Médicale (U1074). Z.L. is supported by a stipend from the Chinese Scientific Council (CSC).

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H.-J.D. conceived and supervised the project. Z.L., M.-H.T. and H.-J.D. designed experiments. M.-H.T., F.B. and R.P. had significant input into the experimental design. Z.L. performed most of the experiments. R.P. conducted the EBER sequence alignment. Z.L., M.-H.T. and A.S. performed the mouse experiments. S.W.T. contributed the NPC43 and NP460hTert cell lines. Z.L., M.-H.T. and H.-J.D. analysed the data. Z.L. and H.-J.D. wrote the manuscript.

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Correspondence to Henri-Jacques Delecluse.

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Supplementary Information

Supplementary Figs. 1–27 and Supplementary Tables 1 and 3–6.

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Supplementary Table 2

Complete RNA microarray.

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Li, Z., Tsai, M., Shumilov, A. et al. Epstein–Barr virus ncRNA from a nasopharyngeal carcinoma induces an inflammatory response that promotes virus production. Nat Microbiol 4, 2475–2486 (2019). https://doi.org/10.1038/s41564-019-0546-y

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