1887

Abstract

Epstein–Barr virus (EBV; human herpesvirus 4) is an oncogenic herpesvirus implicated in the pathogenesis of several human malignancies. A number of recent studies indicate that EBV can manipulate the local microenvironment by excreting viral and cellular components in nanovesicles called exosomes. In this study, we investigated the impact of EBV-derived exosomes on apoptosis of recipient cells and the molecular pathway involved in this process. Exosomes from EBV-infected but not from non-infected cells induced apoptosis in a number of different cell types, including B-cells, T-cells and epithelial cells. However, this phenomenon was not universal and the Burkitt's lymphoma-derived B-cell line BJAB was found to be resistant to apoptosis. Exosomes from both type I and type III EBV latently infected cells induced apoptosis in a dose- and time-dependent manner. Moreover, cells exposed to EBV exosomes did not form colonies in soft agar assays. We further show that fluorescently labelled exosomes derived from EBV-infected cells are taken up by non-infected cells and induce apoptosis via the extrinsic pathway. Inhibition of caspase-3/7/8 blocks EBV exosome-mediated apoptosis. Furthermore, our data indicate that EBV exosomes trigger apoptosis through the Fas ligand (FasL)-mediated extrinsic pathway, as FasL was present in EBV exosomal fractions and anti-FasL antibodies could block EBV exosome-mediated apoptosis. Together, these data support the notion that EBV hijacks the exosome pathway to excrete viral and cellular components that can modulate its microenvironment.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000313
2015-12-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/12/3646.html?itemId=/content/journal/jgv/10.1099/jgv.0.000313&mimeType=html&fmt=ahah

References

  1. Abrahams V. M. , Straszewski S. L. , Kamsteeg M. , Hanczaruk B. , Schwartz P. E. , Rutherford T. J. , Mor G. . ( 2003;). Epithelial ovarian cancer cells secrete functional Fas ligand. Cancer Res 63: 5573–5581 [PubMed].
    [Google Scholar]
  2. Abusamra A. J. , Zhong Z. , Zheng X. , Li M. , Ichim T. E. , Chin J. L. , Min W. -P. . ( 2005;). Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood Cells Mol Dis 35: 169–173.[CrossRef]
    [Google Scholar]
  3. Ahmed W. , Philip P. S. , Tariq S. , Khan G. . ( 2014;). Epstein–Barr virus-encoded small RNAs (EBERs) are present in fractions related to exosomes released by EBV-transformed cells. PLoS One 9: e99163 [CrossRef] [PubMed].
    [Google Scholar]
  4. Amoroso R. , Fitzsimmons L. , Thomas W. A. , Kelly G. L. , Rowe M. , Bell A. I. . ( 2011;). Quantitative studies of Epstein–Barr virus-encoded microRNAs provide novel insights into their regulation. J Virol 85: 996–1010 [CrossRef] [PubMed].
    [Google Scholar]
  5. Andreola G. , Rivoltini L. , Castelli C. , Huber V. , Perego P. , Deho P. , Squarcina P. , Accornero P. , Lozupone F. , other authors . ( 2002;). Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles. J Exp Med 195: 1303–1316 [CrossRef] [PubMed].
    [Google Scholar]
  6. Colombo M. , Raposo G. , Théry C. . ( 2014;). Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30: 255–289 [CrossRef] [PubMed].
    [Google Scholar]
  7. Dellis O. , Arbabian A. , Papp B. , Rowe M. , Joab I. , Chomienne C. . ( 2011;). Epstein–Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells. J Biol Chem 286: 18583–18592 [CrossRef] [PubMed].
    [Google Scholar]
  8. Eldh M. , Lötvall J. , Malmhäll C. , Ekström K. . ( 2012;). Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods. Mol Immunol 50: 278–286 [CrossRef] [PubMed].
    [Google Scholar]
  9. Flanagan J. , Middeldorp J. , Sculley T. . ( 2003;). Localization of the Epstein–Barr virus protein LMP 1 to exosomes. J Gen Virol 84: 1871–1879 [CrossRef] [PubMed].
    [Google Scholar]
  10. Greening D. W. , Xu R. , Ji H. , Tauro B. J. , Simpson R. J. . ( 2015;). A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol 1295: 179–209 [CrossRef] [PubMed].
    [Google Scholar]
  11. Gregorovic G. , Bosshard R. , Karstegl C. E. , White R. E. , Pattle S. , Chiang A. K. S. , Dittrich-Breiholz O. , Kracht M. , Russ R. , Farrell P. J. . ( 2011;). Cellular gene expression that correlates with EBER expression in Epstein–Barr virus-infected lymphoblastoid cell lines. J Virol 85: 3535–3545 [CrossRef] [PubMed].
    [Google Scholar]
  12. Guasparri I. , Keller S. A. , Cesarman E. . ( 2004;). KSHV vFLIP is essential for the survival of infected lymphoma cells. J Exp Med 199: 993–1003 [CrossRef] [PubMed].
    [Google Scholar]
  13. Gutzeit C. , Nagy N. , Gentile M. , Lyberg K. , Gumz J. , Vallhov H. , Puga I. , Klein E. , Gabrielsson S. , other authors . ( 2014;). Exosomes derived from Burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in B cells. J Immunol 192: 5852–5862 [CrossRef] [PubMed].
    [Google Scholar]
  14. Holler N. , Tardivel A. , Kovacsovics-Bankowski M. , Hertig S. , Gaide O. , Martinon F. , Tinel A. , Deperthes D. , Calderara S. , other authors . ( 2003;). Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 23: 1428–1440 [CrossRef] [PubMed].
    [Google Scholar]
  15. Hood J. L. , San R. S. , Wickline S. A. . ( 2011;). Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res 71: 3792–3801 [CrossRef] [PubMed].
    [Google Scholar]
  16. Inal J. M. , Kosgodage U. , Azam S. , Stratton D. , Antwi-Baffour S. , Lange S. . ( 2013;). Blood/plasma secretome and microvesicles. Biochim Biophys Acta 1834: 2317–2325 [CrossRef] [PubMed].
    [Google Scholar]
  17. Iwakiri D. , Eizuru Y. , Tokunaga M. , Takada K. . ( 2003;). Autocrine growth of Epstein–Barr virus-positive gastric carcinoma cells mediated by an Epstein–Barr virus-encoded small RNA. Cancer Res 63: 7062–7067 [PubMed].
    [Google Scholar]
  18. Izquierdo-Useros N. , Naranjo-Gómez M. , Erkizia I. , Puertas M. C. , Borràs F. E. , Blanco J. , Martinez-Picado J. . ( 2010;). HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse?. PLoS Pathog 6: e1000740 [CrossRef] [PubMed].
    [Google Scholar]
  19. Kayagaki N. , Kawasaki A. , Ebata T. , Ohmoto H. , Ikeda S. , Inoue S. , Yoshino K. , Okumura K. , Yagita H. . ( 1995;). Metalloproteinase-mediated release of human Fas ligand. J Exp Med 182: 1777–1783 [CrossRef] [PubMed].
    [Google Scholar]
  20. Kelly G. L. , Milner A. E. , Baldwin G. S. , Bell A. I. , Rickinson A. B. . ( 2006;). Three restricted forms of Epstein–Barr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells. Proc Natl Acad Sci U S A 103: 14935–14940 [CrossRef] [PubMed].
    [Google Scholar]
  21. Keryer-Bibens C. , Pioche-Durieu C. , Villemant C. , Souquère S. , Nishi N. , Hirashima M. , Middeldorp J. , Busson P. . ( 2006;). Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9. BMC Cancer 6: 283 [CrossRef] [PubMed].
    [Google Scholar]
  22. Khan G. , Hashim M. J. . ( 2014;). Global burden of deaths from Epstein–Barr virus attributable malignancies 1990–2010. Infect Agent Cancer 9: 38 [CrossRef] [PubMed].
    [Google Scholar]
  23. Khan G. , Miyashita E. M. , Yang B. , Babcock G. J. , Thorley-Lawson D. A. . ( 1996;). Is EBV persistence in vivo a model for B cell homeostasis?. Immunity 5: 173–179 [CrossRef] [PubMed].
    [Google Scholar]
  24. Kim J. W. , Wieckowski E. , Taylor D. D. , Reichert T. E. , Watkins S. , Whiteside T. L. . ( 2005;). Fas ligand-positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res 11: 1010–1020 [PubMed].
    [Google Scholar]
  25. Klibi J. , Niki T. , Riedel A. , Pioche-Durieu C. , Souquere S. , Rubinstein E. , Le Moulec S. , Guigay J. , Hirashima M. , other authors . ( 2009;). Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein–Barr virus-infected nasopharyngeal carcinoma cells. Blood 113: 1957–1966 [CrossRef] [PubMed].
    [Google Scholar]
  26. Klinker M. W. , Lizzio V. , Reed T. J. , Fox D. A. , Lundy S. K. . ( 2014;). Human B cell-derived lymphoblastoid cell lines constitutively produce Fas ligand and secrete MHCII+FasL+ killer exosomes. Front Immunol 5: 144 [CrossRef] [PubMed].
    [Google Scholar]
  27. Krammer P. H. . ( 2000;). CD95's deadly mission in the immune system. Nature 407: 789–795 [CrossRef] [PubMed].
    [Google Scholar]
  28. Longnecker R. , Kieff E. , Cohen J. I. . ( 2013;). Epstein–Barr virus. . In Fields Virology , 6th edn., pp. 1898–1959. Edited by Knipe D. M. , Howley P. M. . Philadelphia, PA: Lippincott Williams & Wilkins;.
    [Google Scholar]
  29. Marquitz A. R. , Mathur A. , Chugh P. E. , Dittmer D. P. , Raab-Traub N. . ( 2014;). Expression profile of microRNAs in Epstein–Barr virus-infected AGS gastric carcinoma cells. J Virol 88: 1389–1393 [CrossRef] [PubMed].
    [Google Scholar]
  30. Meckes D. G. Jr . ( 2015;). Exosomal communication goes viral. J Virol 89: 5200–5203 [CrossRef] [PubMed].
    [Google Scholar]
  31. Meckes D. G. Jr , Shair K. H. Y. , Marquitz A. R. , Kung C. -P. , Edwards R. H. , Raab-Traub N. . ( 2010;). Human tumor virus utilizes exosomes for intercellular communication. Proc Natl Acad Sci U S A 107: 20370–20375 [CrossRef] [PubMed].
    [Google Scholar]
  32. Meckes D. G. Jr , Gunawardena H. P. , Dekroon R. M. , Heaton P. R. , Edwards R. H. , Ozgur S. , Griffith J. D. , Damania B. , Raab-Traub N. . ( 2013;). Modulation of B-cell exosome proteins by gamma herpesvirus infection. Proc Natl Acad Sci U S A 110: E2925–E2933 [CrossRef] [PubMed].
    [Google Scholar]
  33. Menezes J. , Leibold W. , Klein G. , Clements G. . ( 1975;). Establishment and characterization of an Epstein–Barr virus (EBC)-negative lymphoblastoid B cell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt's lymphoma. Biomedicine 22: 276–284 [PubMed].
    [Google Scholar]
  34. Miller G. , Lipman M. . ( 1973;). Release of infectious Epstein–Barr virus by transformed marmoset leukocytes. Proc Natl Acad Sci U S A 70: 190–194 [CrossRef] [PubMed].
    [Google Scholar]
  35. Mrizak D. , Martin N. , Barjon C. , Jimenez-Pailhes A. -S. , Mustapha R. , Niki T. , Guigay J. , Pancré V. , de Launoit Y. , other authors . ( 2015;). Effect of nasopharyngeal carcinoma-derived exosomes on human regulatory T cells. J Natl Cancer Inst 107: 363–376 [CrossRef] [PubMed].
    [Google Scholar]
  36. Nadkarni J. S. , Nadkarni J. J. , Clifford P. , Manolov G. , Fenyö E. M. , Klein E. . ( 1969;). Characteristics of new cell lines derived from Burkitt lymphomas. Cancer 23: 64–79 [PubMed].[CrossRef]
    [Google Scholar]
  37. Nanbo A. , Inoue K. , Adachi-Takasawa K. , Takada K. . ( 2002;). Epstein–Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma. EMBO J 21: 954–965 [CrossRef] [PubMed].
    [Google Scholar]
  38. Nanbo A. , Kawanishi E. , Yoshida R. , Yoshiyama H. . ( 2013;). Exosomes derived from Epstein–Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells. J Virol 87: 10334–10347 [CrossRef] [PubMed].
    [Google Scholar]
  39. Okabayashi S. , Kimura N. . ( 2010;). LGI3 interacts with flotillin-1 to mediate APP trafficking and exosome formation. Neuroreport 21: 606–610 [CrossRef] [PubMed].
    [Google Scholar]
  40. Pegtel D. M. , Cosmopoulos K. , Thorley-Lawson D. A. , van Eijndhoven M. A. J. , Hopmans E. S. , Lindenberg J. L. , de Gruijl T. D. , Würdinger T. , Middeldorp J. M. . ( 2010;). Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107: 6328–6333 [CrossRef] [PubMed].
    [Google Scholar]
  41. Pratt Z. L. , Kuzembayeva M. , Sengupta S. , Sugden B. . ( 2009;). The microRNAs of Epstein–Barr virus are expressed at dramatically differing levels among cell lines. Virology 386: 387–397 [CrossRef] [PubMed].
    [Google Scholar]
  42. Raposo G. , Stoorvogel W. . ( 2013;). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200: 373–383 [CrossRef] [PubMed].
    [Google Scholar]
  43. Rio D. C. , Clark S. G. , Tjian R. . ( 1985;). A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction. Science 227: 23–28 [CrossRef] [PubMed].
    [Google Scholar]
  44. Ruf I. K. , Lackey K. A. , Warudkar S. , Sample J. T. . ( 2005;). Protection from interferon-induced apoptosis by Epstein–Barr virus small RNAs is not mediated by inhibition of PKR. J Virol 79: 14562–14569 [CrossRef] [PubMed].
    [Google Scholar]
  45. Schneider U. , Schwenk H. U. , Bornkamm G. . ( 1977;). Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer 19: 621–626 [CrossRef] [PubMed].
    [Google Scholar]
  46. Schneider P. , Holler N. , Bodmer J. L. , Hahne M. , Frei K. , Fontana A. , Tschopp J. . ( 1998;). Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med 187: 1205–1213 [CrossRef] [PubMed].
    [Google Scholar]
  47. Shisler J. , Yang C. , Walter B. , Ware C. F. , Gooding L. R. . ( 1997;). The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis. J Virol 71: 8299–8306 [PubMed].
    [Google Scholar]
  48. Sinclair A. J. , Farrell P. J. . ( 1995;). Host cell requirements for efficient infection of quiescent primary B lymphocytes by Epstein–Barr virus. J Virol 69: 5461–5468 [PubMed].
    [Google Scholar]
  49. Stenqvist A. -C. , Nagaeva O. , Baranov V. , Mincheva-Nilsson L. . ( 2013;). Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus. J Immunol 191: 5515–5523 [CrossRef] [PubMed].
    [Google Scholar]
  50. Suda T. , Hashimoto H. , Tanaka M. , Ochi T. , Nagata S. . ( 1997;). Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing. J Exp Med 186: 2045–2050 [CrossRef] [PubMed].
    [Google Scholar]
  51. Tanaka M. , Suda T. , Takahashi T. , Nagata S. . ( 1995;). Expression of the functional soluble form of human Fas ligand in activated lymphocytes. EMBO J 14: 1129–1135 [PubMed].
    [Google Scholar]
  52. Tanaka M. , Itai T. , Adachi M. , Nagata S. . ( 1998;). Downregulation of Fas ligand by shedding. Nat Med 4: 31–36 [CrossRef] [PubMed].
    [Google Scholar]
  53. Théry C. , Amigorena S. , Raposo G. , Clayton A. . ( 2006;). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30: 3.22.1–3.22.29.
    [Google Scholar]
  54. Verweij F. J. , van Eijndhoven M. A. J. , Hopmans E. S. , Vendrig T. , Wurdinger T. , Cahir-McFarland E. , Kieff E. , Geerts D. , van der Kant R. , other authors . ( 2011;). LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation. EMBO J 30: 2115–2129 [CrossRef] [PubMed].
    [Google Scholar]
  55. Verweij F. J. , van Eijndhoven M. A. J. , Middeldorp J. , Pegtel D. M. . ( 2013;). Analysis of viral microRNA exchange via exosomes in vitro and in vivo . Methods Mol Biol 1024: 53–68 [CrossRef] [PubMed].
    [Google Scholar]
  56. Villanueva M. T. . ( 2014;). Microenvironment: small containers, important cargo. Nat Rev Cancer 14: 764–765 [CrossRef] [PubMed].
    [Google Scholar]
  57. Wieckowski E. U. , Visus C. , Szajnik M. , Szczepanski M. J. , Storkus W. J. , Whiteside T. L. . ( 2009;). Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes. J Immunol 183: 3720–3730 [CrossRef] [PubMed].
    [Google Scholar]
  58. Witwer K. W. , Buzás E. I. , Bemis L. T. , Bora A. , Lässer C. , Lötvall J. , Nolte-'t Hoen E. N. , Piper M. G. , Sivaraman S. , other authors . ( 2013;). Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2: 2 [CrossRef] [PubMed].
    [Google Scholar]
  59. Yang L. , Aozasa K. , Oshimi K. , Takada K. . ( 2004;). Epstein–Barr virus (EBV)-encoded RNA promotes growth of EBV-infected T cells through interleukin-9 induction. Cancer Res 64: 5332–5337 [CrossRef] [PubMed].
    [Google Scholar]
  60. Yang L. , Wu X. , Wang D. , Luo C. , Chen L. . ( 2013;). Renal carcinoma cell-derived exosomes induce human immortalized line of Jurkat T lymphocyte apoptosis in vitro . Urol Int 91: 363–369 [CrossRef] [PubMed].
    [Google Scholar]
  61. Young L. S. , Rickinson A. B. . ( 2004;). Epstein–Barr virus: 40 years on. Nat Rev Cancer 4: 757–768 [CrossRef] [PubMed].
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000313
Loading
/content/journal/jgv/10.1099/jgv.0.000313
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error