High Levels of EBV-Encoded RNA 1 (EBER1) Trigger Interferon and Inflammation-Related Genes in Keratinocytes Expressing HPV16 E6/E7

Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV–modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or infiltrating dendritic cells may play a role in the inflammation-to-oncogenesis transition of HPV-associated cervical cancer through modulation of innate immune signals.

[1]  D. Pegtel,et al.  Extracellular Vesicles Exploit Viral Entry Routes for Cargo Delivery , 2016, Microbiology and Molecular Reviews.

[2]  T. Wurdinger,et al.  Sensing of latent EBV infection through exosomal transfer of 5′pppRNA , 2016, Proceedings of the National Academy of Sciences.

[3]  Tineke Vendrig,et al.  Possible contributing role of Epstein-Barr virus (EBV) as a cofactor in human papillomavirus (HPV)-associated cervical carcinogenesis. , 2015, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[4]  Lifang Yang,et al.  Nasopharyngeal carcinoma progression is mediated by EBER-triggered inflammation via the RIG-I pathway. , 2015, Cancer letters.

[5]  S. H. van der Burg,et al.  The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation , 2015, Nature Communications.

[6]  C. Mathers,et al.  Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012 , 2015, International journal of cancer.

[7]  R. Cobucci,et al.  Link between chronic inflammation and human papillomavirus-induced carcinogenesis (Review) , 2015, Oncology letters.

[8]  D. Iwakiri.  Epstein-Barr Virus-Encoded RNAs: Key Molecules in Viral Pathogenesis , 2014, Cancers.

[9]  G. Khan,et al.  Epstein-Barr Virus-Encoded Small RNAs (EBERs) Are Present in Fractions Related to Exosomes Released by EBV-Transformed Cells , 2014, PloS one.

[10]  J. Berkhof,et al.  Clinical Validation of the HPV-Risk Assay, a Novel Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus DNA by Targeting the E7 Region , 2014, Journal of Clinical Microbiology.

[11]  C. Meijer,et al.  Differential In Vitro Immortalization Capacity of Eleven, Probable High-Risk Human Papillomavirus Types , 2013, Journal of Virology.

[12]  M. Ammendolia,et al.  Exosomes released in vitro from Epstein-Barr virus (EBV)-infected cells contain EBV-encoded latent phase mRNAs. , 2013, Cancer letters.

[13]  H. Yoshiyama,et al.  Exosomes Derived from Epstein-Barr Virus-Infected Cells Are Internalized via Caveola-Dependent Endocytosis and Promote Phenotypic Modulation in Target Cells , 2013, Journal of Virology.

[14]  H. Gunawardena,et al.  Modulation of B-cell exosome proteins by gamma herpesvirus infection , 2013, Proceedings of the National Academy of Sciences.

[15]  M. Schiffman,et al.  Updating the natural history of human papillomavirus and anogenital cancers. , 2012, Vaccine.

[16]  M. Okano,et al.  Acute or Chronic Life-Threatening Diseases Associated With Epstein-Barr Virus Infection , 2012, The American journal of the medical sciences.

[17]  H. Varmus,et al.  Epstein-Barr Virus: An Important Vaccine Target for Cancer Prevention , 2011, Science Translational Medicine.

[18]  Jaap M Middeldorp,et al.  Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion. , 2011, Biochimica et biophysica acta.

[19]  A. Rickinson,et al.  Immune defence against EBV and EBV-associated disease. , 2011, Current opinion in immunology.

[20]  Mei-Ru Chen.  Epstein–Barr Virus, the Immune System, and Associated Diseases , 2011, Front. Microbio..

[21]  Susanne Gabrielsson,et al.  Exosomes Containing Glycoprotein 350 Released by EBV-Transformed B Cells Selectively Target B Cells through CD21 and Block EBV Infection In Vitro , 2011, The Journal of Immunology.

[22]  N. Raab-Traub,et al.  Human tumor virus utilizes exosomes for intercellular communication , 2010, Proceedings of the National Academy of Sciences.

[23]  T. D. de Gruijl,et al.  Functional delivery of viral miRNAs via exosomes , 2010, Proceedings of the National Academy of Sciences.

[24]  A. Molinari,et al.  Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells* , 2009, The Journal of Biological Chemistry.

[25]  K. Kawa,et al.  Epstein-Barr virus (EBV)–encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3 , 2009, The Journal of experimental medicine.

[26]  S. Franceschi,et al.  Infections and cancer: established associations and new hypotheses. , 2009, Critical reviews in oncology/hematology.

[27]  B. Aggarwal,et al.  Targeting Inflammatory Pathways for Prevention and Therapy of Cancer: Short-Term Friend, Long-Term Foe , 2009, Clinical Cancer Research.

[28]  M. Samanta,et al.  Epstein–Barr virus-encoded small RNA induces IL-10 through RIG-I-mediated IRF-3 signaling , 2008, Oncogene.

[29]  T. Kanda,et al.  EB virus‐encoded RNAs are recognized by RIG‐I and activate signaling to induce type I IFN , 2006, The EMBO journal.

[30]  Xavier Castellsagué,et al.  Chapter 1: HPV in the etiology of human cancer. , 2006, Vaccine.

[31]  G. Taraboletti,et al.  Bioavailability of VEGF in tumor-shed vesicles depends on vesicle burst induced by acidic pH. , 2006, Neoplasia.

[32]  J. Kutok,et al.  Spectrum of Epstein-Barr virus-associated diseases. , 2006, Annual review of pathology.

[33]  H. Yoshiyama,et al.  Epstein-Barr Virus-Encoded Poly(A)− RNA Confers Resistance to Apoptosis Mediated through Fas by Blocking the PKR Pathway in Human Epithelial Intestine 407 Cells , 2005, Journal of Virology.

[34]  T. Kanda,et al.  Critical Role of Epstein-Barr Virus (EBV)-Encoded RNA in Efficient EBV-Induced B-Lymphocyte Growth Transformation , 2005, Journal of Virology.

[35]  Simon C Watkins,et al.  Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. , 2004, Blood.

[36]  Jaap M Middeldorp,et al.  Localization of the Epstein-Barr virus protein LMP 1 to exosomes. , 2003, The Journal of general virology.

[37]  A. Nanbo,et al.  Epstein–Barr virus RNA confers resistance to interferon‐α‐induced apoptosis in Burkitt's lymphoma , 2002 .

[38]  A. Nanbo,et al.  The role of EBERs in oncogenesis. , 2001, Seminars in cancer biology.

[39]  C. Meijer,et al.  Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q. , 1996, Oncogene.

[40]  C. Melief,et al.  B lymphocytes secrete antigen-presenting vesicles , 1996, The Journal of experimental medicine.

[41]  M. Clemens,et al.  Expression of genes for the Epstein-Barr virus small RNAs EBER-1 and EBER-2 in Daudi Burkitt's lymphoma cells: effects of interferon treatment. , 1992, The Journal of general virology.

[42]  A. Nanbo,et al.  Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma. , 2002, The EMBO journal.