EBER2 RNA-induced transcriptome changes identify cellular processes likely targeted during Epstein Barr Virus infection
- Sebastian Eilebrecht†1,
- François-Xavier Pellay†2,
- Peter Odenwälder1,
- Guillaume Brysbaert2,
- Bernd-Joachim Benecke1 and
- Arndt Benecke2Email author
© Benecke et al; licensee BioMed Central Ltd. 2008
Received: 02 August 2008
Accepted: 28 October 2008
Published: 28 October 2008
Little is known about the physiological role of the EBER1 and 2 nuclear RNAs during Epstein Barr viral infection. The EBERs are transcribed by cellular RNA Polymerase III and their strong expression results in 106 to 107 copies per EBV infected cell, making them reliable diagnostic markers for the presence of EBV. Although the functions of most of the proteins targeted by EBER RNAs have been studied, the role of EBERs themselves still remains elusive.
The cellular transcription response to EBER2 expression using the wild-type and an internal deletion mutant was determined. Significant changes in gene expression patterns were observed. A functional meta-analysis of the regulated genes points to inhibition of stress and immune responses, as well as activation of cellular growth and cytoskeletal reorganization as potential targets for EBER2 RNA. Different functions can be assigned to different parts of the RNA.
These results provide new avenues to the understanding of EBER2 and EBV biology, and set the grounds for a more in depth functional analysis of EBER2 using transcriptome activity measurements.
Epstein-Barr virus (EBV) is a member of the herpesvirus family present in almost the entire human adult population [1–3] and has been found to be associated with oncogenesis of Burkitt's lymphoma, B- and T-cell leukemia/lymphomas, nasopharyngeal carcinoma, breast cancer and gastric cancer [4–7]. In EBV infected cells, several viral genes are expressed of which the six nuclear antigens (EBNAs 1–6), the three membrane proteins (LMP-1, -2A and -2B) and the small non translated EBER1 and EBER2 RNAs are the most abundant [1, 3].
The EBERs are transcribed by cellular RNA Polymerase III (polIII) and their strong expression results in 106 to 107 copies per EBV infected cell [8, 9], making them reliable diagnostic markers for the presence of EBV. EBERs are located in the nucleus  and have been shown to bind to several cellular proteins, such as the La antigen [9, 11–14], the EBER-associated protein (EAP, now referred to as ribosomal protein L22) [15, 16], and the interferon-inducible protein kinase R (PKR) [17, 18]. The binding of EBER1 to PKR blocks the PKR pathway resulting in the resistance of the cell to Fas-mediated apoptosis . Although the functions of most of the proteins targeted by EBER RNAs have been studied, the role of EBERs themselves still remains elusive.
EBER-induced interleukin (IL)-10 expression in Burkitt's lymphoma (BL) cells has been demonstrated . IL-10 is suggested to be an autocrine growth factor for BL cells , and hence potentially links EBER expression to hyperplastic transformation. Furthermore, a recent report indicates that rather EBER2 than EBER1 plays a central role in B-cell growth transformation . Given this indication of a transcriptional response to EBER expression, as well as the indication of the functional implication of EBER2 in cellular transformation, we used microarrays to assess transcriptome changes following expression of EBER2 in HEK 293 cells.
Results and discussion
For microarray analysis, HEK293 cells were transfected with pUC18 vector or the EBER2/EBER2-L2 constructs by calcium phosphate co-precipitation technique using the initial constructs to avoid secondary effects by the co-expression of GFP. Total RNA was extracted 48 h post-transfection with an RNeasy Midi Kit (Qiagen), and analyzed for quality with an Agilent 2100 Bioanalyser. RNA amplification, labeling, hybridization and detection were done following the protocols supplied by Applied Biosystems using 2 μg of total RNA as input. Labeled cRNAs were then hybridized and detected on HGS Version 2.0 AB1700 microarrays containing probes for 29362 validated human genes. The AB1700 technology has been demonstrated to cover an increased signal dynamic range, display higher sensitivity and provide more robust gene expression estimates when compared to the leading competing technologies .
Statistically significant changes in the HEK293 transcriptome upon EBER2 or EBER2-L2 expression are observed (Figure 3A). In the former case a total of 1218 probes are found to detect differential gene expression at p < 0.01, of which 137 are stimulated, and 1081 are repressed in their expression when compared to mock (pUC18) transfected cells. Thus EBER2 expression has a significant impact on the cellular transcriptome. Likewise, the EBER2-L2 has a very pronounced effect on cellular transcription. A total of 6269 probes are found to return statistically significant (p < 0.01) differential signals, with the vast majority of genes being repressed in their expression (6230 probes). Comparisons of both target gene sets (Additional Files 2, 3) reveal that out of the 1218 EBER2 targets 962 (or 79%) are also found to be EBER2-L2 targets (Venn-Diagrams, Figure 3A; Additional File 4). The clear majority of genes specific to EBER2 are up-regulated in their expression indicating that positive transcription regulation requires integrity of, and may be dependent directly on the L2 structure. The important number of genes negatively affected in their transcription by EBER2-L2 is believed to be a consequence of functionally unproductive titration of cellular components leading to global repression of RNA processing in the cell. Alternatively, albeit very unlikely, we can not formally rule out the possibility that EBER2 RNA or degradation products of the RNA interfere with the transcriptome activity measurements. Such findings have to be considered when using EBER2 RNA sequences as a vehicle for the expression of shRNAs, as recently proposed by Choy and colleagues .
The strong negative regulation by EBER2-L2 is explained by over-representation (p < 1.00 E-9) of genes related to mRNA maturation and protein synthesis in this dataset (Figure 3D). EBER2-L2 likely interferes in a non-physiological manner with the production and/or assembly of the splicing and translation apparatuses, as the same processes are not overrepresented when the EBER2 target gene set is analyzed. Furthermore, both EBER2 and EBER2-L2 might be processed by the RNA editing/degradation machinery and give rise to molecules inferring with either physiological processes, or, albeit highly unlikely, the transcriptome analysis itself.
The EBER2 specific target gene repertoire also reveals down-regulation of three highly related interferon inducible trans-membrane proteins IFITM1, 2, 3, and induction of several cellular growth regulators such as FGF receptor 3 and TGFβ receptor 3 . The regulation of the latter is observed only with EBER2 and therefore is directly assignable to L2 integrity (Figure 4). The main categories of EBER2 targeted genes related to cellular growth, reorganization, and stress thereby seem to coincide well with previous advances in the understanding of EBER2 biology . The results here should therefore provide a novel, complementary avenue to the understanding of EBER RNAs and EBV physiopathology.
These investigations establish the feasibility of recording transcriptome dynamics downstream of non-translated small nuclear RNAs. Furthermore, they do not only point to a role of EBER2 in regulating the activity of cellular pathways related to growth and the cytoskeleton, as well as negative interference into interferon mediated immune responses and cellular stress, but also provide a basis for the identification of the molecular targets of EBER2 RNA and the understanding of the physiopathology of EBV, in general. Regulating the activity of growth, cytoskeletal dynamics as well as inhibition of cellular stress seems in tune with recent advances in the understanding of EBER2 biology in the context of B-cell transformation . Finally, these data should also prove very helpful in the interpretation of results obtained using shRNA-EBER fusion constructs as pioneered by Choy and colleagues . Given its high expression levels and structural stability, EBER2, like other pol-III transcribed and/or viral RNAs are being proposed as vehicles for the over-expression of knock-down small interfering RNA species. The fact that the backbone might, as shown here in the case of the EBER2, have profound effects on the cellular transcriptome will certainly have to be studied during the validation of such hybrid molecules.
F.X.P. is recipient of a fellowship from the Nausicaa Combat Sa Leucémie Association. This work was funded through support from the European Hematology Association – José Carreras Foundation, the Institut des Hautes Études Scientifiques, the Centre National de la Recherche Scientifique (CNRS), and the Région Nord (all to A.B.). A.B.'s laboratory also acknowledges funding from the Agence Nationale de Recherche sur le SIDA, and the Agence Nationale de la Recherche (ISPA, 07-PHYSIO-013-02) towards the development of transcriptome analysis methodology.
The transcriptome profiles used for this study have been deposited in the GEO database with Accession No.: GSE11093. The additional files are also available from the authors' website in the web sources/supplementary data section at http://seg.ihes.fr.
- Epstein MA, Achong BG, Editors: The EPSTEIN-BARR Virus: Recent Advances. William Heinemann Medical Books. 1986Google Scholar
- Klein G: Viral latency and transformation: the strategy of Epstein-Barr virus. Cell. 1989, 58: 5-8.View ArticlePubMedGoogle Scholar
- Young LS, Rickinson AB: Epstein-Barr Virus: 40 years on. Nat Rev Cancer. 2004, 4: 757-68.View ArticlePubMedGoogle Scholar
- Herrmann K, Niedobitek G: Epstein-Barr virus-associated carcinomas: facts and fiction. J Pathol. 2003, 199: 140-5.View ArticlePubMedGoogle Scholar
- Pattle SB, Farrell PJ: The role of Epstein-Barr virus in cancer. Expert Opin Biol Ther. 2006, 6: 1193-205.View ArticlePubMedGoogle Scholar
- Lippincott Williams & Wilkins, Rickinson AB, Kieff E: Epstein-Barr virus. Field's Virology. Edited by: Kinpes DM, Howley PM. 2001, Lippincott Williams & Wilkins, II: 2575-2627.Google Scholar
- Serraino D, Piselli P, Angeletti C, Scuderi M, Ippolito G, Capobianchi MR: Infection with Epstein-Barr virus and cancer: an epidemiological review. J Biol Regul Homeost Agents. 2005, 19: 63-70.PubMedGoogle Scholar
- Dumpelmann E, Mittendorf H, Benecke BJ: Efficient transcription of the EBER2 gene depends on the structural integrity of the RNA. RNA. 2003, 9: 432-42.PubMed CentralView ArticlePubMedGoogle Scholar
- Lerner MR, Andrews NC, Miller G, Steitz JA: 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. 1981, 78: 805-9.PubMed CentralView ArticlePubMedGoogle Scholar
- Chang KL, Chen YY, Shibata D, Weiss LM: Description of an in situ hybridization methodology for detection of Epstein-Barr virus RNA in paraffin-embedded tissues, with a survey of normal and neoplastic tissues. Diagn Mol Pathol. 1992, 1: 246-55.View ArticlePubMedGoogle Scholar
- Bhat RA, Thimmappaya B: Two small RNAs encoded by Epstein-Barr virus can functionally substitute for the virus-associated RNAs in the lytic growth of adenovirus 5. Proc Natl Acad Sci USA. 1983, 80: 4789-93.PubMed CentralView ArticlePubMedGoogle Scholar
- Glickman JN, Howe JG, Steitz JA: Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J Virol. 1988, 62: 902-11.PubMed CentralPubMedGoogle Scholar
- Howe JG, Shu MD: Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs. J Virol. 1988, 62: 2790-8.PubMed CentralPubMedGoogle Scholar
- Toczyski DP, Steitz JA: EAP, a highly conserved cellular protein associated with Epstein-Barr virus small RNAs (EBERs). EMBO J. 1991, 10: 459-66.PubMed CentralPubMedGoogle Scholar
- Fok V, Mitton-Fry RM, Grech A, Steitz JA: Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22. RNA. 2006, 12: 872-82.PubMed CentralView ArticlePubMedGoogle Scholar
- Toczyski DP, Matera AG, Ward DC, Steitz JA: The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes. Proc Natl Acad Sci USA. 1994, 91: 3463-7.PubMed CentralView ArticlePubMedGoogle Scholar
- Clarke PA, Schwemmle M, Schickinger J, Hilse K, Clemens MJ: Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI. Nucleic Acids Res. 1991, 19: 243-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Clemens MJ: The small RNAs of Epstein-Barr virus. Mol Biol Rep. 1993, 17: 81-92.View ArticlePubMedGoogle Scholar
- Nanbo A, Yoshiyama H, Takada K: 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. J Virol. 2005, 79: 12280-5.PubMed CentralView ArticlePubMedGoogle Scholar
- Kitagawa N, Goto M, Kurozumi K, Maruo S, Fukayama M, Naoe T, Yasukawa M, Hino K, Suzuki T, Todo S, Takada K: Epstein-Barr virus-encoded poly(A)(-) RNA supports Burkitt's lymphoma growth through interleukin-10 induction. EMBO J. 2000, 19: 6742-50.PubMed CentralView ArticlePubMedGoogle Scholar
- Fan SQ, Ma J, Zhou J, Xiong W, Xiao BY, Zhang WL, Tan C, Li XL, Shen SR, Zhou M, Zhang QH, Ou YJ, Zhuo HD, Fan S, Zhou YH, Li GY: Differential expression of Epstein-Barr virus-encoded RNA and several tumor-related genes in various types of nasopharyngeal epithelial lesions and nasopharyngeal carcinoma using tissue microarray analysis. Hum Pathol. 2006, 37: 593-605.View ArticlePubMedGoogle Scholar
- Wu Y, Maruo S, Yajima M, Kanda T, Takada T: Epstein-Barr virus (EBV)-encoded RNA 2 (EBER2) but not EBER1 plays a critical role in EBV-induced B-cell growth transformation. J Virol. 2007, 81: 11236-45.PubMed CentralView ArticlePubMedGoogle Scholar
- Choy EY, Kok KH, Tsao SW, Jin DY: Utility of Epstein-Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs. Gene Ther. 2008, 15: 191-202.View ArticlePubMedGoogle Scholar
- Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH: Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci USA. 2004, 101: 7287-92.PubMed CentralView ArticlePubMedGoogle Scholar
- Noth S, Brysbaert G, Pellay FX, Benecke A: High-sensitivity transcriptome data structure and implications for analysis and biologic interpretation. Genomics Proteomics Bioinformatics. 2006, 4: 212-29.View ArticlePubMedGoogle Scholar
- Tzima E: Role of small GTPases in endothelial cytoskeletal dynamics and the shear stress response. Circ Res. 2006, 98: 176-85.View ArticlePubMedGoogle Scholar
- Hall A: Rho GTPases and the control of cell behaviour. Biochem Soc Trans. 2005, 33: 891-5.View ArticlePubMedGoogle Scholar
- Goley ED, Welch MD: The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol. 2006, 7: 713-26.View ArticlePubMedGoogle Scholar
- Wade RH: Microtubules: an overview. Methods Mol Med. 2007, 137: 1-16.View ArticlePubMedGoogle Scholar
- Geiger TR, Martin JM: The Epstein-Barr virus-encoded LMP-1 oncoprotein negatively affects Tyk2 phosphorylation and interferon signaling in human B cells. J Virol. 2006, 80: 11638-50.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.