Epstein-Barr Virus Induces Cellular Transcription Factors to Allow Active Expression of EBER Genes by RNA Polymerase III

The EBER genes of Epstein-Barr virus (EBV) are transcribed by RNA polymerase (pol) III to produce untranslated RNAs that are implicated in oncogenesis. These EBER transcripts are the most highly expressed viral gene products in EBV-transformed cells. We have identified changes to the cellular transcription machinery that may contribute to the high levels of EBER RNA. These include phosphorylation of ATF2, which interacts with EBER promoters. A second is induction of TFIIIC, a pol III-specific factor that activates EBER genes; all five subunits of TFIIIC are overexpressed in EBV-positive cells. In addition, EBV induces BDP1, a subunit of the pol III-specific factor TFIIIB. Although BDP1 is the only TFIIIB subunit induced by EBV, its induction is sufficient to stimulate EBER expression in vivo, implying a limiting function. The elevated levels of BDP1 and TFIIIC in EBV-positive cells stimulate production of tRNA, 7SL, and 5S rRNA. Abnormally high expression of these cellular pol III products may contribute to the ability of EBV to enhance growth potential. The EBER genes of Epstein-Barr virus (EBV) are transcribed by RNA polymerase (pol) III to produce untranslated RNAs that are implicated in oncogenesis. These EBER transcripts are the most highly expressed viral gene products in EBV-transformed cells. We have identified changes to the cellular transcription machinery that may contribute to the high levels of EBER RNA. These include phosphorylation of ATF2, which interacts with EBER promoters. A second is induction of TFIIIC, a pol III-specific factor that activates EBER genes; all five subunits of TFIIIC are overexpressed in EBV-positive cells. In addition, EBV induces BDP1, a subunit of the pol III-specific factor TFIIIB. Although BDP1 is the only TFIIIB subunit induced by EBV, its induction is sufficient to stimulate EBER expression in vivo, implying a limiting function. The elevated levels of BDP1 and TFIIIC in EBV-positive cells stimulate production of tRNA, 7SL, and 5S rRNA. Abnormally high expression of these cellular pol III products may contribute to the ability of EBV to enhance growth potential. Epstein-Barr virus (EBV) 2The abbreviations used are: EBV, Epstein-Barr virus; ATF, activating transcription factor; BL, Burkitt lymphoma; CRE, cAMP-response element; ChIP, chromatin immunoprecipitation; EBER, Epstein-Barr virus-encoded RNA; NPC, nasopharyngeal carcinoma; pol, RNA polymerase; RT-PCR, reverse transcriptase-PCR; pol, RNA polymerase. 2The abbreviations used are: EBV, Epstein-Barr virus; ATF, activating transcription factor; BL, Burkitt lymphoma; CRE, cAMP-response element; ChIP, chromatin immunoprecipitation; EBER, Epstein-Barr virus-encoded RNA; NPC, nasopharyngeal carcinoma; pol, RNA polymerase; RT-PCR, reverse transcriptase-PCR; pol, RNA polymerase. is the causative agent of infectious mononucleosis and is closely associated with a range of malignant diseases, including Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), and gastric carcinomas (1Young L.S. Rickinson A.B. Nature Rev. Cancer. 2004; 4: 757-768Crossref PubMed Scopus (1587) Google Scholar). The EBV genome contains two small, well conserved adjacent genes that are transcribed by RNA polymerase (pol) III, called EBER1 and EBER2 (2Rosa M.D. Gottlieb E. Lerner M. Steitz J.A. Mol. Cell. Biol. 1981; 1: 785-796Crossref PubMed Scopus (206) Google Scholar, 3Arrand J.R. Young L.S. Tugwood J.D. J. Virol. 1989; 63: 983-986Crossref PubMed Google Scholar). Their transcripts are the most highly expressed viral products during latent infection and immortalization of human B lymphocytes by EBV, accumulating in ∼107 copies/cell (4Arrand J.R. Rymo L. J. Virol. 1982; 41: 376-389Crossref PubMed Google Scholar). As such, EBERs are used diagnostically to establish the EBV status of tumors by in situ hybridization (5Khan G. Coates P.J. Kangro H.O. Slavin G. J. Clin. Pathol. 1992; 45: 616-620Crossref PubMed Scopus (96) Google Scholar, 6Arrand J.R. Epstein-Barr Virus Rep. 2000; 7: 145-149Google Scholar). Deletion of the EBER genes does not prevent EBV from infecting or immortalizing B lymphocytes in culture (7Swaminathan S. Tomkinson B. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1546-1550Crossref PubMed Scopus (164) Google Scholar), but this may reflect a redundancy of function within the large viral genome. Indeed, EBERs can stimulate DNA and protein synthesis when transfected into normal cord blood lymphocytes in the absence of other viral genes (8Zeuthen J. J. Cancer Res. Clin. Oncol. 1983; 106: 1-11Crossref PubMed Scopus (9) Google Scholar, 9Zeuthen J. Adv. Viral Oncol. 1983; 3: 183-200Google Scholar). Transfection of EBER genes into NIH3T3 cells or EBV-negative BL lines can allow them to form colonies in soft agar (10Laing K.G. Matys V. Clemens M.J. Biochem. Soc. Trans. 1995; 23: 311Crossref PubMed Scopus (10) Google Scholar, 11Laing K.G. Elia A. Jeffrey I. Matys V. Tilleray V.J. Souberbielle B. Clemens M.J. Virology. 2002; 297: 253-269Crossref PubMed Scopus (25) Google Scholar). Furthermore, the EBER-transfected BL cells can produce tumors in mice (12Komano J. Maruo S. Kurozumi K. Oda T. Takada K. J. Virol. 1999; 73: 9827-9831Crossref PubMed Google Scholar, 13Ruf I.K. Rhyne P.W. Yang C. Cleveland J.L. Sample J.T. J. Virol. 2000; 74: 10223-10228Crossref PubMed Scopus (104) Google Scholar, 14Yamamoto N. Takizawa T. Iwanaga Y. Shimizu N. Yamamoto N. FEBS Lett. 2000; 484: 153-158Crossref PubMed Scopus (67) Google Scholar). Because EBER transcripts are not translated, these remarkable discoveries provided the first evidence of oncogenic RNA. The massive expression of EBER genes is a striking feature of EBV-transformed cells, yet little is known about how it is achieved. Several observations suggest that it is not simply due to inherent strength of the EBER promoters but instead requires EBV-induced changes to the cellular environment. For example, stable transfection of EBER1 alone allowed maximal expression of ∼105 transcripts/cell, whereas latent infection with the EBV genome can result in ∼107 EBER1 transcripts/cell (11Laing K.G. Elia A. Jeffrey I. Matys V. Tilleray V.J. Souberbielle B. Clemens M.J. Virology. 2002; 297: 253-269Crossref PubMed Scopus (25) Google Scholar, 15Lerner M.R. Andrews M.C. Miller G. Steitz J.A. Science. 1981; 211: 400-402Crossref PubMed Scopus (373) Google Scholar). However, significant EBER expression only becomes apparent 36 h after infection, following the appearance of other EBV latent gene products (16Rooney C. Howe J.G. Speck S.H. Miller G. J. Virol. 1989; 63: 1531-1539Crossref PubMed Google Scholar). Furthermore, when latently infected cells switch to lytic viral replication, EBER gene transcription decreases dramatically (17Greifenegger N. Jager M. Kunz-Schughart L.A. Wolf H. Schwarzmann F. J. Virol. 1998; 72: 9323-9328Crossref PubMed Google Scholar). These data point to a strong influence of trans-acting factors in controlling the EBERs. We present evidence that the high levels of EBER expression in latently infected tumor cells reflect changes to the host transcription machinery. Thus, the pol III-specific transcription factors TFIIIC and BDP1 are both overexpressed in EBV-positive cells of lymphoid or epithelial origin. This is associated with a specific increase in levels of some pol III products. We show that induction of TFIIIC may be mediated, in part, by ATF-2, which undergoes an activating phosphorylation in response to EBV. ATF-2 also interacts with the EBER genes, which may further enhance their transcription. These combined effects can explain the high levels of EBER RNA that are diagnostic of EBV-associated tumors (5Khan G. Coates P.J. Kangro H.O. Slavin G. J. Clin. Pathol. 1992; 45: 616-620Crossref PubMed Scopus (96) Google Scholar, 6Arrand J.R. Epstein-Barr Virus Rep. 2000; 7: 145-149Google Scholar). Cell Culture and Extraction—HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen). Hone1, Ad/AH, and Akata cells were cultured in RPMI (Invitrogen). All media were supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. G418 (300 μg/ml) was included in the media of EBV-positive HeLa, Ad/AH, and Akata cells. Whole cell protein extracts were prepared as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Antibodies and Western Blotting—Western blotting was carried out as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Ab7 against TFIIIC220 was a generous gift from Dr. Arnie Berk (19Shen Y. Igo M. Yalamanchili P. Berk A.J. Dasgupta A. Mol. Cell. Biol. 1996; 16: 4163-4171Crossref PubMed Scopus (53) Google Scholar). We have described previously antibodies 4286 against TFIIIC110 (20Sutcliffe J.E. Brown T.R.P. Allison S.J. Scott P.H. White R.J. Mol. Cell. Biol. 2000; 20: 9192-9202Crossref PubMed Scopus (64) Google Scholar), 128 against BRF1 (21Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), and 2663 against BDP1 (21Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar). Antibody 3238 against TFIIIC102 was raised by immunizing rabbits with synthetic peptide MSGFSPELIDYLEGK (human TFIIIC102 residues 1–15Lerner M.R. Andrews M.C. Miller G. Steitz J.A. Science. 1981; 211: 400-402Crossref PubMed Scopus (373) Google Scholar) coupled to keyhole limpet hemocyanin. Antibody 1898–64 was prepared by affinity purification of anti-serum 1898 against TFIIIC90 (22Felton-Edkins Z.A. White R.J. J. Biol. Chem. 2002; 277: 48182-48191Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) using synthetic peptide GMGNADDEQQEEGTSC (human TFIIIC90 residues 613–627). Antibodies 58C9 against TBP, M-19 against TAFI48, L-19 against TFIIIC63, N-96 against total ATF-2, F-1 against ATF2 phosphorylated at Thr-71, and C-11 against actin were from Santa Cruz Biotechnology. RNA Extraction and RT-PCR—RNA extraction and reverse transcription were performed as previously described (23Larminie C.G.C. Sutcliffe J.E. Tosh K. Winter A.G. Felton-Edkins Z.A. White R.J. Mol. Cell. Biol. 1999; 19: 4927-4934Crossref PubMed Scopus (37) Google Scholar). Primers and cycling parameters have been described for BRF1 (23Larminie C.G.C. Sutcliffe J.E. Tosh K. Winter A.G. Felton-Edkins Z.A. White R.J. Mol. Cell. Biol. 1999; 19: 4927-4934Crossref PubMed Scopus (37) Google Scholar), 5S rRNA, tRNATyr, 7SL, TFIIIC subunits, and ARPP P0 (24Winter A.G. Sourvinos G. Allison S.J. Tosh K. Scott P.H. Spandidos D.A. White R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12619-12624Crossref PubMed Scopus (100) Google Scholar), U6, BDP1, and TBP (22Felton-Edkins Z.A. White R.J. J. Biol. Chem. 2002; 277: 48182-48191Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and 7SK and MRP (25Daly N.L. Arvanitis D.A. Fairley J.A. Gomez-Roman N. Morton J.P. Graham S.V. Spandidos D.A. White R.J. Oncogene. 2005; 24: 880-888Crossref PubMed Scopus (34) Google Scholar). RT-PCR of EBER1 RNA used primers 5′-CCTAGAGGTTTTGCTAGG-3′ and 5′-GACCACCAGCTGGTACTT-3′ to give a 143-bp product; cycling parameters were 95 °C for 150 s, followed by 25 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s and then 5 min at 72 °C. Chromatin Immunoprecipitation—ChIP assays were performed as previously described (26Gomez-Roman N. Grandori C. Eisenman R.N. White R.J. Nature. 2003; 421: 290-294Crossref PubMed Scopus (316) Google Scholar) using antibodies Ab7 against TFIIIC220, 3238 against TFIIIC102, 1H4 against EBNA1 (27Murray P.G. Niedobitek G. Kremmer E. Grasser F. Reynolds G.M. Cruchley A. Williams D.M. Muller-Lantzsch N. Young L.S. J. Pathol. 1996; 178: 44-47Crossref PubMed Scopus (41) Google Scholar), M-19 against TAFI48, H-79 against c-Jun, and N-96 against ATF-2 (Santa Cruz Biotechnology). Amplification of the EBER1 gene region used the same primers and cycling parameters as above. Primers and PCR conditions have been described for ARPP P0, tRNALeu, and 5S rRNA genes (24Winter A.G. Sourvinos G. Allison S.J. Tosh K. Scott P.H. Spandidos D.A. White R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12619-12624Crossref PubMed Scopus (100) Google Scholar, 26Gomez-Roman N. Grandori C. Eisenman R.N. White R.J. Nature. 2003; 421: 290-294Crossref PubMed Scopus (316) Google Scholar). Primers 5′-GTTTGCAGTTCCCCTGGTTAC-3′ and 5′-CTTCGTCCAACAACGACTCC-3′ were used for the amplification of the TFIIIC220 promoter region, and the primer pair 5′-TCTCCCCTTTTTGACACTGC-3′ and 5′-AGGGGGAGGAGTAATTGTGG-3′ was used for amplification of the TFIIIC110 promoter region. For both TFIIIC220 and TFIIIC110, amplification conditions were 94 °C for the initial denaturation, followed by 27 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s and finally 4 min at 72 °C. Promoter Constructs and Mutagenesis—The TFIIIC220 primers above were used to amplify from HeLa genomic DNA a fragment that was cloned into pGEM-T easy vector (Promega) to give pGEM-220. Reporter construct pGL-220 was then made by digesting pGEM-220 with SpeI and NcoI and subcloning the resultant fragment into pGL3-Basic vector (Promega) treated with NcoI and NheI. pGL-220 contains a 261-bp fragment extending from 167 bp upstream of the predicted TFIIIC220 transcription start site to 94 bp downstream. PCR mutagenesis (28Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar) was used to introduce substitutions into the CRE of the TFIIIC220 promoter in pGEM-220, with 5′-CGGGGAGTGTGGTCATGCCGGC-3′ and 5′-GCCGGCATGACCACACTCCCCG-3′ overlapping oligonucleotides and SP6 and 5′-GGGCGTATCTCTTCATAGCC-3′ flanking primers. The fragment was then treated with SpeI and NcoI restriction endonucleases and cloned into NheI- and NcoI-digested pGL3-Basic vector to give pGL220mt. PCR mutagenesis was also used to mutate the EBER2 CRE in construct E2–160 (29Howe J.G. Shu M.-D. Cell. 1989; 57: 825-834Abstract Full Text PDF PubMed Scopus (124) Google Scholar), with SP6 and T7 flanking primers and 5′-CAACCGCAGCGTAGCTGTTTACCAGC-3′ and 5′-GCTGCGGTTGTAGGCGGGGTTAAGCG-3′ as overlapping oligonucleotides. PCR product containing the mutated CRE was treated with BamHI and EcoRI and then cloned back into the original plasmid digested with the same restriction enzymes. The full-length BDP1 cDNA was PCR subcloned from a modified pSBET construct generously provided by Nouria Hernandez (30Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar) and inserted into a modified version of the pCDNA3 expression vector encoding two N-terminal hemagglutinin tags to give pCDNA3HA.Bdp1. Transfection Assays—HeLa cells were transfected in Opti-MEM medium (Invitrogen) using Lipofectamine (Invitrogen) transfection reagent. Cells were incubated for 6 h in transfection mixture and then in fresh medium for an additional 40 h before harvesting. For luciferase assays, extracts were made in passive lysis buffer (Promega) according to the manufacturer's instructions. Luciferase levels were then quantified using a Luminoskan Ascent Luminometer (Labsystems) using the dual-luciferase assay kit (Promega). Firefly luciferase activity was normalized against Renilla luciferase activity from cotransfected plasmid Ubi-Renilla (31Kassel O. Schneider S. Heilbock C. Litfin M. Gottlicher M. Herrlich P. Genes Dev. 2004; 18: 2518-2528Crossref PubMed Scopus (87) Google Scholar). For RT-PCR analysis, RNA was extracted 48 h after transfection using TRI reagent (Sigma) according to the manufacturer's instructions. Protein for Western blots was extracted as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Transcription and Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays used gel-purified oligonucleotide probes that were 5′ end-labeled with polynucleotide kinase and nuclear “mini-extracts” prepared as described (32Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). Mini-extract (6 μg) was preincubated for 15 min at room temperature with 0.5 μg of poly(dI-dC) and unlabeled competitor oligonucleotides, as specified, in 12.5 mm Hepes, pH 7.9, 5 mm MgCl2, 15% glycerol, 1 mm dithiothreitol, 1 mm EDTA, 50 μg/ml bovine serum albumin, 0.025% Nonidet P-40. Probe (1 ng) was added and the incubation continued for a further 15 min before loading onto a 5% non-denaturing polyacrylamide gel, which was run in 0.5× TBE (Tris borate-EDTA) at 4 °C. In vitro transcription assays were carried out as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Partially purified pol III and TFIIIB fractions were prepared by sequential chromatography of HeLa nuclear extract on phosphocellulose and A25 DEAE-Sephadex as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). TFIIIC was partially purified by sequential chromatography of HeLa nuclear extract on phosphocellulose and heparin-Sepharose CL-6B as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Further TFIIIC purification was achieved using a DNA affinity column carrying the B-block promoter sequence of the VA1 gene as previously described (18White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). EBV Can Stimulate Expression of Cellular pol III Transcripts and TFIIIC in HeLa Cells—Several DNA tumor viruses have been found to stimulate transcription of cellular pol III templates (reviewed by Ref. 33White R.J. Landes Bioscience, Austin. 2002; www.eurekah.comGoogle Scholar). To test whether this is also the case for EBV, we used RT-PCR to assay expression of pol III transcripts. Representatives were examined of each of the three types of promoter arrangement that are used by pol III (Fig. 1A). 5S rRNA genes have type 1 promoters comprising A- and C-blocks embedded within the transcribed region; tRNA genes have type 2 promoters composed of A- and B-blocks that are also located within the transcribed region; 7SK, MRP, and U6 snRNA genes have type 3 promoters that involve TATA boxes and proximal sequence elements that are located upstream of the transcription start site (33White R.J. Landes Bioscience, Austin. 2002; www.eurekah.comGoogle Scholar, 34Geiduschek E.P. Kassavetis G.A. J. Mol. Biol. 2001; 310: 1-26Crossref PubMed Scopus (302) Google Scholar, 35Schramm L. Hernandez N. Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (438) Google Scholar). EBER gene promoters can be considered a hybrid, with internal A- and B-blocks that are typical of type 2 promoters, as well as upstream motifs, including a TATA box, that contribute to transcription (29Howe J.G. Shu M.-D. Cell. 1989; 57: 825-834Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 36Howe J.G. Shu M.-D. Mol. Cell. Biol. 1993; 13: 2655-2665Crossref PubMed Scopus (24) Google Scholar, 37Niller H.H. Salamon D. Ilg K. Koroknai A. Banati F. Bauml G. Rucker O.L. Schwarzmann F. Wolf H. Minarovits J. Med. Sci. Monit. 2003; 9: 1-9Google Scholar). The cellular 7SL genes have an upstream promoter arrangement similar to the EBER genes, as well as important internal promoter sequences (36Howe J.G. Shu M.-D. Mol. Cell. Biol. 1993; 13: 2655-2665Crossref PubMed Scopus (24) Google Scholar, 38Ullu E. Weiner A.M. Nature. 1985; 318: 371-374Crossref PubMed Scopus (124) Google Scholar, 39Kleinert H. Gladen A. Geisler M. Benecke B.-J. J. Biol. Chem. 1988; 263: 11511-11515Abstract Full Text PDF PubMed Google Scholar, 40Muller J. Benecke B.-J. Biochem. Cell Biol. 1999; 77: 431-438Crossref PubMed Scopus (13) Google Scholar). Levels of 5S rRNA, tRNA, and 7SL RNA were found to be significantly elevated in EBV-positive HeLa cells when normalized to the control mRNA encoding acidic ribosomal phosphoprotein P0 (ARPP P0), a pol II product (Fig. 1B). This suggests that EBV, like several other DNA tumor viruses, may stimulate pol III transcription. However, none of the type 3 promoter products examined (U6, 7SK, and MRP RNA) showed evidence of induction. Thus, the stimulatory effect of EBV on cellular class III gene expression shows clear selectivity. TFIIIC is a pol III-specific transcription factor that is required by type 1 and 2 promoters, but not by type 3 (33White R.J. Landes Bioscience, Austin. 2002; www.eurekah.comGoogle Scholar, 34Geiduschek E.P. Kassavetis G.A. J. Mol. Biol. 2001; 310: 1-26Crossref PubMed Scopus (302) Google Scholar, 35Schramm L. Hernandez N. Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (438) Google Scholar). Because EBV induces types 1 and 2, but not type 3, we examined whether it is regulating TFIIIC. Western blotting demonstrated that all five subunits of TFIIIC are expressed at elevated levels in HeLa cells infected with EBV, whereas actin appears unchanged (Fig. 1C). The mRNA encoding the TFIIIC220 subunit is overexpressed in the EBV-positive HeLa cells (Fig. 1D), but there is little consistent change in levels of the mRNAs for the other four subunits of TFIIIC (two distinct transcripts are detected from the TFIIIC102 gene, probably due to alternative splicing). We conclude that EBV can induce TFIIIC and that this may, under some circumstances, involve a level of post-transcriptional control that has not been reported previously. EBV Induces TFIIIC Expression in Several Cell Types—In addition to HeLa, we examined whether EBV can activate the pol III machinery in other cell types. Two models of EBV-induced carcinomas were tested, the Hone1 cell line, which was isolated from an NPC, and the Ad/AH line, which is derived from an adenocarcinoma of the nasopharynx (41Takimoto T. Ogura H. Ohno S. Umeda R. Hatano M. J. Natl. Cancer Inst. 1984; 73: 711-715PubMed Google Scholar). As in HeLa cells, EBV infection of Ad/AH and Hone1 cells stimulates expression of 5S rRNA, tRNA, and 7SL RNA, but not 7SK or MRP RNA (Fig. 2A). U6 RNA levels increase slightly in Ad/AH cells, but not in Hone1. Viral induction of pol III transcription therefore shows specificity. In both these cell lines, it is accompanied by a clear increase in the mRNAs encoding all five subunits of TFIIIC (Fig. 2B). Elevated TFIIIC expression may therefore be a common feature of EBV-infected carcinoma cells. As a model of Burkitt lymphoma, we used the Japanese BL-derived Akata line alongside a matched EBV-negative subclone that was isolated from parental Akata cells by limiting dilution (42Shimizu N. Tanabe-Tochikura A. Kuroiwa Y. Takada K. J. Virol. 1994; 68: 6069-6073Crossref PubMed Google Scholar). As in the carcinoma models, expression of 5S rRNA, 7SL RNA, and tRNA is elevated specifically in EBV-positive Akata cells when compared with the EBV-negative derivative, whereas type III promoters show no evidence of activation (Fig. 2C). Similarly, the virally infected Akata cells overexpress mRNAs encoding the five TFIIIC subunits (Fig. 2D). EBV can therefore induce TFIIIC expression and endogenous pol III products in cells of disparate origin, representing virally induced lymphomas and carcinomas. TFIIIC Binds to EBER Genes in Vivo—The DNA sequences recognized by TFIIIC are the A- and B-block internal promoter elements found in most pol III-transcribed genes, including tRNA and EBER genes (33White R.J. Landes Bioscience, Austin. 2002; www.eurekah.comGoogle Scholar, 35Schramm L. Hernandez N. Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (438) Google Scholar). However, as mentioned above, EBER promoters also have upstream elements that are important for transcription (29Howe J.G. Shu M.-D. Cell. 1989; 57: 825-834Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 43Wensing B. Stuhler A. Jenkins P. Hollyoake M. Karstegl C.E. Farrell P.J. J. Virol. 2001; 75: 6235-6241Crossref PubMed Scopus (27) Google Scholar). Deletion of the EBER2 B-block was found to ablate expression both in vitro and in vivo (29Howe J.G. Shu M.-D. Cell. 1989; 57: 825-834Abstract Full Text PDF PubMed Scopus (124) Google Scholar), but interpretation of this result is complicated by its location within the transcribed region, as loss of expression might reflect destabilization of the mutated transcript. Indeed, genomic footprinting revealed little evidence for TFIIIC occupancy at EBER1 or EBER2 (37Niller H.H. Salamon D. Ilg K. Koroknai A. Banati F. Bauml G. Rucker O.L. Schwarzmann F. Wolf H. Minarovits J. Med. Sci. Monit. 2003; 9: 1-9Google Scholar). We therefore considered it important to establish whether EBER genes utilize TFIIIC in vivo. To this end, we carried out ChIPs to assay occupancy in EBV-infected HeLa cells (Fig. 3A). As positive control, we confirmed that the viral EBNA1 protein is bound in the vicinity of the EBER locus, as expected due to the proximity of its oriP recognition site within the viral genome (43Wensing B. Stuhler A. Jenkins P. Hollyoake M. Karstegl C.E. Farrell P.J. J. Virol. 2001; 75: 6235-6241Crossref PubMed Scopus (27) Google Scholar). Clear evidence for the presence of TFIIIC was obtained using antibodies against TFIIIC220 and TFIIIC102. An antibody against the pol I factor TAFI48 provided a negative control. Additional evidence of specificity was provided by the use of EBV-negative HeLa cells, which lack EBER genes and therefore give no signal. These data provide evidence that TFIIIC does indeed interact with the EBER genes in vivo. In contrast, TFIIIC was not detected at the pol II-transcribed acidic ribosomal phosphoprotein P0 gene (Fig. 3B). The ChIP assay was also used to assess whether EBV infection changes the amount of TFIIIC that is bound to endogenous class III genes. This revealed that TFIIIC occupancy of chromosomal tRNA genes is significantly elevated in the EBV-positive cells (Fig. 3A). As expected, neither EBNA1 nor TAFI48 were detected at these genes. We conclude that the elevated expression of TFIIIC following EBV infection can increase its occupancy of cellular target genes. ATF2 Activation May Increase Transcription of the EBER and TFIIIC220 Genes—In addition to the internal promoter that provides a binding site for TFIIIC, both the EBER genes also have a consensus ATF recognition site (CRE) located ∼50 bp upstream of the transcription start site (Fig. 4A). Deletion or point mutation of these sequences compromises expression of EBER1 and EBER2 in HeLa and BL cells (29Howe J.G. Shu M.-D. Cell. 1989; 57: 825-834Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 43Wensing B. Stuhler A. Jenkins P. Hollyoake M. Karstegl C.E. Farrell P.J. J. Virol. 2001; 75: 6235-6241Crossref PubMed Scopus (27) Google Scholar). We observed a similar effect in extracts of the Hone1 NPC line. Thus, point mutation of the CRE caused a marked reduction in EBER2 transcription relative to the wild type (Fig. 4B). This was the case for both EBV-positive and EBV-negative Hone1 cell extracts. Fig. 4B also shows that extracts from EBV-positive cells give elevated pol III transcription relative to matched extracts from EBV-negative cells. This difference is not restricted to the EBER2 gene but is also seen with other pol III templates, including the adenoviral VA1 gene, which has a TFIIIC-dependent type 2 promoter (Fig. 4C). The relative weakness of the EBER2 promoter is apparent, giving much less transcription than the powerful VA1 template (Fig. 4C). The occupancy of the EBER CRE motifs has been confirmed in vivo by genomic footprinting (37Niller H.H. Salamon D. Ilg K. Koroknai A. Banati F. Bauml G. Rucker O.L. Schwarzmann F. Wolf H. Minarovits J. Med. Sci. Monit. 2003; 9: 1-9Google Scholar). However, the footprint did not show which CRE-binding protein(s) occupies these motifs. Several members of the AP-1 and ATF families have been shown to interact with CRE sequences (44Eferl R. Wagner E.F. Nat. Rev. Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1576) Google Scholar). We found by ChIP that ATF2 associates with the EBER genes, but not with 5S rRNA genes (Fig. 4D). Only background amplification was observed with negative control antibody against TAFI48. Our data do not address the full constellation of factors that may bind the CRE in EBER promoters but do provide evidence for the presence of ATF2 in vivo. ATF2 can be activated by mitogen-activated protein kinases that phosphorylate residue Thr-71 within its transactivation domain (45Gupta S. Campbell D. Derijard B. Davis R.L. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar, 46van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (569) Google Scholar). EBV has been shown to activate mitogen-activated protein kinase pathways and trigger hyperphosphorylation and activation of ATF2 (1Young L.S. Rickinson A.B. Nature Rev. 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