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The Epstein‐Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis‐element of the terminal protein 1 gene promoter.

The Epstein‐Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis‐element of the... The EMBO Journal vol. 12 no. 1 pp. 167 - 175, 1993 The Epstein Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter Ursula Zimber-Strobi, Elisabeth transformed cell lines. In Kremmer1, lymphoblastoid these immortalized cell Friedrich lines the virus is maintained as an Grasser2, Gabriele Marschall, molecule episomal at number et Gerhard Laux high copy (Lindahl al., 1976) and only a few and Georg W.Bornkamm viral genes are expressed. These genes code for six nuclear Institut fOir Klinische Molekularbiologie und Tumorgenetik, Institut fiir antigens, 3c and and EBNA1, 2, 3a, 3b, -LP, three Immunologie, im Forschungszentrum fiir Umwelt und Gesundheit, membrane the latent proteins, membrane protein (LMP) and GSF, Marchioninistrasse 25, 8000 Munchen and 2lnstitut fOir terminal and also proteins (TPs) 1 2, denoted LMP2A Medizinische by Mikrobiologie, Universititsklinikum, Haus 47, 6650 Homburg/Saar, and B review see Kieff and Germany (for Liebowitz, These 1990). gene seem to be for products important induction and Communicated by W.Schaffner maintenance of B-cell immortalization. However, the individual functions of these gene products in latent infection The Epstein-Barr virus protein EBNA2 acts as a and growth transformation are only poorly transcriptional activator of cellular and understood. viral genes and EBNA2, one of the best plays a crucial role in studied latent gene products, is the immortalization of human absolutely necessary for B-cell immortalization. primary B-cells by EBV. We have One viral shown previously that mutant EBNA2 (P3HR1) carrying a 6.6 kb deletion transactivates the promoters (Bornkamm of the latent et membrane al., 1982; Jeang and Hayward, that antigens LM1P, TP1 and TP2. 1983) removes The promoter EBNA2 and of of the TPI part EBNA-LP, has lost the ability to gene was chosen as a model system to study transform primary B-cells. the molecular Reintroduction of EBNA2 into mechanism of EBNA2 mediated the P3HRl virus transactivation. To by homologous recombination reconstituted identify an EBNA2 dependent cis- the full acting transforming capacity (Cohen et al., element, various TPI promoter-reporter 1989; gene Hammerschmidt and Sudgen, In constructs were in 1989). natural EBV transfected the absence and isolates, presence two alleles of the of an EBNA2 EBNA2 gene exist coding for two expression vector into the proteins established B- (EBNA2A and cell line EBNA2B) with 57% homology BL41-P3HR1. We were able (Dambaugh to delineate an 81 et al., 1984; Adldinger et bp EBNA2 al., 1985; Zimber et al., responsive region between 1986). -258 and -177 There have been some reports that relative to the TPI EBV strains RNA start site. The element carrying worked EBNA2A could transform primary B-cells more in either efficiently orientation and could mediate EBNA2 than those containing an EBNA2B allele (Rickinson dependent transactivation on et a heterologous al., promoter. Cohen et 1987; al., 1989). The EBNA2 gene codes for a Electrophoretic mobility shift assays revealed three 487 amino acid phoshorylated polypeptide (Grasser et specific protein-DNA complexes al., formed with sequences 1991). Characteristic features of the of the EBNA2 primary structure of the responsive element. Two of these were not protein include a proline stretch of -40 amino cell type but the acids near specific, third was detected in only the N-terminus, a negatively charged C-terminus and EBNA2 positive cell a extracts. Gel-shift analysis in the glycine/arginine-rich region. It has recently been shown presence of EBNA2 that specific monoclonal antibodies EBNA2 acts as a transcriptional activator: it stimulates the revealed that EBNA2 is a component of the third transcription of the cellular genes CD21, CD23 and complex. Thus, these c-fgr experiments demonstrate that (Calender et al., 1987; EBNA2 Wang et al., 1987; Abbot et interacts with an EBNA2 al., responsive cis-element 1990; Cordier et al., 1990; Knutson, as well as of the 1990) the TPI promoter. expression of the viral words: genes LMP, TPI and TP2 Key DNA -protein - (Fahraeus interaction/EBNA2/Epstein et al., 1990; Ghosh and Barr 1 Kieff, 1990; Zimber-Strobl et virus/terminal protein /transactivation al., 1991). Although a transactivation domain has been mapped in the C-terminus of EBNA2 (Cohen and Kieff, 1991), it is not yet known whether EBNA2 transactivates several Introduction genes by interacting with EBNA2 responsive cis-elements Epstein-Barr virus a human or whether it does so (EBV), widespread by modulating the expression or herpesvirus, is the of infectious modification of transcription factors. A consensus aetiological agent sequence mononucleosis, self-limiting lymphoproliferative disease. in the promoter of the region EBNA2-regulated genes has The virus is associated with two human not been described until malignancies, now. namely Burkitt's B-cell and lymphoma (a To elucidate the mechanism of EBNA2 neoplasia) mediated which is of nasopharyngeal carcinoma, composed transactivation, we focused our interest on the TPI poorly promoter differentiated cells. EBV has a dual it TP epithelial region. The gene is transcribed across the tropism: terminal repeats infects primary B-cells and cells. Virus of the EBV resting epithelial genome (Laux et al., 1988; Sample et al., 1989). production occurs in whereas B-cells A functional only epithelial cells, transcription unit can only be created if the linear molecule are infected. In vitro infection of B- is circularized. The TATA-box of the TPI latently resting lymphocytes leads to the of virus- promoter is located at the hand outgrowth permanently right end of the linear viral 1 67 © Oxford Press University U.Zimber-Strobl et al. III -45 .zzzL- rzzzzzzzzzz4llll - 124 - 177 - 258 %acetylation Stimulation BL41-P3HR1/ BL41-P3HR1 BL41-B95-8 CAT-constructs "IlllzzzriI -45 2.1 2.1 1 0.8 1.4 1.75 -124 -177 1.5 1.2 0.8 -258 1.0 10.5 10.5 - 804 7.2 10.4 -432 0.7 0.3 5.3 17.7 -543 .. 77777M,,,,{,,,,zz//d A -1 47/-258 RLU 10000 - 1 E I1 I1 0 l n.,I tHi I1 -124 -177 -258 -432 -543 -804 A -147/ transfected -45 LUC-constructs -258 fold induction 1,1 1,2 25,6 50,9 57,2 71 1,7 0,72 EBNA2 by constructs in the absence and of EBNA2. Schematic of the Fig. 1. Transfection of different TPI promoter-reporter gene presence (A) representation The restriction used to construct the recombinant plasmids are indicated. The positions of the TPJ TPJ promoter-reporter gene constructs. enzymes denote their 5'-end relative to the TPJ transcription start site. Open boxes represent the reporter gene, hatched boxes the TPI promoter fragments CAT activities after transfection of the TPI promoter-CAT constructs into EBNA2 negative BL41-P3HR1 cells and EBNA2 promoter region. (B) BL41-B95-8 cells. Median activities of two independent experiments and the fold inductions by EBNA2 are indicated. (C) Relative light positive after of constructs in the absence and of EBNA2 expression vectors into units (RLU) measured transfections TPI promoter-LUC presence BL41-P3HR1 cells. transfections without EBNA2; hatched boxes, cotransfection with the EBNA2 expression vector pU294-6, coding Open boxes, for wild-type EBNA2; dotted and black boxes, cotransfections with EBNA2 expression vectors p604 and pU812/9 respectively, coding for mutated EBNA2. The median activities of two to five independent experiments and standard deviations are indicated. at 166 469 of the B95-8 virus DNA Results genome position sequence (Baer et al., 1984). Identification of an EBNA2 responsive cis-element In this report we describe an element that mediates EBNA2 to the In addition within the TP1 promoter region transactivation by TPI promoter. we demonstrate specific protein-DNA interactions within EBNA2 transactivated a TPJ promoter-CAT construct this and evidence that EBNA2 itself is a containing 804 bp upstream of the TPI cap site (Zimber- region provide Strobl et component of this protein-DNA complex. al., 1991). Plasmids containing progressive 168 EBNA2 interaction with TP1 promoter A B B. * ..* *---------@@ - EBNA2 - - - - Mut - WT Mut WT Mut W1 MLIt W7 VT EDNA2 2 .glob.- CMV- CAT- TATA LTP conStructs 135bp1t 135bp 2 81 DP 2 8l0E mrinimal CMV- CAT- zf ... 2 5 0,7 100 acetylation I/. LTR corstructs promoter 1,2 17 0.9 1 8 37 1.0 3 22 1.8 0.5 2 0.2 0.2 0,2 100 % acetylation Fig. 2. 81 bp (-177 to -258) of the TPI promoter are sufficient to mediate transactivation by EBNA2. (A) Results of CAT assays of transfections of CAT constructs containing the EBNA2 responsive region (pTPl-CAT/135bp and pTP1-CAT/81bp) in front of the TPI minimal promoter (pTPI-CAT/-45) without EBNA2 (-), with pU294-6 coding for wild-type EBNA2 (WT) and with p604 coding for mutated EBNA2 (Mut) into cells. The element was cloned in the correct (135bp/l, 81bp/1) and inverse (135bp/2, 81bp/2) orientations. (B) CAT assays after BL41-P3HR1 transfections of CAT constructs containing the TPI promoter region from position - 124 to -258 in front of the ,3-globin minimal promoter into without EBNA2 (-) and with the EBNA2 expression vector pU294-6 (+). The element is cloned in either orientation (f135bp/1, BL41-P3HR1 cells inverse). correct; ,B135bp/2, truncations of the 5' region of the TPI promoter linked to LUC constructs containing at least 258 bp of the TPI 5' flanking region were inducible by EBNA2 (by factors of the reporter genes chloramphenicol acetyltransferase (CAT) three smallest constructs were 25-71), whereas the or luciferase (LUC) were constructed (Figure IA) to identify EBNA2. cis-element within this promoter. unresponsive to an EBNA2 responsive that a specific EBNA2 responsive constructs were transiently transfected into the EBNA2 In order to demonstrate These we removed the TPI promoter BL41-P3HR1 cell line and into EBNA2 positive element has been identified, negative BL41-B95-8 cells. A cytomegalovirus enhancer-long region between positions -258 and -147 in the TPI terninal construct (CMV-LTR-CAT) was used promoter reporter gene construct pTP1-LUC/-543, leaving repeat-CAT intact. This construct as a positive control. the boundaries of the TPI promoter into BL41-P3HR1 cells together with the Transfections of the TPI promoter-CAT constructs into was transfected vectors mentioned above. As shown in BL41-P3HR1 cells resulted in very low or undetectable CAT EBNA2 expression IC, induction of LUC activities by EBNA2 could activities 1B). Transfections of constructs containing Figure (Figure not be observed after transfection of the TPI promoter LUC -45, -124 and -177 bp upstream of the TPI cap site into construct lacking the TI' promoter region between -258 BL41-B95-8 cells resulted in low CAT activities similar to into BL41-P3HRI cells. -147. This was confirmed by transfection of the same those obtained with transfections and activities increased after construct with the CAT reporter gene (data not shown). However, CAT significantly of CAT constructs containing sequences These data show that sequences between -258 and -147 transfections mediated transactivation. are required for EBNA2 of position -177 relative to the TPI cap site upstream (Figure 1B). These results suggested that an EBNA2 element is orientation The EBNA2 responsive responsive region is located between -177 and -258. active on a heterologous promoter in order to independent and We performed cotransfection experiments element was cloned in front of the TPI minimal the increased TPI promoter activity in The putative prove that and the CAT reporter gene to study cells is mediated by EBNA2. TPI promoter promoter (+60/-45) BL41-B95-8 the EBNA2 responsive region is active even when constructs driving the reporter gene luciferase were whether deletion from its TPI promoter region. Two DNA transfected into BIAl-P3HRI cells either without EBNA2 separated cognate constructs were made carrying a larger (-124 to -258) and expression vector or with or with an EBNA2A (pU294-6) to in both orientations. a smaller (-177 -258) EBNA2A vectors carrying mutations within the fragment expression the EBNA2 responsive element The EBNA2 mutation In these constructs suggested EBNA2 open reading frame (ORF). vector sequences. The distance of the insertion after one-third of the EBNA2 is surrounded by contains a linker p604 box is to the native from the TPI TATA comparable to a truncated EBNA2 In the element leading gene product. ORF, All DNA constructs were transfected in the absence amino acids 248-382 of situation. EBNA2 mutation pU812/9, vectors into of EBNA2 expression are deleted. The EBNA2 and its mutants are and presence EBNA2 gene and for cells. p604, coding wild-type the control of the natural promoter of EBNA2. BL41-P3HR1 pU294-6 under were used as EBNA2 and truncated EBNA2, respectively, relative units of the LUC assays The obtained light (RLU) vectors. Transfection of all constructs EBNA2 expression data described above IC). Only confirmed the CAT (Figure 169 U.Zimber-Strobi et al. .,PI~~~~k ..wsU+bStWof~~~~~~~~~~~~~~ I':, . -- Bf n. ^^~~~~~~~- - 0 -i to fa mm . is .. I _ ^ Fig. 3. Analysis of DNA-protein interactions within the TPI free promoter. Autoradiogram of an EMSA is shown made with nuclear oligo with a extracts of BL41-P3HRI cells which were incubated 32p- labelled fragment spanning the TPI promoter region from position -12 to -258 relative to the start. TPI transcription Competition studies were with the EBNA2 performed oligonucleotides covering Fig. 4. EMSAs using 054bp (EBV positions 166 236-166 289 responsive region identified in the CAT and LUC The analyses. according to the EBV sequence of Baer et al., 1984) as radioactively positions of the in the EBV are described in oligonucleotides genome labelled probe and nuclear extracts of the promyelocyte line HL60, Materials and methods. Unlabelled oligonucleotides were added to the cervical carcinoma line HeLa, the EBV negative B-cell line BL41 and gel-shift reaction in a 100-fold molar excess. four EBV B-cell lines: positive BL41-P3HRI (EBNA2 negative), HH514 (EBNA2 negative), Jijoye (EBNA2B) and M-ABA (EBNA2A). resulted in a CAT in the significant higher activity presence of the EBNA2 vector of the expression pU294-6, regardless to - 177), four overlapping oligonucleotides [01 orientation of the element relative to the promoter (-262/-230), 02 (-239/-209), 03 (-219/-181) and 04 (Figure 2A). (-196/-158)] covering these sequences were synthesized To determine whether the EBNA2 responsive region and added to BL41-P3HR1 extracts. I and III Complexes contains that can a be with 01 02 sequences regulate heterologous could competed and covering sequences promoter, we cloned the TPI from between promoter region positions -262 and -209. None of the complexes could be -124 to -258 upstream of the 3-globin minimal promoter the competed by adjacent oligonucleotides 03 and 04. (Westin et al., 1987). Cotransfections of the EBNA2 In order to the interaction of TPI study promoter vector in expression pU294-6 resulted significantly increased sequences from position -262 to -209 with cellular proteins CAT activities in the of presence reporter plasmids carrying in more detail, the 54 bp fragment was end-labelled and the EBNA2 responsive region (Figure 2B). In this assay, incubated with nuclear extracts of several cell lines (HL60, the element conferred EBNA2 inducibility in an orientation M-ABA HeLa, BL41, BL4l-P3HR1, HH514, Jijoye, and fashion as has been independent demonstrated by the TPI Raji) (Figures 4 and Similar were SA). complexes formed promoter constructs. with the 54 bp fragment as with the 247 bp fragment used From these data we conclude that an 81 bp region within in the EMSA described above. Again one prominent (I) and the TPI promoter harbours an EBNA2 responsive cis- two weaker complexes (II and III) could be detected. which will element, we call EBNA2RE. EBNA2RE is active I -III were formed in Complexes all cell extracts tested and in both orientations and able to regulate a heterologous therefore seem to be cell-type unspecific. Notably, additional promoter. and could be complexes (IVA IVB) detected with extracts of the EBNA2 cell lines M-ABA positive Jijoye, and Raji, Specific DNA -protein interactions within the EBNA2 which were not found with extracts of EBNA2 cell negative responsive region lines. Gel-retardation assays were performed to identify specific The of specificity the DNA-protein interactions was DNA-protein interactions within the TPI promoter. A in analysed competition experiments following addition of DNA fragment spanning the TPI promoter from position the unlabelled 54 bp oligonucleotide to EMSAs with Raji, -12 to -258 was end-labelled and incubated with nuclear HH514, Jijoye and M-ABA extracts (Figure 5A and B). extracts of BL41-P3HR1 cells. The result of the Only complex II could not be competed and appears to be electrophoretic mobility shift (EMSA) is shown in Figure 3. unspecific. Additionally, we tested the ability of 0 1 and 02 EMSA revealed one prominent DNA-protein interaction to prevent formation of protein-DNA complexes formed (complex I) and two weaker complexes (II and III). To with the 54 bp oligonucleotide and nuclear proteins of Raji investigate whether some of these complexes are formed with cells. All specific complexes could be competed by 0 1 and sequences located in the EBNA2 responsive region (-258 02 with the same efficiency as by 054bp (Figure 5A). The 170 EBNA2 interaction with TP1 promoter 54bp 01 02 llrn - a -.. __. a- -IV - - a -a S IVA --. -- IVb _ d :1 * I- III 'l1~' b ;t' £ * *. X _ free oligo free ol!go 262 - 209 ~ ~ -:1 ~ -- -- -2-2 Fig. 5. Competition assays demonstrate the specificity of complexes I, III and IVA. (A) Autoradiogram of an EMSA. Nuclear extracts of Raji cells (EBNA2A positive B-cell line) were incubated with the labelled oligonucleotide 054bp. Unlabelled oligonucleotides 054bp, 01, 02 and 1 lmut were added to the reaction mixture in 10-, 50- and 100-fold excesses. The DNA-protein complexes were separated on a 5% polyacrylamide gel. (B) Nuclear extracts of HH514, Jijoye and M-ABA cells (indicated by 1, 2 and 3 respectively) were incubated with the radioactively labelled 054bp oligonucleotide. For competition unlabelled oligonucleotide 054bp was added in 100-fold excess to the reaction mixture. The resulting products were on a 4% polyacrylamide gel. (C) Schematic representation of the oligonucleotides used in the EMSA. The indicated positions correspond to separated the 7PJ transcription start site. An 11 bp motif duplicated in 054bp is marked by dotted boxes, the 10 bp overlapping region of 01 and 02 by hatched boxes. two share a 10 bp overlapping region and low affinity for EBNA2B in oligonucleotides EBNA2A but has only a very a common motif of 11 bp (Figure 5C). To test whether the et in preparation). Two immunoprecipitation (Kremmer al., 1 -DNA interaction, nonspecific antibodies, raised against murine CD45, of the I bp motif was involved in the protein 1 imut, Figure 5C). same as the anti-EBNA2-Rl and anti-EBNA2-R2 these sequences were truncated (oligo isotype the truncated oligonucleotide has as a control. The presence of the anti- As shown in Figure 5A, antibodies were used lost the to prevent formation of the EBNA2-R1 resulted in a supershift of complex IVB completely ability antibody DNA-protein complexes. This suggests that the 11 bp motif in and IVA in M-ABA extracts (Figure 6A). Jijoye complex EBNA2 EBNA2A is important for protein -DNA interaction within the contrast, anti-EBNA2-R2, which recognizes By but EBNA2B with a low affinity, responsive region. with a high affinity very of IVA but not of IVB. led to a supershift complex complex control antibodies had no effect on the mobility of EBNA2 is present in DNA -protein complexes formed Both IVA and IVB. with the EBNA2 responsive region complexes lines the with two further Differences between EBNA2 positive and negative cell We have repeated experiments in EMSAs described above were low anti-EBNA2-R3 and anti- detected the mobility EBNA2 monoclonals, IVA and IVB. To test whether EBNA2 was EBNA2-R1 1. Both antibodies recognized EBNA2A, complexes EBNA2-R3. We in of the complexes described above, DNA whereas EBNA2B was recognized only by present any as with the antibodies described above. were performed with HH5 14, Jijoye and M- got the same results binding assays EBNA2B and caused a of but ABA extracts (EBNA2 negative, positive Both antibodies supershift complex IVa, only IVb was able to (data EBNA2A respectively) in the presence and absence anti-EBNA2-R3 supershift complex positive, EBNA2A. The not of monoclonal antibodies raised against shown). is able to detect EBNA2A that IVA contains EBNA2A monoclonal antibody EBNA2-R1 These results imply complex the other anti-EBNA2-R2 IVB EBNA2B. None of as well as EBNA2B, whereas recognizes and complex complexes 171 et al. U.Zimber-Strobi .- !; .I- Er F ! ii: i.: :.Z -, A'.. A BI X f..i... a as i". 6. EBNA2 is a component of complex IV. (A) Effect of monoclonal EBNA2 antibodies on TPJ promoter DNA-protein complexes. Gel-shift Fig. antibodies. and anti-EBNA2-R2 are monoclonal rat antibodies raised against EBNA2A. were incubated with the indicated Anti-EBNA2-Rl reactions as well as anti-EBNA2-R2 EBNA2A with the same affinity as anti-EBNA2-Rl, but only Anti-EBNA2-RI recognizes EBNA2A EBNA2B; recognizes low Incubation reactions were on a 4% gel. Only complexed DNA is visible on EBNA2B with a very affinity. separated polyacrylamide recognizes since the free was run off the 2 and 3 indicate gel-shift reactions with extracts of cell lines HH514 the autoradiogram oligonucleotide gel. 1, and M-ABA respectively. Anti-mouse CD45 IgGI and IgG2a are two control monoclonal (EBNA2 negative), Jijoye (EBNA2B) (EBNA2A) subclasses as anti-EBNA2-RI and anti-EBNA2-R2, respectively, and recognize neither EBNA2A nor EBNA2B. (B) Effect antibodies of the same Ig EBNA2A on the reconstitution of IV. Gel-shift reactions were incubated with the indicated nuclear extracts (NE). 3 Al of in vitro translated complex RNA or BMV RNA and 1 1d of monoclonal antibodies or anti- with EBNA2 supernatant (anti-EBNA2-R3 reticulocyte lysate (IVT) programmed were added as indicated above. The reactions were preincubated for 5 min at room temperature before adding the radioactively mouse CD45) The DNA complexes were analysed on a 4% polyacrylamide gel. The free oligonucleotide was run off the labelled oligonucleotide 054bp. protein gel. monoclonal EBNA2 Discussion were affected by the antibodies, do not contain EBNA2. suggesting that they of EBNA2 to B-cell that EBNA2 is really a component of the It is assumed that the contribution We confirmed -DNA IV the ability of in vitro immortalization is conferred by its ability to transactivate protein complex by studying The EBNA2 ORF process EBNA2 to form this complex. viral and cellular genes. In order to understand the synthesized The in vitro it to was translated in a reticulocyte lysate. of immortalization at a molecular level, is essential of EBNA2 transactivation. EBNA2 was added to the EBNA2 negative elucidate the mechanism mediated synthesized were As We chose the TPI promoter as a model system for BL41-P3HR1 extracts and EMSAs performed. translated EBNA2 formed EBNA2 transcription. As shown shown in 6B the in vitro studying dependent Figure In addition of previously, TPI transcription was dependent on the presence a more slowly migrating complex. contrast, in vitro translated of EBNA2 in Burkitt's lymphoma cells (Zimber-Strobl et al., a control reticulocyte lysate containing mosaic virus RNA did not induce this Our first aim was to identify a cis-acting element brome (BMV) 1991). in vitro translated That the complex induced by mediating EBNA2 responsiveness. Therefore we transfected complex. EBNA2 was confirmed EBNA2 did contain the added by different TPJ promoter-reporter gene constructs in the the effect of monoclonal antibodies. Anti- presence and absence of EBNA2. By this approach we were assaying induced able to an between positions - 177 EBNA2-R3 but not the control antibody (anti-CD45) identify 81 bp element of formed An additional and -258 which is sufficient to confer EBNA2 a supershift the newly complex. in the of nuclear extracts, responsiveness. complex (*) appeared presence EBNA2 and monoclonal anti-EBNA2 antibodies in this The EBNA2 responsive element (EBNA2RE) works in and This complex was not always reproducible either orientation and is able to regulate a heterologous experiment. in vitro is therefore not further considered. Besides the promoter. These results imply that EBNA2 responsiveness translated EBNA2 diminished formation of I and complexes is conferred by an enhancer-like cis-element. This is or not is The III. Whether this effect specific still unclear. observation is in agreement with previous reports. EBNA2 EBNA2 was not able to bind DNA in the responsive elements were also identified within the LMP, in vitro translated absence of nuclear that EBNA2 interacts BamHI-C and CD23 promoters and it has been reported that extracts, suggesting with DNA not directly but indirectly. work in an orientation independent fashion and regulate they 172 EBNA2 interaction with TP1 promoter a heterologous promoter (Sung et al., 1991; Tsang et al., study whether one of the retarded bands contained EBNA2. Addition of EBNA2 antibodies resulted in a supershift of 1991; Wang et al., 1991; Woisetschlaeger et al., 1991). complexes IVA and IVB. An antibody recognizing only We performed EMSAs to identify specific protein-DNA a supershift of complex IVA only, interactions within the TPJ promoter. One prominent EBNA2A induced an antibody specific for EBNA2A and EBNA2B DNA-protein complex (I) and two weaker complexes (II whereas III) were detected with a radioactively labelled fragment retarded migration of both complexes IVA and IVB. This and the TPI promoter from position -12 to -258 indicates that EBNA2B is a component of complex IVB and spanning of the EBNA2RE). It was possible to compete EBNA2A a component of complex IVA. To prove that (5'-end specifically complexes I and III with two oligonucleotides EBNA2 is indeed a component of complex IV, we added in vitro translated EBNA2 to EBNA2 negative BL41-P3HR1 spanning the region -262 to -230 and -239 to -209. EMSAs. The in vitro translated extracts and performed When extracts were mixed with a radioactive oligonucleotide the formation of a more slowly migrating from position -262 to -209 and EMSAs were performed, EBNA2 induced By adding monoclonal antibodies we showed that the same pattern of complex formation occurred. These complex. indicate that protein(s) interact with sequences EBNA2-induced complex contained the added EBNA2. experiments the located in the EBNA2RE. The complex containing the in vitro translated EBNA2 Formation of complexes I, II and III appeared to be slightly faster than complex IVa visible in M-ABA migrated be caused by different independent of EBNA2 and even independent of the cellular extracts. This phenomenon could and in vitro synthesized EBNA2. of B-cells, since they were detected in all cell modifications of the in vivo background 54 bp fragment is sufficient to bind EBNA2, lines studied. This implies binding of a universal transcription Although the We were not able to identify this factor by comparing it is not able to mediate EBNA2 responsiveness (data not factor. the sequence with known consensus sequences of shown), suggesting that sequences between -209 and -177 transcription factors (Ghosh, 1990). The only obvious are necessary for EBNA2 dependent transactivation. It is cognate nuclear factor binding sequence in the 54 bp conceivable that an additional protein binds to these responsiveness. fragment is one SPI consensus sequence at position -240. sequences and contributes to EBNA2 for the first time that EBNA2 is Addition of a 500-fold excess of an unlabelled Here we have shown in complexes of specific DNA sequences and oligonucleotide representing the SPI binding site failed to present Our experiments suggest that EBNA2 interacts compete the shift (data not shown). It is therefore unlikely protein(s). that this SPI site plays an essential role in the observed with DNA indirectly via protein -protein interactions. Such shown to be responsible for DNA -protein interaction. interactions have been VP16 (Lillie and Green, 1989; Formation of all complexes except complex II was transactivation by EIA and Green, 1990) transactivators of competed by an excess of unlabelled 01 as well as 02. These Stern et al., 1989; Liu and share a 10 bp overlapping region and simplex virus, respectively. VP16 two oligonucleotides adenovirus and herpes of 11 bp. The 11 bp motif seems to play binds to the universal transcription factor Oct 1, juxtaposing a common motif transactivation domain of VP16 to the promoter. It seems an essential role in DNA -protein interactions, since an the likely that EBNA2 also interacts with a transcription factor, oligonucleotide containing the complete 10 bp overlap, but in complexI and/or III. We are in the two truncated 11 bp motifs, has lost the ability to inhibit the perhaps contained the factors interacting with the protein binding of the 54 bp oligonucleotide. process of identifying of the TPI promoter, which should further Realizing the importance of the 11 bp motif we searched EBNA2RE EBNA2 mediated transactivation. for this sequence in the EBNA2 responsive regions of the elucidate and LMP promoters which were shown to be BamHI-C EBNA2. Sung et al. (1991) reported an EBNA2 activated by Materials and methods responsive region of 98 bp within the BamHI-C promoter between positions -367 and -465. In the LMP promoter, conditions Cell lines and culture described an element of 30 bp between Raji and BL41 have been described elsewhere (Crawford Tsang et al. (1991) Cell lines M-ABA, et 1979; Pulvertaft, 1965; Lenoir et al., 1985). HH514 is a single cell al., -205 which is essential although not positions -234 and clone of Jijoye, carrying a deletion in the EBNA2 gene (Hinuma et al., 1967; for EBNA2 responsiveness. Remarkably, the sufficient BL41-B95-8 were obtained after Miller et al., 1974). BL41-P3HR1 and promoter contains, at position -371, a 10 bp BamHI-C cell line BL41 with virus infection of the EBV negative Burkitt's lymphoma that is identical to the 11 bp motif, and the LMP et al., 1987). HL60 is sequence strains P3HR1 and B95-8, respectively (Calender line described by Collins et al. (1978). a promyelocyte cell contains, at position -218, a sequence that shares promoter in RPMI 1640 medium supplemented with 10% All cell lines were grown 11 bp motif. Recently it was shown 7 bp (GTGGGAA) of the 100 U/mI and 100 streptomycin. Cultures fetal calf serum, penicillin [tg/mn that parts of the 10 bp motif of the by footprint analyses at 37°C in an atmosphere of 5% CO2. Cells were diluted were incubated interact with a DNA binding protein (Jin BamHI-C promoter medium twice a week. 1:3 with fresh 1992). Taking this into account, it seems likely and Speck, Plasmids that the 11 bp motif plays an essential role in EBNA2 TPJ (Zimber-Strobl promoter-CAT construct described previously The mediated transactivation. SnaI Pvull (-177), et was cut with either SacH (-45), (-124), al., 1991) DNA complexes? Is EBNA2 a component of the -protein or and ends were filled NruI HindU (-432) Nsil (-543) protruding (-258), EBNA2 and negative cell After with PstI the fragments containing As mentioned above, positive in with T4 polymerase. digestion were isolated and inserted into the filled in HindIII the TPI promoter region showed almost identical binding patterns in EMSA. extracts communica- and PstI sites of the vector pBLCAT5 (P.Herrlich, personal or could be a slowly migrating band (IVA IVB) Only very - 147/-258 was constructed BclI and NruI digestion by tion). pTPl-CAT/A and which was detected in M-ABA Jijoye extracts, Raji, and pTPI-CAT/81bp were of pTPI-CAT/-543. pTPI-CAT/135bp EBNA2 negative cell extracts. We mixed +NruI of -CAT/-543. missing in all SmaI+NruI and PvuII digestion pTP1 generated by were isolated and cloned into the StuI 135 and 81 monoclonal antibodies in the EMSA to The fragments EBNA2 specific bp bp 173 U.Zimber-Strobi et al. R2 (rat IgG2a), R3 (rat IgG2a) and RI RI and R3 site of pTP1-CAT/-45. The 135 bp fragment was also cloned into the filled 1 (rat IgG2a). recognize EBNA2A with thesame as whereas R2 and RI in SalI site of (-glob-CAT containing the ,B-globin minimal promoter in affinity EBNA2B, 1 recognize EBNA2A, but have a very low affinity (R2) or no to EBNA2B. front of the CAT reporter gene (Laux et al., in preparation). All LUC affinity (RI 1) Anti-mouse CD45 or rat were used as control constructs were obtained by digesting pBLLUC5 (Laux et al., in preparation) (rat IgG 1 IgG2a) isotypic antibodies. with BamHI and KpnI. The fragment containing the luc gene was isolated and ligated to the corresponding pTP1-CAT construct also digested with In vitro translation BamHI and KpnI. pGa206/6 was digested with NaeI. The 4.5 kb the The EBNA2 expression vector pU294-6 contains an EBV fragment from fragment containing EBNA2 ORF and regulatory sequences was and transcribed position 35 448 to 54360 according to the B95-8 EBV sequence (Baer et al., prepared using T3 RNA polymerase and a Boehringer in vitro transcription kit. The resultant 1984). The clone was generated by deleting the NotI repeats of pM780-28 RNA was translated in vitro using a Promega reticulocyte lysate translation described by Polack et al. (1984). pU812/9 was generated by digesting translation were Western kit. The products assayed by SDS -PAGE and pU294-6 with SphI and religation. p604 was kindly provided by W.Hammerschmidt. It was constructed by inserting an XbaI linker in p135.15 blotting. (Hammerschmidt and Sudgen, 1989) at position 48 950 according to the Radioactively labelled probes B95-8 EBV sequence published by Baer et al. (1984). The CMV-LTR- pTPl-CAT/-543 was digested with NruI and Hindmi. The isolated CAT construct was obtained from I.S.Y.Chen (Cann et al., 1988). fragment with Klenow in The plasmid used in the in vitro translation of EBNA2 was constructed was labelled polymerase the presence of [32P]dCTP. The 054bp was synthesized with 5'-protruding which were filled in with by inserting the genomic B95-8 EBV sequence from position 48 039 to 54 ends, Klenow in the of 360 in a Bluescript vector digested with HindIII and BamHI. Just in front polymerase presence [32P]dCTP. of the translation start of EBNA2, an EcoRI site was inserted by site directed mutagenesis (pBSmutC). pBSmutC was cut with HindIII and EcoRI and an oligonucleotide representing a ribosome binding site was inserted Acknowledgements (pGa206/6). We thank W.Hammerschmidt for the and for critical providing p604 plasmid reading of the and for the Oligonucleotides manuscript B.Kempkes providing plasmid pBSmutC. This work was supported Die Deutsche The positions of oligonucleotides used in relation to the EBV genomic by Forschungsgemein- schaft (Forschergruppe Virus-Zellwechselwirkung) and Fonds der sequence according to Baer et al. (1984) are as follows: 01, 166 236-166 Chemischen Industrie. 268; 02, 166 259-166 289; 03, 166 279-166 317; 04, 166 302-166 340;054bp, 166 236-166 289; and I I mut, 166 252-166 272. Transfection of the cells References Electroporation of cells was carried out by the method of Cann et al. (1988). Briefly, 107 cells were washed once and resuspended in 0.25ml fresh ice- Ricksten,A., Gordon,J., Wang,F., Abbot,S.D., Rowe,M., Cadwallader,K., Rymo,L. and Rickinson,A.B. (1990) J. Virol., 64, cold RPMI 1640 supplemented with 10% fetal calf serum. The cells were 2126-2134. placed on ice in a Gene Pulser cuvette, and 20 of the corresponding and Delius,H., Clarke,J. Bornkamm,G.W. /tg Freese,U.K., Adldinger,H.K., DNA was added. Cells were electroporated with a Bio-Rad Gene Pulser (1985) Virology, 141, 221-234. at 250 V and 960 At 10min after electroporation, cells were resuspended et 207-211. Baer,R. ItF. al. (1984) Nature, 310, in 10ml of prewarmed RPMI with 20% fetal calf serum. and Zimber,U. (1982) J. Bornkamm,G.W., Hudewentz,J., Freese,U.K. Virol., 43, 952-968. CAT assay Banchereau,J., Vuillaume,M. and Calender,A., Billaud,M., Aubry,J.P., CAT assays were carried out as described previously (Zimber-Strobl et al., G.M. Proc. Acad. 84, Natl. Sci. USA, 8060-8064. Lenoir, (1987) 1991). CAT activities were quantified by densitometrically scanning the 3, Oncogene, 123-128. Cann,A.J., Chen,I.S.Y. (1988) Koyanagi,J., signals of an autoradiogram with the Cybertech system (Appligene). and J. 5880-5885. Cohen,J. Kieff,E. Virol., 65, (1991) and E. Proc. Natl. Mannick,J. Kieff, (1989) Acad. Cohen,J.I., Wang,F., LUC assays Sci. USA, 86, 9558-9562. Cells were harvested 40 h after transfection, washed once in ice-cold PBS and Gallo,R.C. (1978) Proc. Collins,S.J., Ruscetti,F.W., Gallagher,R.E. Natl. Acad. Sci. USA, 75, 2458-2462. and resuspended in 100 91 mM K2HPO4, 9 mM KH2PO4, 1 mM DTT, IA J. 1% Triton X-100, pH 7.8. Debris was removed by centrifugation at 14 000 g 1002-1013. Cordier,M. Virol., 64, (1990) for 10 min. 10y1 of the supernatant wasmixed with 350 25 mM glycyl- Achong,B.G., Finerty,S. Crawford,D.H., M.A., Bomkamm,G.W., ,u Epstein glycine, pH 7.8; 5 mM ATP; 15 mM MgSO4 and 100 1 mM luciferin; and Thompson, J. (1979) Int. J. Cancer, 24, 294-302. j1 0.5 M Tris-HCI, pH 7.8. The bioluminescence (in RLU) was measured (1984) Proc. Natl. Chamnankit,L. and Kieff,E. Dambaugh,T., Hennessy,K., with a Lumat LB9501 (Berthold, Wildbach). Acad. Sci. USA, 81, 7632-7636. and Nucleic Acids Res., (1983) Lebovitz,R.M. Roeder,R.G. Dignam,J.D., Nuclear extract preparation 11, 1475-1489. Nuclear extracts were prepared by a modification of the method of Dignam and Rymo,L. (1990) Fahraeus,R.. Jansson,A., Sjoblom,A. Ricksten,A., Proc. Natl. Acad. Sci. USA, 87, et al. (1983). The pellet (3-5x 107 cells) was washed once in ice-cold 7390-7394. PBS, resuspended with 3-4 vol 10 mM HEPES pH 7.9; 10 mM KCI, Nucleic Acids Ghosh,D. 1749-1756. (1990) Res., 18, 1.5 mM MgCl2; 5 mM DTT; 0.5 mM PMSF (buffer A) and incubated and J. 1855-1858. Ghosh,D 64, Kieff,E. (1990) Virol., on ice for 1 h. Lysis of cells was achieved by 10-20 strokes with a Dounce J. and Muller-Lantzsch,N. (1991) Virol., Grasser,F.A., Haiss,P., Gottel,S. 65, 3779-3788. homogenizer, microscopically controlled in the presence of trypan blue. Nuclei were pelleted for 10 s at maximal speed in an Eppendorf minifuge, and 340, 393-397. Hammerschmidt,W. Nature, (1989) Sugden,B. washed once in buffer A, resuspended in 3 vol of buffer B (20 mM HEPES and Wudarski,D.J., Blakeslee,J.R.J. Hinuma,Y., Konn,M., Yamaguchi,J., pH 7.9; 20% glycerine; 420 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; Grace,J.T.J. (1967) J. Virol., 1, 1045-1051. 5 mM DTT; 0.5 mM PMSF) and incubated on ice for 30 min. Nuclei were and 135-148. J. Virol., 48, Jeang,K.T. Hayward,S.D. (1983) removed by centrifugation at 14 000 g for 20 min. The supematant was and J. Jin,X.W. 66, 2846-2852. (1992) Virol., Speck,S.H. stored in liquid nitrogen. and In Knipe,D. (eds), Virology. Kieff,E. Liebowitz,D. Fields,B. and (1990) Raven Press, New York, pp. 1889-1920. Gel-shift analysis J. 2530-2536. Knutson,J.C. (1990) Virol., 64, Binding reactions were performed in a volume of 20 td, containing 5 and J., 7, 769-774. Laux,G., EMBO /O Perricaudet,M. Farrell,P.J. (1988) binding buffer (41 mM HEPES pH 7.9; 200 mM KCI; 4 mM EDTA; 1.6% Sci. and IARC Lenoir,G.M., Vuillaume,M. Bonnardel,C. (1985) Publ., Ficoll; 4mM DTT; 0.5 mM PMSF), 2 yl poly(dIdC) (2 mg/ml), 2 BSA 60, 309-318. Id (20 mg/ml), 5 jLg protein extract and 0.2-1 ng radioactively labelled DNA. and 39-44. Lillie,J.W. Green,M.R. Nature, 338, (1989) After incubation at room temperature for 30 min, the reaction products were Lindahl,T., Kaschka-Dierich,C. Adams,A., Bornkamm,G.W., Bjursell,G., separated in a 5% polyacrylamide gel. In competition and supershift and Jehn,U. (1976) J. Mol. Biol., 102, 511-530. Cell, 61, 1217-1224. and Green,M. (1990) experiments either unlabelled oligonucleotide or 1 a1 tissue culture supemratant Liu,F. monoclonal antibody was added to the reaction mixture. The Miller,G., Robinson,J., Heston, L. and Lipman,M. (1974) Proc. Nat!. Acad. containing were used as anti-EBNA2 monoclonal antibodies: RI (rat IgGI), Sci USA, 71, 4006-4010. following 174 EBNA2 interaction with TP1 promoter Polack,A., Hartl,G., Zimber,U., Freese,U.K., Laux,G., Takaki,K., Hohn,B., Gissmann,L. and Bomkamm,G.W. (1984) Gene, 27, 279-288. Pulvertaft,R.J.V. (1964) Lancet, 1, 238-240. Rickinson,A.B., Young,L.S. and Rowe,M. (1987) J. Virol., 61, 1310-1317. Sample,J., Liebowitz,D. and Kieff,E. (1989) J. Virol., 63, 933-937. Stern,S., Tanaka,M. and Herr,W. (1989) Nature, 341, 624-630. Sung,N.S., Kenney,S., Gutsch,D. and Pagano,J.S. (1991) J. Virol., 65, 6765 -6771. Tsang,S.F., Wang,F., Kenneth,M.I. and E. Kieff, (1991) J. Virol., 65, 6765-6771. Wang,F., Gregory,C.D., Rowe,M., Rickinson,A.B., Wang,D., Birkenbach,M., Kikutani,H., Kishimoto,T. and Kieff,E. (1987) Proc. Natl. Acad. Sci. USA, 84, 3452-3456. Wang,F., Tsang,S.F., Kishimoto,T. and Kieff,E. J. Hitoshi,K., (1991) Virol., 65, 4101-4106. Westin,G., Gerster,T., Muller,M.M., Schaffner,G. and Schaffner,W. (1987) Nucleic Acids Res., 15, 6787-6789. Woisetschlaeger,M., Jin,X.W., Yandava,C.N., Furmanski,L.A., Strominger,J.L. and Proc. Acad. Sci. Speck,S.H (1991) Natl. USA, 88, 3942 -3946. Zimber,U. et al. (1986) Virology, 154, 56-66. Zimber-Strobl,U., Suentzenich,K.O., Laux,G., Eick,D., Cordier,M., Calender,A., Billaud,M., and J. Lenoir,G.M. Bornkamm,G.W. (1991) Virol., 65, 415-423. Received on July 20, 1992; revised on September 1992 10, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The Epstein‐Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis‐element of the terminal protein 1 gene promoter.

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Copyright © European Molecular Biology Organization 1993
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10.1002/j.1460-2075.1993.tb05642.x
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Abstract

The EMBO Journal vol. 12 no. 1 pp. 167 - 175, 1993 The Epstein Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter Ursula Zimber-Strobi, Elisabeth transformed cell lines. In Kremmer1, lymphoblastoid these immortalized cell Friedrich lines the virus is maintained as an Grasser2, Gabriele Marschall, molecule episomal at number et Gerhard Laux high copy (Lindahl al., 1976) and only a few and Georg W.Bornkamm viral genes are expressed. These genes code for six nuclear Institut fOir Klinische Molekularbiologie und Tumorgenetik, Institut fiir antigens, 3c and and EBNA1, 2, 3a, 3b, -LP, three Immunologie, im Forschungszentrum fiir Umwelt und Gesundheit, membrane the latent proteins, membrane protein (LMP) and GSF, Marchioninistrasse 25, 8000 Munchen and 2lnstitut fOir terminal and also proteins (TPs) 1 2, denoted LMP2A Medizinische by Mikrobiologie, Universititsklinikum, Haus 47, 6650 Homburg/Saar, and B review see Kieff and Germany (for Liebowitz, These 1990). gene seem to be for products important induction and Communicated by W.Schaffner maintenance of B-cell immortalization. However, the individual functions of these gene products in latent infection The Epstein-Barr virus protein EBNA2 acts as a and growth transformation are only poorly transcriptional activator of cellular and understood. viral genes and EBNA2, one of the best plays a crucial role in studied latent gene products, is the immortalization of human absolutely necessary for B-cell immortalization. primary B-cells by EBV. We have One viral shown previously that mutant EBNA2 (P3HR1) carrying a 6.6 kb deletion transactivates the promoters (Bornkamm of the latent et membrane al., 1982; Jeang and Hayward, that antigens LM1P, TP1 and TP2. 1983) removes The promoter EBNA2 and of of the TPI part EBNA-LP, has lost the ability to gene was chosen as a model system to study transform primary B-cells. the molecular Reintroduction of EBNA2 into mechanism of EBNA2 mediated the P3HRl virus transactivation. To by homologous recombination reconstituted identify an EBNA2 dependent cis- the full acting transforming capacity (Cohen et al., element, various TPI promoter-reporter 1989; gene Hammerschmidt and Sudgen, In constructs were in 1989). natural EBV transfected the absence and isolates, presence two alleles of the of an EBNA2 EBNA2 gene exist coding for two expression vector into the proteins established B- (EBNA2A and cell line EBNA2B) with 57% homology BL41-P3HR1. We were able (Dambaugh to delineate an 81 et al., 1984; Adldinger et bp EBNA2 al., 1985; Zimber et al., responsive region between 1986). -258 and -177 There have been some reports that relative to the TPI EBV strains RNA start site. The element carrying worked EBNA2A could transform primary B-cells more in either efficiently orientation and could mediate EBNA2 than those containing an EBNA2B allele (Rickinson dependent transactivation on et a heterologous al., promoter. Cohen et 1987; al., 1989). The EBNA2 gene codes for a Electrophoretic mobility shift assays revealed three 487 amino acid phoshorylated polypeptide (Grasser et specific protein-DNA complexes al., formed with sequences 1991). Characteristic features of the of the EBNA2 primary structure of the responsive element. Two of these were not protein include a proline stretch of -40 amino cell type but the acids near specific, third was detected in only the N-terminus, a negatively charged C-terminus and EBNA2 positive cell a extracts. Gel-shift analysis in the glycine/arginine-rich region. It has recently been shown presence of EBNA2 that specific monoclonal antibodies EBNA2 acts as a transcriptional activator: it stimulates the revealed that EBNA2 is a component of the third transcription of the cellular genes CD21, CD23 and complex. Thus, these c-fgr experiments demonstrate that (Calender et al., 1987; EBNA2 Wang et al., 1987; Abbot et interacts with an EBNA2 al., responsive cis-element 1990; Cordier et al., 1990; Knutson, as well as of the 1990) the TPI promoter. expression of the viral words: genes LMP, TPI and TP2 Key DNA -protein - (Fahraeus interaction/EBNA2/Epstein et al., 1990; Ghosh and Barr 1 Kieff, 1990; Zimber-Strobl et virus/terminal protein /transactivation al., 1991). Although a transactivation domain has been mapped in the C-terminus of EBNA2 (Cohen and Kieff, 1991), it is not yet known whether EBNA2 transactivates several Introduction genes by interacting with EBNA2 responsive cis-elements Epstein-Barr virus a human or whether it does so (EBV), widespread by modulating the expression or herpesvirus, is the of infectious modification of transcription factors. A consensus aetiological agent sequence mononucleosis, self-limiting lymphoproliferative disease. in the promoter of the region EBNA2-regulated genes has The virus is associated with two human not been described until malignancies, now. namely Burkitt's B-cell and lymphoma (a To elucidate the mechanism of EBNA2 neoplasia) mediated which is of nasopharyngeal carcinoma, composed transactivation, we focused our interest on the TPI poorly promoter differentiated cells. EBV has a dual it TP epithelial region. The gene is transcribed across the tropism: terminal repeats infects primary B-cells and cells. Virus of the EBV resting epithelial genome (Laux et al., 1988; Sample et al., 1989). production occurs in whereas B-cells A functional only epithelial cells, transcription unit can only be created if the linear molecule are infected. In vitro infection of B- is circularized. The TATA-box of the TPI latently resting lymphocytes leads to the of virus- promoter is located at the hand outgrowth permanently right end of the linear viral 1 67 © Oxford Press University U.Zimber-Strobl et al. III -45 .zzzL- rzzzzzzzzzz4llll - 124 - 177 - 258 %acetylation Stimulation BL41-P3HR1/ BL41-P3HR1 BL41-B95-8 CAT-constructs "IlllzzzriI -45 2.1 2.1 1 0.8 1.4 1.75 -124 -177 1.5 1.2 0.8 -258 1.0 10.5 10.5 - 804 7.2 10.4 -432 0.7 0.3 5.3 17.7 -543 .. 77777M,,,,{,,,,zz//d A -1 47/-258 RLU 10000 - 1 E I1 I1 0 l n.,I tHi I1 -124 -177 -258 -432 -543 -804 A -147/ transfected -45 LUC-constructs -258 fold induction 1,1 1,2 25,6 50,9 57,2 71 1,7 0,72 EBNA2 by constructs in the absence and of EBNA2. Schematic of the Fig. 1. Transfection of different TPI promoter-reporter gene presence (A) representation The restriction used to construct the recombinant plasmids are indicated. The positions of the TPJ TPJ promoter-reporter gene constructs. enzymes denote their 5'-end relative to the TPJ transcription start site. Open boxes represent the reporter gene, hatched boxes the TPI promoter fragments CAT activities after transfection of the TPI promoter-CAT constructs into EBNA2 negative BL41-P3HR1 cells and EBNA2 promoter region. (B) BL41-B95-8 cells. Median activities of two independent experiments and the fold inductions by EBNA2 are indicated. (C) Relative light positive after of constructs in the absence and of EBNA2 expression vectors into units (RLU) measured transfections TPI promoter-LUC presence BL41-P3HR1 cells. transfections without EBNA2; hatched boxes, cotransfection with the EBNA2 expression vector pU294-6, coding Open boxes, for wild-type EBNA2; dotted and black boxes, cotransfections with EBNA2 expression vectors p604 and pU812/9 respectively, coding for mutated EBNA2. The median activities of two to five independent experiments and standard deviations are indicated. at 166 469 of the B95-8 virus DNA Results genome position sequence (Baer et al., 1984). Identification of an EBNA2 responsive cis-element In this report we describe an element that mediates EBNA2 to the In addition within the TP1 promoter region transactivation by TPI promoter. we demonstrate specific protein-DNA interactions within EBNA2 transactivated a TPJ promoter-CAT construct this and evidence that EBNA2 itself is a containing 804 bp upstream of the TPI cap site (Zimber- region provide Strobl et component of this protein-DNA complex. al., 1991). Plasmids containing progressive 168 EBNA2 interaction with TP1 promoter A B B. * ..* *---------@@ - EBNA2 - - - - Mut - WT Mut WT Mut W1 MLIt W7 VT EDNA2 2 .glob.- CMV- CAT- TATA LTP conStructs 135bp1t 135bp 2 81 DP 2 8l0E mrinimal CMV- CAT- zf ... 2 5 0,7 100 acetylation I/. LTR corstructs promoter 1,2 17 0.9 1 8 37 1.0 3 22 1.8 0.5 2 0.2 0.2 0,2 100 % acetylation Fig. 2. 81 bp (-177 to -258) of the TPI promoter are sufficient to mediate transactivation by EBNA2. (A) Results of CAT assays of transfections of CAT constructs containing the EBNA2 responsive region (pTPl-CAT/135bp and pTP1-CAT/81bp) in front of the TPI minimal promoter (pTPI-CAT/-45) without EBNA2 (-), with pU294-6 coding for wild-type EBNA2 (WT) and with p604 coding for mutated EBNA2 (Mut) into cells. The element was cloned in the correct (135bp/l, 81bp/1) and inverse (135bp/2, 81bp/2) orientations. (B) CAT assays after BL41-P3HR1 transfections of CAT constructs containing the TPI promoter region from position - 124 to -258 in front of the ,3-globin minimal promoter into without EBNA2 (-) and with the EBNA2 expression vector pU294-6 (+). The element is cloned in either orientation (f135bp/1, BL41-P3HR1 cells inverse). correct; ,B135bp/2, truncations of the 5' region of the TPI promoter linked to LUC constructs containing at least 258 bp of the TPI 5' flanking region were inducible by EBNA2 (by factors of the reporter genes chloramphenicol acetyltransferase (CAT) three smallest constructs were 25-71), whereas the or luciferase (LUC) were constructed (Figure IA) to identify EBNA2. cis-element within this promoter. unresponsive to an EBNA2 responsive that a specific EBNA2 responsive constructs were transiently transfected into the EBNA2 In order to demonstrate These we removed the TPI promoter BL41-P3HR1 cell line and into EBNA2 positive element has been identified, negative BL41-B95-8 cells. A cytomegalovirus enhancer-long region between positions -258 and -147 in the TPI terninal construct (CMV-LTR-CAT) was used promoter reporter gene construct pTP1-LUC/-543, leaving repeat-CAT intact. This construct as a positive control. the boundaries of the TPI promoter into BL41-P3HR1 cells together with the Transfections of the TPI promoter-CAT constructs into was transfected vectors mentioned above. As shown in BL41-P3HR1 cells resulted in very low or undetectable CAT EBNA2 expression IC, induction of LUC activities by EBNA2 could activities 1B). Transfections of constructs containing Figure (Figure not be observed after transfection of the TPI promoter LUC -45, -124 and -177 bp upstream of the TPI cap site into construct lacking the TI' promoter region between -258 BL41-B95-8 cells resulted in low CAT activities similar to into BL41-P3HRI cells. -147. This was confirmed by transfection of the same those obtained with transfections and activities increased after construct with the CAT reporter gene (data not shown). However, CAT significantly of CAT constructs containing sequences These data show that sequences between -258 and -147 transfections mediated transactivation. are required for EBNA2 of position -177 relative to the TPI cap site upstream (Figure 1B). These results suggested that an EBNA2 element is orientation The EBNA2 responsive responsive region is located between -177 and -258. active on a heterologous promoter in order to independent and We performed cotransfection experiments element was cloned in front of the TPI minimal the increased TPI promoter activity in The putative prove that and the CAT reporter gene to study cells is mediated by EBNA2. TPI promoter promoter (+60/-45) BL41-B95-8 the EBNA2 responsive region is active even when constructs driving the reporter gene luciferase were whether deletion from its TPI promoter region. Two DNA transfected into BIAl-P3HRI cells either without EBNA2 separated cognate constructs were made carrying a larger (-124 to -258) and expression vector or with or with an EBNA2A (pU294-6) to in both orientations. a smaller (-177 -258) EBNA2A vectors carrying mutations within the fragment expression the EBNA2 responsive element The EBNA2 mutation In these constructs suggested EBNA2 open reading frame (ORF). vector sequences. The distance of the insertion after one-third of the EBNA2 is surrounded by contains a linker p604 box is to the native from the TPI TATA comparable to a truncated EBNA2 In the element leading gene product. ORF, All DNA constructs were transfected in the absence amino acids 248-382 of situation. EBNA2 mutation pU812/9, vectors into of EBNA2 expression are deleted. The EBNA2 and its mutants are and presence EBNA2 gene and for cells. p604, coding wild-type the control of the natural promoter of EBNA2. BL41-P3HR1 pU294-6 under were used as EBNA2 and truncated EBNA2, respectively, relative units of the LUC assays The obtained light (RLU) vectors. Transfection of all constructs EBNA2 expression data described above IC). Only confirmed the CAT (Figure 169 U.Zimber-Strobi et al. .,PI~~~~k ..wsU+bStWof~~~~~~~~~~~~~~ I':, . -- Bf n. ^^~~~~~~~- - 0 -i to fa mm . is .. I _ ^ Fig. 3. Analysis of DNA-protein interactions within the TPI free promoter. Autoradiogram of an EMSA is shown made with nuclear oligo with a extracts of BL41-P3HRI cells which were incubated 32p- labelled fragment spanning the TPI promoter region from position -12 to -258 relative to the start. TPI transcription Competition studies were with the EBNA2 performed oligonucleotides covering Fig. 4. EMSAs using 054bp (EBV positions 166 236-166 289 responsive region identified in the CAT and LUC The analyses. according to the EBV sequence of Baer et al., 1984) as radioactively positions of the in the EBV are described in oligonucleotides genome labelled probe and nuclear extracts of the promyelocyte line HL60, Materials and methods. Unlabelled oligonucleotides were added to the cervical carcinoma line HeLa, the EBV negative B-cell line BL41 and gel-shift reaction in a 100-fold molar excess. four EBV B-cell lines: positive BL41-P3HRI (EBNA2 negative), HH514 (EBNA2 negative), Jijoye (EBNA2B) and M-ABA (EBNA2A). resulted in a CAT in the significant higher activity presence of the EBNA2 vector of the expression pU294-6, regardless to - 177), four overlapping oligonucleotides [01 orientation of the element relative to the promoter (-262/-230), 02 (-239/-209), 03 (-219/-181) and 04 (Figure 2A). (-196/-158)] covering these sequences were synthesized To determine whether the EBNA2 responsive region and added to BL41-P3HR1 extracts. I and III Complexes contains that can a be with 01 02 sequences regulate heterologous could competed and covering sequences promoter, we cloned the TPI from between promoter region positions -262 and -209. None of the complexes could be -124 to -258 upstream of the 3-globin minimal promoter the competed by adjacent oligonucleotides 03 and 04. (Westin et al., 1987). Cotransfections of the EBNA2 In order to the interaction of TPI study promoter vector in expression pU294-6 resulted significantly increased sequences from position -262 to -209 with cellular proteins CAT activities in the of presence reporter plasmids carrying in more detail, the 54 bp fragment was end-labelled and the EBNA2 responsive region (Figure 2B). In this assay, incubated with nuclear extracts of several cell lines (HL60, the element conferred EBNA2 inducibility in an orientation M-ABA HeLa, BL41, BL4l-P3HR1, HH514, Jijoye, and fashion as has been independent demonstrated by the TPI Raji) (Figures 4 and Similar were SA). complexes formed promoter constructs. with the 54 bp fragment as with the 247 bp fragment used From these data we conclude that an 81 bp region within in the EMSA described above. Again one prominent (I) and the TPI promoter harbours an EBNA2 responsive cis- two weaker complexes (II and III) could be detected. which will element, we call EBNA2RE. EBNA2RE is active I -III were formed in Complexes all cell extracts tested and in both orientations and able to regulate a heterologous therefore seem to be cell-type unspecific. Notably, additional promoter. and could be complexes (IVA IVB) detected with extracts of the EBNA2 cell lines M-ABA positive Jijoye, and Raji, Specific DNA -protein interactions within the EBNA2 which were not found with extracts of EBNA2 cell negative responsive region lines. Gel-retardation assays were performed to identify specific The of specificity the DNA-protein interactions was DNA-protein interactions within the TPI promoter. A in analysed competition experiments following addition of DNA fragment spanning the TPI promoter from position the unlabelled 54 bp oligonucleotide to EMSAs with Raji, -12 to -258 was end-labelled and incubated with nuclear HH514, Jijoye and M-ABA extracts (Figure 5A and B). extracts of BL41-P3HR1 cells. The result of the Only complex II could not be competed and appears to be electrophoretic mobility shift (EMSA) is shown in Figure 3. unspecific. Additionally, we tested the ability of 0 1 and 02 EMSA revealed one prominent DNA-protein interaction to prevent formation of protein-DNA complexes formed (complex I) and two weaker complexes (II and III). To with the 54 bp oligonucleotide and nuclear proteins of Raji investigate whether some of these complexes are formed with cells. All specific complexes could be competed by 0 1 and sequences located in the EBNA2 responsive region (-258 02 with the same efficiency as by 054bp (Figure 5A). The 170 EBNA2 interaction with TP1 promoter 54bp 01 02 llrn - a -.. __. a- -IV - - a -a S IVA --. -- IVb _ d :1 * I- III 'l1~' b ;t' £ * *. X _ free oligo free ol!go 262 - 209 ~ ~ -:1 ~ -- -- -2-2 Fig. 5. Competition assays demonstrate the specificity of complexes I, III and IVA. (A) Autoradiogram of an EMSA. Nuclear extracts of Raji cells (EBNA2A positive B-cell line) were incubated with the labelled oligonucleotide 054bp. Unlabelled oligonucleotides 054bp, 01, 02 and 1 lmut were added to the reaction mixture in 10-, 50- and 100-fold excesses. The DNA-protein complexes were separated on a 5% polyacrylamide gel. (B) Nuclear extracts of HH514, Jijoye and M-ABA cells (indicated by 1, 2 and 3 respectively) were incubated with the radioactively labelled 054bp oligonucleotide. For competition unlabelled oligonucleotide 054bp was added in 100-fold excess to the reaction mixture. The resulting products were on a 4% polyacrylamide gel. (C) Schematic representation of the oligonucleotides used in the EMSA. The indicated positions correspond to separated the 7PJ transcription start site. An 11 bp motif duplicated in 054bp is marked by dotted boxes, the 10 bp overlapping region of 01 and 02 by hatched boxes. two share a 10 bp overlapping region and low affinity for EBNA2B in oligonucleotides EBNA2A but has only a very a common motif of 11 bp (Figure 5C). To test whether the et in preparation). Two immunoprecipitation (Kremmer al., 1 -DNA interaction, nonspecific antibodies, raised against murine CD45, of the I bp motif was involved in the protein 1 imut, Figure 5C). same as the anti-EBNA2-Rl and anti-EBNA2-R2 these sequences were truncated (oligo isotype the truncated oligonucleotide has as a control. The presence of the anti- As shown in Figure 5A, antibodies were used lost the to prevent formation of the EBNA2-R1 resulted in a supershift of complex IVB completely ability antibody DNA-protein complexes. This suggests that the 11 bp motif in and IVA in M-ABA extracts (Figure 6A). Jijoye complex EBNA2 EBNA2A is important for protein -DNA interaction within the contrast, anti-EBNA2-R2, which recognizes By but EBNA2B with a low affinity, responsive region. with a high affinity very of IVA but not of IVB. led to a supershift complex complex control antibodies had no effect on the mobility of EBNA2 is present in DNA -protein complexes formed Both IVA and IVB. with the EBNA2 responsive region complexes lines the with two further Differences between EBNA2 positive and negative cell We have repeated experiments in EMSAs described above were low anti-EBNA2-R3 and anti- detected the mobility EBNA2 monoclonals, IVA and IVB. To test whether EBNA2 was EBNA2-R1 1. Both antibodies recognized EBNA2A, complexes EBNA2-R3. We in of the complexes described above, DNA whereas EBNA2B was recognized only by present any as with the antibodies described above. were performed with HH5 14, Jijoye and M- got the same results binding assays EBNA2B and caused a of but ABA extracts (EBNA2 negative, positive Both antibodies supershift complex IVa, only IVb was able to (data EBNA2A respectively) in the presence and absence anti-EBNA2-R3 supershift complex positive, EBNA2A. The not of monoclonal antibodies raised against shown). is able to detect EBNA2A that IVA contains EBNA2A monoclonal antibody EBNA2-R1 These results imply complex the other anti-EBNA2-R2 IVB EBNA2B. None of as well as EBNA2B, whereas recognizes and complex complexes 171 et al. U.Zimber-Strobi .- !; .I- Er F ! ii: i.: :.Z -, A'.. A BI X f..i... a as i". 6. EBNA2 is a component of complex IV. (A) Effect of monoclonal EBNA2 antibodies on TPJ promoter DNA-protein complexes. Gel-shift Fig. antibodies. and anti-EBNA2-R2 are monoclonal rat antibodies raised against EBNA2A. were incubated with the indicated Anti-EBNA2-Rl reactions as well as anti-EBNA2-R2 EBNA2A with the same affinity as anti-EBNA2-Rl, but only Anti-EBNA2-RI recognizes EBNA2A EBNA2B; recognizes low Incubation reactions were on a 4% gel. Only complexed DNA is visible on EBNA2B with a very affinity. separated polyacrylamide recognizes since the free was run off the 2 and 3 indicate gel-shift reactions with extracts of cell lines HH514 the autoradiogram oligonucleotide gel. 1, and M-ABA respectively. Anti-mouse CD45 IgGI and IgG2a are two control monoclonal (EBNA2 negative), Jijoye (EBNA2B) (EBNA2A) subclasses as anti-EBNA2-RI and anti-EBNA2-R2, respectively, and recognize neither EBNA2A nor EBNA2B. (B) Effect antibodies of the same Ig EBNA2A on the reconstitution of IV. Gel-shift reactions were incubated with the indicated nuclear extracts (NE). 3 Al of in vitro translated complex RNA or BMV RNA and 1 1d of monoclonal antibodies or anti- with EBNA2 supernatant (anti-EBNA2-R3 reticulocyte lysate (IVT) programmed were added as indicated above. The reactions were preincubated for 5 min at room temperature before adding the radioactively mouse CD45) The DNA complexes were analysed on a 4% polyacrylamide gel. The free oligonucleotide was run off the labelled oligonucleotide 054bp. protein gel. monoclonal EBNA2 Discussion were affected by the antibodies, do not contain EBNA2. suggesting that they of EBNA2 to B-cell that EBNA2 is really a component of the It is assumed that the contribution We confirmed -DNA IV the ability of in vitro immortalization is conferred by its ability to transactivate protein complex by studying The EBNA2 ORF process EBNA2 to form this complex. viral and cellular genes. In order to understand the synthesized The in vitro it to was translated in a reticulocyte lysate. of immortalization at a molecular level, is essential of EBNA2 transactivation. EBNA2 was added to the EBNA2 negative elucidate the mechanism mediated synthesized were As We chose the TPI promoter as a model system for BL41-P3HR1 extracts and EMSAs performed. translated EBNA2 formed EBNA2 transcription. As shown shown in 6B the in vitro studying dependent Figure In addition of previously, TPI transcription was dependent on the presence a more slowly migrating complex. contrast, in vitro translated of EBNA2 in Burkitt's lymphoma cells (Zimber-Strobl et al., a control reticulocyte lysate containing mosaic virus RNA did not induce this Our first aim was to identify a cis-acting element brome (BMV) 1991). in vitro translated That the complex induced by mediating EBNA2 responsiveness. Therefore we transfected complex. EBNA2 was confirmed EBNA2 did contain the added by different TPJ promoter-reporter gene constructs in the the effect of monoclonal antibodies. Anti- presence and absence of EBNA2. By this approach we were assaying induced able to an between positions - 177 EBNA2-R3 but not the control antibody (anti-CD45) identify 81 bp element of formed An additional and -258 which is sufficient to confer EBNA2 a supershift the newly complex. in the of nuclear extracts, responsiveness. complex (*) appeared presence EBNA2 and monoclonal anti-EBNA2 antibodies in this The EBNA2 responsive element (EBNA2RE) works in and This complex was not always reproducible either orientation and is able to regulate a heterologous experiment. in vitro is therefore not further considered. Besides the promoter. These results imply that EBNA2 responsiveness translated EBNA2 diminished formation of I and complexes is conferred by an enhancer-like cis-element. This is or not is The III. Whether this effect specific still unclear. observation is in agreement with previous reports. EBNA2 EBNA2 was not able to bind DNA in the responsive elements were also identified within the LMP, in vitro translated absence of nuclear that EBNA2 interacts BamHI-C and CD23 promoters and it has been reported that extracts, suggesting with DNA not directly but indirectly. work in an orientation independent fashion and regulate they 172 EBNA2 interaction with TP1 promoter a heterologous promoter (Sung et al., 1991; Tsang et al., study whether one of the retarded bands contained EBNA2. Addition of EBNA2 antibodies resulted in a supershift of 1991; Wang et al., 1991; Woisetschlaeger et al., 1991). complexes IVA and IVB. An antibody recognizing only We performed EMSAs to identify specific protein-DNA a supershift of complex IVA only, interactions within the TPJ promoter. One prominent EBNA2A induced an antibody specific for EBNA2A and EBNA2B DNA-protein complex (I) and two weaker complexes (II whereas III) were detected with a radioactively labelled fragment retarded migration of both complexes IVA and IVB. This and the TPI promoter from position -12 to -258 indicates that EBNA2B is a component of complex IVB and spanning of the EBNA2RE). It was possible to compete EBNA2A a component of complex IVA. To prove that (5'-end specifically complexes I and III with two oligonucleotides EBNA2 is indeed a component of complex IV, we added in vitro translated EBNA2 to EBNA2 negative BL41-P3HR1 spanning the region -262 to -230 and -239 to -209. EMSAs. The in vitro translated extracts and performed When extracts were mixed with a radioactive oligonucleotide the formation of a more slowly migrating from position -262 to -209 and EMSAs were performed, EBNA2 induced By adding monoclonal antibodies we showed that the same pattern of complex formation occurred. These complex. indicate that protein(s) interact with sequences EBNA2-induced complex contained the added EBNA2. experiments the located in the EBNA2RE. The complex containing the in vitro translated EBNA2 Formation of complexes I, II and III appeared to be slightly faster than complex IVa visible in M-ABA migrated be caused by different independent of EBNA2 and even independent of the cellular extracts. This phenomenon could and in vitro synthesized EBNA2. of B-cells, since they were detected in all cell modifications of the in vivo background 54 bp fragment is sufficient to bind EBNA2, lines studied. This implies binding of a universal transcription Although the We were not able to identify this factor by comparing it is not able to mediate EBNA2 responsiveness (data not factor. the sequence with known consensus sequences of shown), suggesting that sequences between -209 and -177 transcription factors (Ghosh, 1990). The only obvious are necessary for EBNA2 dependent transactivation. It is cognate nuclear factor binding sequence in the 54 bp conceivable that an additional protein binds to these responsiveness. fragment is one SPI consensus sequence at position -240. sequences and contributes to EBNA2 for the first time that EBNA2 is Addition of a 500-fold excess of an unlabelled Here we have shown in complexes of specific DNA sequences and oligonucleotide representing the SPI binding site failed to present Our experiments suggest that EBNA2 interacts compete the shift (data not shown). It is therefore unlikely protein(s). that this SPI site plays an essential role in the observed with DNA indirectly via protein -protein interactions. Such shown to be responsible for DNA -protein interaction. interactions have been VP16 (Lillie and Green, 1989; Formation of all complexes except complex II was transactivation by EIA and Green, 1990) transactivators of competed by an excess of unlabelled 01 as well as 02. These Stern et al., 1989; Liu and share a 10 bp overlapping region and simplex virus, respectively. VP16 two oligonucleotides adenovirus and herpes of 11 bp. The 11 bp motif seems to play binds to the universal transcription factor Oct 1, juxtaposing a common motif transactivation domain of VP16 to the promoter. It seems an essential role in DNA -protein interactions, since an the likely that EBNA2 also interacts with a transcription factor, oligonucleotide containing the complete 10 bp overlap, but in complexI and/or III. We are in the two truncated 11 bp motifs, has lost the ability to inhibit the perhaps contained the factors interacting with the protein binding of the 54 bp oligonucleotide. process of identifying of the TPI promoter, which should further Realizing the importance of the 11 bp motif we searched EBNA2RE EBNA2 mediated transactivation. for this sequence in the EBNA2 responsive regions of the elucidate and LMP promoters which were shown to be BamHI-C EBNA2. Sung et al. (1991) reported an EBNA2 activated by Materials and methods responsive region of 98 bp within the BamHI-C promoter between positions -367 and -465. In the LMP promoter, conditions Cell lines and culture described an element of 30 bp between Raji and BL41 have been described elsewhere (Crawford Tsang et al. (1991) Cell lines M-ABA, et 1979; Pulvertaft, 1965; Lenoir et al., 1985). HH514 is a single cell al., -205 which is essential although not positions -234 and clone of Jijoye, carrying a deletion in the EBNA2 gene (Hinuma et al., 1967; for EBNA2 responsiveness. Remarkably, the sufficient BL41-B95-8 were obtained after Miller et al., 1974). BL41-P3HR1 and promoter contains, at position -371, a 10 bp BamHI-C cell line BL41 with virus infection of the EBV negative Burkitt's lymphoma that is identical to the 11 bp motif, and the LMP et al., 1987). HL60 is sequence strains P3HR1 and B95-8, respectively (Calender line described by Collins et al. (1978). a promyelocyte cell contains, at position -218, a sequence that shares promoter in RPMI 1640 medium supplemented with 10% All cell lines were grown 11 bp motif. Recently it was shown 7 bp (GTGGGAA) of the 100 U/mI and 100 streptomycin. Cultures fetal calf serum, penicillin [tg/mn that parts of the 10 bp motif of the by footprint analyses at 37°C in an atmosphere of 5% CO2. Cells were diluted were incubated interact with a DNA binding protein (Jin BamHI-C promoter medium twice a week. 1:3 with fresh 1992). Taking this into account, it seems likely and Speck, Plasmids that the 11 bp motif plays an essential role in EBNA2 TPJ (Zimber-Strobl promoter-CAT construct described previously The mediated transactivation. SnaI Pvull (-177), et was cut with either SacH (-45), (-124), al., 1991) DNA complexes? Is EBNA2 a component of the -protein or and ends were filled NruI HindU (-432) Nsil (-543) protruding (-258), EBNA2 and negative cell After with PstI the fragments containing As mentioned above, positive in with T4 polymerase. digestion were isolated and inserted into the filled in HindIII the TPI promoter region showed almost identical binding patterns in EMSA. extracts communica- and PstI sites of the vector pBLCAT5 (P.Herrlich, personal or could be a slowly migrating band (IVA IVB) Only very - 147/-258 was constructed BclI and NruI digestion by tion). pTPl-CAT/A and which was detected in M-ABA Jijoye extracts, Raji, and pTPI-CAT/81bp were of pTPI-CAT/-543. pTPI-CAT/135bp EBNA2 negative cell extracts. We mixed +NruI of -CAT/-543. missing in all SmaI+NruI and PvuII digestion pTP1 generated by were isolated and cloned into the StuI 135 and 81 monoclonal antibodies in the EMSA to The fragments EBNA2 specific bp bp 173 U.Zimber-Strobi et al. R2 (rat IgG2a), R3 (rat IgG2a) and RI RI and R3 site of pTP1-CAT/-45. The 135 bp fragment was also cloned into the filled 1 (rat IgG2a). recognize EBNA2A with thesame as whereas R2 and RI in SalI site of (-glob-CAT containing the ,B-globin minimal promoter in affinity EBNA2B, 1 recognize EBNA2A, but have a very low affinity (R2) or no to EBNA2B. front of the CAT reporter gene (Laux et al., in preparation). All LUC affinity (RI 1) Anti-mouse CD45 or rat were used as control constructs were obtained by digesting pBLLUC5 (Laux et al., in preparation) (rat IgG 1 IgG2a) isotypic antibodies. with BamHI and KpnI. The fragment containing the luc gene was isolated and ligated to the corresponding pTP1-CAT construct also digested with In vitro translation BamHI and KpnI. pGa206/6 was digested with NaeI. The 4.5 kb the The EBNA2 expression vector pU294-6 contains an EBV fragment from fragment containing EBNA2 ORF and regulatory sequences was and transcribed position 35 448 to 54360 according to the B95-8 EBV sequence (Baer et al., prepared using T3 RNA polymerase and a Boehringer in vitro transcription kit. The resultant 1984). The clone was generated by deleting the NotI repeats of pM780-28 RNA was translated in vitro using a Promega reticulocyte lysate translation described by Polack et al. (1984). pU812/9 was generated by digesting translation were Western kit. The products assayed by SDS -PAGE and pU294-6 with SphI and religation. p604 was kindly provided by W.Hammerschmidt. It was constructed by inserting an XbaI linker in p135.15 blotting. (Hammerschmidt and Sudgen, 1989) at position 48 950 according to the Radioactively labelled probes B95-8 EBV sequence published by Baer et al. (1984). The CMV-LTR- pTPl-CAT/-543 was digested with NruI and Hindmi. The isolated CAT construct was obtained from I.S.Y.Chen (Cann et al., 1988). fragment with Klenow in The plasmid used in the in vitro translation of EBNA2 was constructed was labelled polymerase the presence of [32P]dCTP. The 054bp was synthesized with 5'-protruding which were filled in with by inserting the genomic B95-8 EBV sequence from position 48 039 to 54 ends, Klenow in the of 360 in a Bluescript vector digested with HindIII and BamHI. Just in front polymerase presence [32P]dCTP. of the translation start of EBNA2, an EcoRI site was inserted by site directed mutagenesis (pBSmutC). pBSmutC was cut with HindIII and EcoRI and an oligonucleotide representing a ribosome binding site was inserted Acknowledgements (pGa206/6). We thank W.Hammerschmidt for the and for critical providing p604 plasmid reading of the and for the Oligonucleotides manuscript B.Kempkes providing plasmid pBSmutC. This work was supported Die Deutsche The positions of oligonucleotides used in relation to the EBV genomic by Forschungsgemein- schaft (Forschergruppe Virus-Zellwechselwirkung) and Fonds der sequence according to Baer et al. (1984) are as follows: 01, 166 236-166 Chemischen Industrie. 268; 02, 166 259-166 289; 03, 166 279-166 317; 04, 166 302-166 340;054bp, 166 236-166 289; and I I mut, 166 252-166 272. Transfection of the cells References Electroporation of cells was carried out by the method of Cann et al. (1988). Briefly, 107 cells were washed once and resuspended in 0.25ml fresh ice- Ricksten,A., Gordon,J., Wang,F., Abbot,S.D., Rowe,M., Cadwallader,K., Rymo,L. and Rickinson,A.B. (1990) J. Virol., 64, cold RPMI 1640 supplemented with 10% fetal calf serum. The cells were 2126-2134. placed on ice in a Gene Pulser cuvette, and 20 of the corresponding and Delius,H., Clarke,J. Bornkamm,G.W. /tg Freese,U.K., Adldinger,H.K., DNA was added. 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Cells were harvested 40 h after transfection, washed once in ice-cold PBS and Gallo,R.C. (1978) Proc. Collins,S.J., Ruscetti,F.W., Gallagher,R.E. Natl. Acad. Sci. USA, 75, 2458-2462. and resuspended in 100 91 mM K2HPO4, 9 mM KH2PO4, 1 mM DTT, IA J. 1% Triton X-100, pH 7.8. Debris was removed by centrifugation at 14 000 g 1002-1013. Cordier,M. Virol., 64, (1990) for 10 min. 10y1 of the supernatant wasmixed with 350 25 mM glycyl- Achong,B.G., Finerty,S. Crawford,D.H., M.A., Bomkamm,G.W., ,u Epstein glycine, pH 7.8; 5 mM ATP; 15 mM MgSO4 and 100 1 mM luciferin; and Thompson, J. (1979) Int. J. Cancer, 24, 294-302. j1 0.5 M Tris-HCI, pH 7.8. The bioluminescence (in RLU) was measured (1984) Proc. Natl. Chamnankit,L. and Kieff,E. Dambaugh,T., Hennessy,K., with a Lumat LB9501 (Berthold, Wildbach). Acad. Sci. USA, 81, 7632-7636. and Nucleic Acids Res., (1983) Lebovitz,R.M. Roeder,R.G. Dignam,J.D., Nuclear extract preparation 11, 1475-1489. 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Id (20 mg/ml), 5 jLg protein extract and 0.2-1 ng radioactively labelled DNA. and 39-44. Lillie,J.W. Green,M.R. Nature, 338, (1989) After incubation at room temperature for 30 min, the reaction products were Lindahl,T., Kaschka-Dierich,C. Adams,A., Bornkamm,G.W., Bjursell,G., separated in a 5% polyacrylamide gel. In competition and supershift and Jehn,U. (1976) J. Mol. Biol., 102, 511-530. Cell, 61, 1217-1224. and Green,M. (1990) experiments either unlabelled oligonucleotide or 1 a1 tissue culture supemratant Liu,F. monoclonal antibody was added to the reaction mixture. The Miller,G., Robinson,J., Heston, L. and Lipman,M. (1974) Proc. Nat!. Acad. containing were used as anti-EBNA2 monoclonal antibodies: RI (rat IgGI), Sci USA, 71, 4006-4010. following 174 EBNA2 interaction with TP1 promoter Polack,A., Hartl,G., Zimber,U., Freese,U.K., Laux,G., Takaki,K., Hohn,B., Gissmann,L. and Bomkamm,G.W. (1984) Gene, 27, 279-288. Pulvertaft,R.J.V. (1964) Lancet, 1, 238-240. 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Zimber-Strobl,U., Suentzenich,K.O., Laux,G., Eick,D., Cordier,M., Calender,A., Billaud,M., and J. Lenoir,G.M. Bornkamm,G.W. (1991) Virol., 65, 415-423. Received on July 20, 1992; revised on September 1992 10,

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Published: Jan 1, 1993

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