Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 8030-8034, September 1992 Medical Sciences A region of VP16 can substitute for a transforming domain of Epstein-Barr virus nuclear protein 2 (herpsvis/trnrpton/raacvaflon) JEFFREY I. COHEN Laboratory of Clinical Investigation, National Institutes of Health, Bethesda, MD 20892 Communicated by Bernard Moss, May 20, 1992

ABSTRACT Epstein-Barr virus (EBV) nuclear protein 2 whether hydrophobic interactions, per se, are essential for (EBNA-2) is essential for EBV-duced B-cell transformationin the function of these other activators. vitro. EBNA-2 contains a 14-amino acid domain that directly To identify critical elements in the transcriptional activa- activates transcription and Is required for transformation. To tion domain of EBNA-2 and to explore their relationship to determine whether another transcriptional activator can sub- similar elements in other acidic activators, we analyzed the stitute for this function, a chimeric virus was constructed that ability of mutated domains to activate transcription and contained a portion of the transcriptional activation domain support B-cell transformation. In addition, we replaced the from the herpes simplex virus VP16 protein inserted in place of transcriptional activation domain of EBNA-2 with a portion the 14-amino acid domain ofEBNA-2. The chimeric virus was of the activation domain of VP16, with which it shares some able to transform B cells efficiently and transactivate expres- structural features, to generate a chimeric EBNA-2-VP16 sion of EBV and B-cell genes. Randomization of the 14-amino gene. The chimeric gene was inserted into the EBV genome, acid sequence in the domain markedly reduced its transcrip- and the recombinant virus was assayed for transforming tional activating activity and the transforming efficiency of the activity in primary B cells. Finally, the chimeric protein was recombinant EBV. Mutation of a trptophan within the 14- analyzed for its ability to transactivate expression of an EBV amino acid domain ofEBNA-2 completely abolished transcrip- and B-cell gene. tional activation and B-cell trasormation. These experiments indicate that EBNA-2 and VP16 activate transcription by MATERIALS AND METHODS imilar mechaniss and that transcriptional activation is re- Cell Lines and DNAs. BJAB cells are derived from an quired for EBV-induced B-cell tansformation. EBV-negative B-cell (16), and P3HR-1 clone 16 cells have a deletion in EBV that includes the EBNA-2 gene Epstein-Barr virus (EBV) transforms B lymphocytes in vitro (17). to proliferate indefinitely and is associated with Burkitt Plasmid GAL4 (13), which encodes the yeast GAL4 DNA lymphoma, Hodgkin disease, and certain other B-cell lym- binding domain, was linearized with BamHI and ligated to phoproliferative disorders (1). Six EBV nuclear proteins double-stranded synthetic oligonucleotides (corresponding (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and to the nucleotide sequence that encodes amino acids 449-483 EBNA-LP) and three latent membrane proteins (LMP-1, of EBNA-2) with Bgl II ends to generate plasmid GAL-E2 LMP-2A, and LMP2-B) are expressed in EBV-transformed B (449-483). Other synthetic oligonucleotides were used to cells in vitro (2). generate GAL-E2 WA454 (alanine substituted for tryptophan EBNA-2 is essential for transformation in vitro (3, 4) and at amino acid 454 of EBNA-2), GAL-E2 WF454 (phenylal- is expressed in tissues ofimmunocompromised patients with anine at 454), GAL-E2 WL454 (leucine at 454), GAL-E2 lymphoproliferative disease (5). EBNA-2 transactivates ex- WY454 (tyrosine at 454), GAL-E2 DN4 (asparagine or glu- pression of the EBV LMP-1 and LMP-2 genes (6, 7) and the tamine substituted for aspartic orglutamic acid, respectively, B-cell CD23, CD21, and c-fgr genes (8-10). Analysis of at amino acids 452, 455, 459, and 462 of EBNA-2), and deletion mutants shows that four domains of EBNA-2 are GAL-E2 R (randomized order of EBNA-2 amino acids 449- essential both for transformation and for transactivation of 462). GAL-E2 (449-462) was described previously (12); LMP-1 (11). Recent studies indicate that one of these four GAL-E2T1 and GAL-E2T2 were made by ligating GAL4 to domains, which encodes a 14-amino acid peptide, can di- aHinfl fragment from plasmid HK-EBNA-2 (3) and pDA1136 rectly activate transcription (12). (18), which contain EBV type 1 and type 2 EBNA-2, respec- The 14-amino acid transcriptional activation domain of tively. Synthetic oligonucleotides (corresponding to the nu- EBNA-2 is highly acidic; it contains six negatively charged cleotide sequence that encodes amino acids 439-451 ofVP16) amino acids. Other transcriptional activation domains have were used to construct GAL-VP and GAL-VP DN1 (aspar- been identified including the adenovirus ElA and the herpes agine substituted for aspartic acid at VP16 amino acid 443). simplex virus (HSV) VP16 proteins (13, 14). Analysis of the Plasmid EBNA-2d449-479 (11) was cut with Bgl II and critical elements of the HSV VP16 activation domain indi- ligated to several of the double-stranded synthetic oligonu- cates that negatively charged amino acids contribute to, but cleotides with Bgl II ends described above. The resulting are not sufficient for, transcriptional activation. Moreover, a plasmids were then cut with HindIII and Rsr I, and the large single critically situated hydrophobic amino acid in the do- fragment was ligated to the large HindIH-Rsr I fragment of main is essential for activation by VP16 (15). Although the cosmid T1EBNA-2 (3), which contains the EcoRI A fiagment distribution of hydrophobic amino acids is conserved among from an EBV type 1 strain, to generate cosmids C-E2, C-E2 several classes of transcriptional activators, it is unknown R, C-E2 WA454, C-E2 VP, and C-E2 VP DN1.

The publication costs ofthis article were defrayed in part by page charge Abbreviations: EBV, Epstein-Barr virus; HSV, herpes simplex payment. This article must therefore be hereby marked "advertisement" virus; EBNA, EBV-encoded nuclear protein; LMP, latent mem- in accordance with 18 U.S.C. §1734 solely to indicate this fact. brane protein; CAT, chloramphenicol acetyltransferase. 8030 Downloaded by guest on September 28, 2021 Medical Sciences: Cohen Proc. Natl. Acad. Sci. USA 89 (1992) 8031 Transfections and Infections. Activator and reporter plas- 2A). The reporter plasmid contained GAL4 binding sites mids were cotransfected into B-lymphoma cells, and cell upstream of the murine mammary tumor virus promoter and lysates were prepared after 48 hr for chloramphenicol ace- the CAT gene (Fig. 2B; ref. 12). Activator and reporter tyltransferase (CAT) assays (12). Transformation assays plasmids were cotransfected into B-lymphoma cells, and the were performed as described (11). Infected cells were plated level of CAT activity in the cells after 48 hr was an indication into 56 wells, and the number of wells containing transform- ofthe ability ofthe EBNA-2 domain to activate transcription. ants was determined 8 weeks after infection. Two indepen- A plasmid with the GAL4 DNA binding domain alone served dent cosmid clones were prepared for each ofthe constructs. as a negative control, whereas a plasmid with the activation Polymerase Chain Reaction. Cellular DNA was amplified domain of EBNA-2 [amino acids 449-462 (12)] fused to the by using primers corresponding to nucleotides 49,590-49,606 GAL4 DNA binding domain was a positive control. and 50,073-50,089 of EBV (19) in a 35-cycle polymerase Previous work has shown that replacement of phenylala- chain reaction. The reaction products were gel purified and nine442 of VP16 with alanine completely inactivates tran- sequenced (20). scriptional activating activity (15). Replacement of tryp- tophan-454 of EBNA-2, which aligns with the phenylalanine of VP16, with alanine (E2 WA454), leucine (E2 WL454), or RESULTS tyrosine (E2 WY 454) reduced the transcriptional activation EBNA-2 and VP16 Transcriptional Activation Domains ofEBNA-2 by 80%o or more (Fig. 3). In contrast, replacement Share Common Structural Features. To identify features that of tryptophan-454 with phenylalanine (E2 WF454) retained EBNA-2 might have in common with other transcriptional over 50% ofthe transcriptional activity. Replacement offour activators, we aligned the amino acid sequence of the acti- of the negatively charged amino acids within the transcrip- vation domain of EBNA-2 from EBV types 1 and 2 with the tional activation domain (E2 DN4) or randomization of the transcriptional activation domain of HSV VP16, using the order of the amino acids in the domain (E2 R) reduced position of the hydrophobic amino acids as a guide (15). transcriptional activation by 80%6. Phenylalanine at position 442 of VP16, critical for transcrip- Replacement of the 14-amino acid transcriptional activa- tional activation by VP16, aligned with tryptophan at position tion domain of EBNA-2 (amino acids 449-462) with the 454 of EBNA-2 (Fig. 1 Upper). Although the amino acid corresponding region ofVP16 (amino acids 439-451) resulted sequences ofthe EBNA-2 and VP16 activation domains share in a 3- to 4-fold increase in the transcriptional activity [Fig. <50%6 amino acid identity, the organization ofthe hydropho- 3, GAL-VP vs. GAL-E2 (449-483)]. Replacement ofaspartic bic amino acids was similar and the negatively charged amino acid443 of VP16 with asparagine (VP DN1) did not affect acids were clustered near the carboxyl terminus of the transcriptional activation of GAL-VP. domain. When the activator constructs were cotransfected with a To determine whether the alignment ofEBNA-2 with VP16 reporter plasmid that lacked GAL4 DNA binding sites (12), was biologically relevant, constructs based on the carboxyl- there was no transcriptional activation (data not shown), terminal 14 amino acids of the domain (Fig. 1 Lower) were which verifies that direct interaction with the reporter plas- tested for their ability to activate transcription in B-lym- mid is required for activating activity. phoma cells. Activator plasmids contained portions of To ensure that the reduced activity ofthe EBNA-2 mutants EBNA-2 fused to the yeast GAL4 DNA binding domain (Fig. was not due to decreased expression or protein instability, we 483 EBNA-2 a __

435 44 \462 Type 1 EBNA-2 L f P ED-lEW 0P P S D P A a D [EX S F 0 *FT TI Type 2 EBNA-2 L M P S [aW 0 P P T L F P A FE M FD FE S IF G *[Al T TE VP16 H E[G PaFelA M A H [DA A [ j[ E0[D MEG [D G [F 425 438 451

E2 [3 Mif~sOF-S2 aE n El T T SE- E2WA454 *MBE S A ME I E- T T m E2DN4 E D IQ S W N II Q T T Q E2R FE IS-DI 1 REm T SFESo T [3M2;I 3 VP I9*UE1 1Ui 1 1 M R G E G VPDN1 FE D D ME N 0 MD M 8 G E G MD FIG. 1. Alignment ofthe amino acid sequence ofthe transcriptional activation region ofEBNA-2 with the activation domain ofherpes simplex VP16. The EBNA-2 gene from EBV strain W91 encodes a protein of483 amino acids with four domains essential for transformation (represented by black bars in the EBNA-2 gene). Amino acids 449-462 activate transcription. (Upper) EBNA-2 sequences from EBV types 1 and 2 (18) were matched with the alignment from Cress and Triezenberg (15) by using the 6 hydrophobic amino acids (reverse-type letters) and negatively charged amino acids (boxed letters) as a visual guide. (Lower) Some of the amino acid sequences used for transcription and transformation assays. The sequence of E2 contains amino acids 449-462 of type 1 EBNA-2 with 3 amino acids (449-451) changed due to a Bgl II linker used in cloning. E2 WA454 is identical to E2 except that the tryptophan at position 454 has been changed to alanine. Additional amino acid substitutions were made at tryptophan-454 (see Materials and Methods). E2 DN4 has four of the negatively charged amino acids (aspartic acid or glutamic acid) replaced (with asparagine or glutamine). E2 R has the same amino acids as E2, but the order ofthe sequence has been randomized. VP contains amino acids 437-451 of VP16 with 2 amino acids (437 and 438) changed due to the Bgl II linker. VP DN1 is identical to VP except that aspartic acid at position 443 has been changed to asparagine. Downloaded by guest on September 28, 2021 8032 Medical Sciences: Cohen Proc. Nati. Acad. Sci. USA 89 (1992) A 1 426 449 462 483 EBNA-2 l GAL GAL41 GAL-E2 (449-483) E2 I GAL-E2 WA454 E2 W-A I E2 I

GAL-E2 WF454 E2 W-F E2 |

GAL-E2 WL454 E2 W-L I E2 I

GAL-E2 WY454 E2 W-Y I E2 ] GAL-E2 DN4 g o E2 I

GAL-E2 R _ i E2 | GAL-VP VP16 I E2 I GAL-VP DN1 D- E2 | FIG. 2. GAL4 fusions with EBNA-2 and VP16 domains. (A) The GAL4 I)NA binding domain (amino acids 1-147) was fised to the EBNA-2 GAL-E2 (449-462) 2o (amino acids 449-462) or VP16 (amino acids 438- 451) sequences shown in the lower part of Fig. 1 GAL-EZT1 Type 1 EBNA-2 followed by the carboxyl portion of EBNA-2 (ami- no acids 463-483). GAL4-E2 (449-46) does not contain the 2 carboxyl portion of EBNA-2, GAL4- GAL-E2T2 Type EBNA-2 I E2T1 has amino acids 426-462 from EBV type 1 EBNA-2 (without the Bgl I linker), and GAL4- E2T2 has the corresponding sequence from EBV type 2 EBNA-2. (B) The reporterconstruct contains 13-GAL4 sites MMTV promoter 13 GAL4 sites followed by the mure mammary B GAL4-MMTV CAT tumor virus (MMTV) promoter and the CAT gene. immunoprecipitated fusion proteins using a GAL4-specific deletion has been restored is transformation-competent (3). antibody. Each ofthe constructs tested, including those with Five EBNA-2 and VP16 constructs (Fig. 1, Lower) were reduced transcriptional activating activity, expressed similar cloned into an EBV cosmid (T1EBNA-2) in place of the amounts of the fusion protein in cells (Fig. 4). nucleotides encoding amino acids 449-462 of the wild-type The Transactivaton Types 1 and 2 EBNA-2 EBNA-2 gene. P3HR-1 cells were transfected with each Have Similar Activities. While the overall amino acid se- cosmid, viral replication was induced, the resultant virus was quences ofEBV types 1 and 2 EBNA-2 have only 56% amino used to infect human umbilical cord mononuclear cells, and acid identity (18) and also differ in their transforming activity transformed clones were selected and assayed. (3) and CD23 transactivating activity (3, 21), EBV types 1 and To ensure that each of the five cosmids could express 2 EBNA-2 have similar LMP-1 transactivating activity (3, 6). EBNA-2, P3HR-1 cells were first transfected with the cosmids Cotransfection of B-lymphoma cells with the reporter plas- and stained 72 hr later with a monoclonal antibody specific for mid and the transcriptional activation domain of EBV type 1 EBNA-2 by using immunofluorescence microscopy (5). Sim- EBNA-2 or the corresponding domain of type 2 EBNA-2 ilar numbers ofcells (2-41%) were positive for EBNA-2 antigen resulted in similar levels of transcriptional activating activity after transfection with each of the five cosmids. (Fig. 3B, GAL-E2T1 vs. GAL-E2T2). Thus, the transforming Virus derived from the chimeric cosmid (C-E2 VP) con- and transactivating differences between EBV types 1 and 2 taining the HSV VP16 sequence in place ofthe 14-amino acid EBNA-2 must reflect differences in amino acid sequence transcriptional activation domain of EBV EBNA-2 exhibited outside of the transcriptional activation domain. a transforming efficiency similar to virus with the corre- The HSV VP16 Transactivatlon Domain Can Substitute for sponding EBV EBNA-2 domain [C-E2 (Table 1)]. The num- a Transforming Domi OfEBNA-2. The 14-amino acid trans- ber of transformed cell clones obtained and the time to activation domain of EBNA-2 is essential for B-cell trans- macroscopic appearance of the clones were similar for both formation by EBV (11). To determine whether another tran- ofthe recombinant viruses. Replacement ofaspartic acid-443 scriptional activator could substitute for this function, we of VP16 with asparagine (C-E2 VP DN1) did not affect constructed a chimeric virus that contains a portion of the transformation. In contrast, randomization of the EBNA-2 transcriptional activation domain from HSV VP16 (amino transcriptional activation sequence (C-E2 R) resulted in virus acids 439-451) inserted in place ofthe 14-amino acid domain that was markedly reduced in transforming efficiency. In of EBNA-2. Previous experiments have shown that cotrans- several experiments only one transformed cell clone was fection of P3HR-1 cells with a cosmid containing EBNA-2 obtained. Replacement of the tryptophan at position 454 of and a plasmid to induce EflV replication results in homolo- EBNA-2 with alanine (C-E2 WA454) yielded virus that failed gous recombination of the P3HR-1 viral genome with the to transform B cells. transfected cosmid DNA. Whereas P3HR-1 cells contain an The levels ofexpression of EBNA-2, LMP-1, and CD23 in EBV that lacks the carboxyl portion of EBNA-LP and the cell clones derived from the two chimeric EBNA-2-VP16 entire EBNA-2 gene and is unable to transform B cells, the viruses (C-E2 VP and C-E2 VP DN1) and the virus containing recombinant EBV in which the EBNA-LP and EBNA-2 the randomized transcriptional activation sequence (C-E2 R) Downloaded by guest on September 28, 2021 Medical Sciences: Cohen Proc. Natl. Acad. Sci. USA 89 (1992) 8033

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:.:: :. :: * .w. FIG. 4. Expression of GAL4-EBNA-2 and GAL4-VP16 con- ... .. structs in transfected cells. COS cells were transfected with activator **2 *I *.'.* * * plasmids (indicated at the top of each lane), cells were labeled with [35S]methionine, and proteins were immunoprecipitated with anti- GAL4 antibody. Relative protein size (in kDa) is indicated in the Fold Induction 1 107 105 23 71 58 center of the of CAT * ;..* gels. FIG. 3. Activation of transcription by GAL4 fusions with adenovirus EMA protein. This region contains the binding EBNA-2 and VP16. Activator and reporter constructs were cotrans- domain for two cellular proteins, the retinoblastoma gene fected into B-lymphoma cells and assayed for CAT activity (12). The product and a 107-kDa protein, in the simian virus 40 and CAT activity for each set of experiments, relative to the activator adenovirus EMAproteins (23, 24). We have shown that a region with GALA alone, is shown at the bottom of each lane. of VP16 with a different biochemical function, transcriptional activation, can substitute for a transforming domain in EBV. were compared to those in cell lines derived from cosmid Analysis of the sequence in the activation domain of T1EBNA-2 or wild-type B95-8 virus. Levels ofEBNA-2 (Fig. EBNA-2 indicates that both hydrophobic and ionic interac- 5A), LMP-1 (FIG. SB), and CD23 (data not shown) were tions play an important role in transcriptional activation. similar in all lymphoblastoid cell clones tested. Replacement of tryptophan-454 with another hydrophobic To verify that the transformed cell lines carried EBNA-2 aromatic amino acid (phenylalanine) retained much of the with sequence, the desired DNAs from transformed cell lines transcriptional activity; however, replacement with polar derived from the four recombinant viruses were amplified by aromatic (tyrosine) or hydrophobic nonaromatic (alanine, the polymerase chain reaction, and sequence analysis con- leucine) amino acids abrogated most of the activity. Simi- firmed that the expected sequence had been inserted into the larly, when phenylalanine442 of VP16 was replaced with EBNA-2 gene. polar aromatic or hydrophobic nonaromatic amino acids, most of the transcriptional activity was lost (15). Replace- DISCUSSION ment offour ofthe negatively charged amino acids within the transcriptional activation domain of EBNA-2 abolished most These experiments indicate that a portion of the transcrip- of the activity. In contrast, when four of the negatively tional activation domain of HSV VP16 can effectively sub- charged amino acids around phenylalanine-442 of VP16 were stitute for a transforming domain of EBNA-2 and preserve replaced, about 60% of the activity was retained (15). the ability of the protein to transactivate gene expression. Randomization of the amino acid sequence in the tran- The domains ofEBNA-2 and VP16 used for complementation scriptional activation domain of EBNA-2 markedly reduced, share <50%o amino acid identity; however, they possess but did not completely abolish, the ability of the protein to certain structural features in common. Both domains contain activate transcription and to contribute to transformation. several negatively charged amino acids, a putative a-helical Randomization of the sequence alters the predicted second- structure, and hydrophobic amino acids that can be aligned ary structure of the domain and changes the position of the in a similar pattern. Since substitution of alanine for a hydrophobic amino acids but does not change the overall hydrophobic amino acid at corresponding sites in EBNA-2 charge ofthe molecule. While the activation domains ofVP16 and VP16 inactivates transcription in both of the proteins, and EBNA-2 have putative a-helical structures, this pre- these proteins may share similar biochemical mechanisms of dicted structure has been shown to be unnecessary for transcriptional activation, despite marked differences in both transcriptional activation by VP16 (15). The marked diminu- their amino acid sequences and the types of cells that EBV tion in transcriptional activation and transformation when the and HSV infect. sequence was randomized reinforces the critical role of the Moran (22) reported that a region of simian virus 40 large order of the hydrophobic amino acids in the sequence. While tumor antigen can substitute for a transforming domain in the the EBNA-2 molecule with the randomized activation do- Downloaded by guest on September 28, 2021 8034 Medical Sciences: Cohen Proc. Natl. Acad. Sci. USA 89 (1992)

Table 1. Transformants obtained from recombinant EBV containing EBNA-2 mutations Time to formation of macroscopic No. of wells containing transformants colonies*, weeks Cosmid Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 1 Exp. 2 Exp. 3 Exp. 4 TlEBNA-2t 27 4 25 37 3 4 3 3 C-E2 10,13 2 21 36 3, 3 5 3 3 C-E2R 0, 0 0 0 1 , - 5 C-E2 WA454 0,0 0 0 0,0 -,- - C-E2 VP 12, 13 8 10 ND 3, 3 3 3 ND C-E2 VP DN1 10, 21 11 11 ND 3, 3 2 3 ND ND, not done. *The time to colony formation was the number of weeks after infection when the first macroscopic colonies were visible. tCosmid T1EBNA-2 contains the EcoRI A fragment from an EBV type 1 strain (3) and is the parent used to derive the other cosmids. main had a markedly reduced ability to activate transcription the transcriptional activating capability of EBNA-2 by inser- in a transient assay, B cells that were successfully trans- tion of a domain of HSV VP16 also restores the transforming formed by the recombinant virus expressed wild-type levels activity of EBV, proves that transcriptional activation by of LMP-1 and CD23. Discordance in the level of transacti- EBNA-2 is essential for EBV-induced B-cell transformation. vating activity in transient transfection assays and in trans- formed cell lines containing recombinant viruses has been Karen Seidel and Masako Moriuchi provided excellent technical seen with other EBNA-2 mutants (11) and may represent assistance, Douglas Last provided anti-GAL4 antibody, Gary Arm- selective advantage ofcells that can express sufficient LMP-1 strong assisted in obtaining umbilical cord mononuclear cells, and and CD23 for outgrowth in culture. Stephen Straus reviewed the manuscript. EBV types 1 and 2 differ markedly in their ability to 1. Miller, G. (1990) in , eds. Fields, B. N., Knipe, D. M., transform primary B cells (25). Experiments with recombi- Chanock, R. M., Hirsch, M. S., Melnick, J. L., Monath, T. P., & nant viruses have shown that a major determinant of the Roizman, B. (Raven, New York), pp. 1921-1958. type-specific differences is the EBNA-2 gene (3). The finding 2. Kieff, E. & Liebowitz, D. (1990) in Virology, eds. Fields, B. N., that the transcriptional activating domain of EBV type 1 Knipe, D. M., Chanock, R. M., Hirsch, M. S., Melnick, J. L., EBNA-2 and its'corresponding domain in type 2 EBNA-2 Monath, T. P., & Roizman, B. (Raven, New York), pp. 1889-1920. have similar activating effects in B cells suggests that 3. Cohen, J. I., Wang, F., Mannick, J. & Kieff, E. (1989) Proc. Natd. EBNA-2 has additional biochemical functions required for Acad. Sci. USA 86, 9558-9562. other of the molecule 4. Hammerschmidt, W. & Sugden, B. (1989) Nature (London) 340, transformation; alternatively, portions 393-397. may contribute to transactivation. The observation that the 5. Young, L., Alfieri, C., Hennessy, K., Evans, H., O'Hara, C., chimeric EBNA-2-VP16 construct transactivates expression Anderson, K. C., Ritz, J., Shapiro, R. S., Rickinson, A., Kieff, E., of LMP-1 and CD23 to levels similar to those seen in & Chohen, J. I. (1989) N. Engl. J. Med. 321, 1080-1085. wild-type virus also suggests that portions of the' EBNA-2 6. Wang, F., Tsang, S. F., Kurilla, M. G., Cohen, J. I. & Kieff, E. molecule other than the transcription activation domain (1990) J. Virol. 64, 3407-3416. examined here interact with viral and cellular promoters or 7. Zimber-Strobl, U. Suentzenich, K. 0., Laux, G., Eick, D., Cordier, with other proteins that interact with these promoters. M., Calender, A., Billaud, M., Lenoir, G. M. & Bornkamm, G. W. Previous studies showed that deletion of the EBNA-2 (1991) J. Virol. 65, 415-423. 8. Wang, F., Gregory, C. D., Rowe, M., Rickinson, A. B., Wang, D., transactivation domain abolishes the transforming activity of Birkenbach, M., Kikutani, H., Kishimoto, T. & Kiiff, E. (1987) EBV (11). Since the EBNA-2 activation domain might con- Proc. Nat!. Acad. Sci. USA 84, 3452-3456. tain sequences important for additional functions besides 9. Wang, F., Gregory, C. D., Sample, C., Rowe, M., Liebowitz, D., transcriptional activation, those experiments did not estab- Muimy, R., Rickinson, A. & Kieff, E. (1990)J. Virol. 64, 2309-2318. lish that transformation correlated strictly with transcrip- 10. Knutson, J. C. (1990) J. Virol. 64, 2530-2536. tional activation. The present observation, that restoration of 11. Cohen, J. I., Wang, F. & Kieff, E. (1991) J. Virol. 65, 2545-2554. 12. Cohen, J. I. & Kieff, E. (1991) J. Virol. 65, 5880-5885. A B 13. Lillie, J. W. & Green, M. R. (1989). Nature (London) 338, 39-44. 14. Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. (1988). Nature C rl (London) 335, 563-564. < 15. Cress, W. D., & Triezenberg, S. J. (1991) Science 251, 87-0. - > a: . i c: m co 16. Menezes, J., Leibold, W., Klein, G. & Clements, G. (1975) Bio- 17 ILu CM, CMi CN L Li Li Li LC CC~~~~~~C medicine 22, 276-284. h- ( c cD Cu cM C%~~~~C 17. Rabson, M. L., Gradoville, L., Heston, L. & Miller, G. (1982) J. Virol. 44, 834-844. 18. Dambaugh, T., Hennessy, K., Chamnankit, L. & Kieff, E. (1984) Proc. Nat!. Acad. Sci. USA 81, 7632-7636. 19. Baer, R., Bankier, A. T., Biggn, M. D., Dei er, P. L., Farrell, 9 P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Satchweil, S. C., 669 Seguin, C., Tuffhell, P. S. & Barrell, B. G. (1984) Nature (London) 310, 207-211. 20. Winship, P. R. (1989) Nucleic Acids Res. 17, 1266. 21. Wang, F., Kikutani, H., Tsang, S. F., Kishimoto, T. & Kieff, E. (1991) J. Virol. 65, 4101-4106. 22. Moran, E. (1988) Nature (London) 334, 168-170. FiG. 5. Immunoblot of EBNA-2 and LMP-1 expression in cells 23. Ewen, M. E., Ludlow, J. W., Marsilo, E., DeCaprio, J. A., Milli- transformed by recombinant EBV. (A) Immunoblot with human kan, R. C., Cheng, S. H., Paucha, E. & Livingston, D. M. (1989) serum. The open arrow indicates EBNA-1, and the closed arrow Cell S, 257-267. indicates EBNA-2. (B) Immunoblot with monoclonal antibody to 24. Whyte, P., Williamson, N. M. & Harlow, E. (1989) Cell 56, 67-75. LMP-1. P3HR-1 lacks the EBNA-2 gene and expresses very low 25. Rickinson, A. B., Young, L. S. & Rowe, M. (1987) J. Virol. 61, levels of LMP-1. Protein size is in kDa. 1310-1317. Downloaded by guest on September 28, 2021