Epstein-Barr Virus Nuclear Protein 3A Domains ... - Journal of Virology

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Feb 7, 2005 - enhancer, are null mutations for LCL growth, whereas EBNA3A ... regulation of transcription through RBP-J /CBF1 is critical for LCL growth.
JOURNAL OF VIROLOGY, Aug. 2005, p. 10171–10179 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.16.10171–10179.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 79, No. 16

Epstein-Barr Virus Nuclear Protein 3A Domains Essential for Growth of Lymphoblasts: Transcriptional Regulation through RBP-J␬/CBF1 Is Critical Seiji Maruo, Eric Johannsen, Diego Illanes, Andrew Cooper, Bo Zhao, and Elliott Kieff* Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 Received 7 February 2005/Accepted 12 May 2005

Experimental reverse genetic replacement of Epstein-Barr virus nuclear antigen 3A (EBNA3A) with a conditional mutant EBNA3A revealed that EBNA3A is critical for continued lymphoblastoid cell (LCL) growth. Wild-type (wt) EBNA3A expressed in the LCLs specifically sustained growth under nonpermissive conditions, whereas EBNA3B or EBNA3C expression had no effect (S. Mauro, E. Johannsen, D. Illanes, A. Cooper, and E. Kieff, J. Virol. 77:10437–10447, 2003). This genetic system and related biochemical assays have now been used to discover that EBNA3A lacking amino acid residues 170 to 240 (⌬170–240), TLGC202 to AAGA202, or ⌬300–386, which are deficient in repression of EBNA2 activation of an RBP-J␬/CBF1-dependent EBV Cp enhancer, are null mutations for LCL growth, whereas EBNA3A ⌬2–124, ⌬410–439, ⌬440–470, ⌬470–500, ⌬500–523, ⌬523–612, and ⌬620–820, which are wt in repression are wt for LCL growth. Thus, EBNA3A regulation of transcription through RBP-J␬/CBF1 is critical for LCL growth. EBNA3A mutants deleted of amino acid residues 240 to 300, 386 to 410, or 827 to 944 were intermediate, null, or intermediate, respectively, for LCL growth despite being wt for RBP-J␬ association and repression. Amino acid residues 240 to 300, 386 to 410, and, particularly, C-terminal residues 827 to 944 are therefore likely to contribute to LCL growth through RBP-J␬-independent mechanisms. Epstein-Barr virus (EBV) causes lymphocyte-proliferative diseases in people with immune system deficiencies, Burkitt’s lymphoma, Hodgkin’s lymphoma, other B- and T-cell lymphomas, anaplastic nasopharyngeal carcinoma, and some gastric carcinomas (for review see references 21 and 38). EBV infection converts primary human B lymphocytes in vitro into continuously proliferating lymphoblastoid cell lines (LCLs) (13, 35). In LCLs, EBV expresses six nuclear proteins (EBNA1, -2, -3A, -3B, -3C, and -LP), three integral membrane proteins (LMP1, -2A, and -2B), two small nonpolyadenylated RNAs (EBER1 and EBER2), and BamA rightward transcripts. EBNA1, -2, -3A, -3C, and -LP, and latent membrane protein 1 (LMP1) are necessary for efficient LCL outgrowth, whereas the rest of the EBV genes are dispensable. EBNA2 and EB nuclear antigen leader protein (EBNALP) are expressed first in primary B-lymphocyte infection and coactivate transcription from cell and viral promoters (1, 2, 45). EBNA2 associates with the sequence-specific DNA binding protein RBP-J␬/CBF-1/CSL and activates transcription from promoters that have nearby RBP-J␬ and PU.1 binding sites (9, 12, 16). EBNA2 activates the cell CD21, CD23, c-fgr, and c-myc promoters and the viral EBNA and LMP promoters and thereby has a key role in EBV conversion of primary human B lymphocytes into LCLs (5, 8, 10, 18). EBNALP strongly coactivates transcription with EBNA2 (11, 31–34). EBNA2 and EBNALP up-regulation of the Cp EBNA promoters leads to EBNA3A, EBNA3B, EBNA3C, and EBNA1 transcription. * Corresponding author. Mailing address: Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115. Phone: (617) 525-4252. Fax: (617) 525-4257. E-mail: [email protected].

When EBNA3A, EBNA3B, and EBNA3C reach steady-state levels, they compete with EBNA2 for binding to RBP-J␬ and regulate virus and cell promoters with EBNA2 (3–7, 17, 19, 22, 24, 26, 27, 36, 37, 42, 44, 46, 47). EBNA3A, EBNA3B, and EBNA3C arose from a tandem triplication of an ancestral gene; the triplication may be unique to Old World primate gamma-1 herpesviruses (15, 39, 40). EBNA3A, EBNA3B, and EBNA3C N-terminal amino acids (aa) 90 to 320 mediate interactions with RBP-J␬ and have 22 to 27% amino acid sequence identity (7, 42, 44, 46). Recombinant EBV reverse genetic experiments indicate that EBNA3A and EBNA3C are critical for EBV conversion of primary B lymphocytes to LCLs (20, 29, 43). Previous studies have used biochemical and functional transcription assays to identify the following domains in EBNA3A: amino acids (aa) 146 to 155 as a nuclear localization signal that is necessary and sufficient for EBNA3A nuclear localization (25), aa 125 to 240 that stably associate with RBP-J␬/CBF-1, the amino acid sequence TLGC202 that is critical for this association (4, 5, 7, 42, 44, 46), aa 1 to 386 that are sufficient for repression of EBNA2 activation of the EBV Cp promoter (5, 7, 26, 36, 44), aa 524 to 666 or aa 627 to 805 that can repress or activate a Gal4-responsive reporter when fused to the Gal4DNA binding domain (3, 4, 7, 14, 36), aa 1 to 523 that when overexpressed can disrupt EBNA2 transcriptional activation through RBP-J␬/CBF-1 in LCLs (5), and aa 857 to 890 that include two (A/V)LDLS motifs that mediate interaction with CtBP (14) (Fig. 1). The significance of these EBNA3A domains for LCL growth remained to be determined and is the objective of these experiments. To determine the importance of EBNA3A domains for growth transformation, we utilized LCLs that are infected with

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FIG. 1. Diagram of EBNA3A domains and mutants with summarized reverse genetic and biochemical data. The diagram indicates EBNA3A aa 146 to 155, which are the essential and sufficient nuclear localization signal (25); aa 90 to 310, which have the highest (22 to 27%) identity to other EBNA3 proteins and are designated “Homology,” T199, L200, and C202 and which are critical for RBP-J␬ association (7); aa 310 to 365, which are charged; and aa 841 to 944, which are acidic. Residues 524 to 666 and 627 to 805 have repressive and activation effects, respectively, on Gal4-responsive promoters when fused to the Gal4 DNA-binding domain (3, 4, 7, 14, 36, 37). The results of wt or mutant EBNA3A association with RBP-J␬, repression of EBNA2 transcriptional activation of the Cp promoter and effects on LCL growth under conditions that are nonpermissive for endogenous EBNA3AHT expression are shown in columns on the right side of the figure. ⴙ, wt phenotypes; ⫺, null; and I, intermediate. The number of transcomplementation experiments (N) performed and the number of different EBV/EBNA3AHT infected LCLs (n) used in experiments are also indicated. The parts of EBNA3A that were deleted without affecting cell growth or with an intermediate effect on cell growth are indicated at the bottom.

recombinant EBV genomes that express a conditional EBNA3A with the last codon fused in frame to the first codon of a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone-binding domain (EBNA3AHT). EBNA3AHT inactivation in EBV/EBNA3AHT-infected LCLs causes growth arrest but, surprisingly, does not affect expression of other EBNAs, LMP1, CD23, or c-myc (29). Wild-type (wt) EBNA3A expression from an oriP plasmid vector transfected into the EBNA3AHT LCLs sustains LCL growth in medium without 4HT, whereas EBNA3B, EBNA3C, or empty vector cannot sustain growth (29). Since wt EBNA3A is specifically required for EBNA3AHT-infected LCL growth in the absence of 4HT, we have used this system to identify the EBNA3A domains that are critical for LCL growth. MATERIALS AND METHODS Cells. BJAB is an EBV-negative B-lymphoma cell line (30). IB4 is an LCL transformed with wt B95 strain EBV (23). Four different EBNA3AHT-infected LCL clones or subclones, 41-3, 41-13, 83, and 163, were used within 8 months of culture from EBV infection. EBNA3AHT-infected LCLs were maintained in RPMI 1640 medium supplemented with 15% fetal bovine serum, L-glutamine,

streptomycin, penicillin, and 200 nM 4HT (Sigma). All other cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, L-glutamine, streptomycin, and penicillin. Viable cell numbers were determined by hemocytometer based on trypan blue exclusion. Plasmids. The plasmids pSG5-FLAG, pSG5-FLAG-EBNA3A (fE3A), pSG5FLAG-EBNA3C (fE3C), pSG5-FLAG-EBNA3A triple alanine substitution (T199A, L200A, and C202A) mutant (AAGA), pSG5-FLAG-EBNA3A aa 1 to 277 (1–277), pSG5-FLAG-EBNA3A aa 1 to 386 (1–386), pSG5-FLAGEBNA3A aa 1 to 523 (1–523), pSG5-FLAG-EBNA3A aa 1 to 826 (⌬827–944), and pSG5FLAG-EBNA3A aa 125 to 944 (⌬2–124) are simian virus 40 (SV40) enhancer- and promoter-driven expression vectors for the indicated FLAGtagged proteins (5, 29). oriP plasmids for expression of FLAG (control), fE3A, fE3C, AAGA, 1–277, 1–386, 1–523, ⌬827–944, or ⌬2–124 under the control of SV40 promoter were made by subcloning the SalI fragments containing SV40 promoter-driven FLAG, fE3A, fE3C, AAA, 1–277, 1–386, 1–523, ⌬827–944, or ⌬2–124 cassettes from pSG5-FLAG, pSG5-fE3A, pSG5-fE3C, pSG5-AAA, pSG5-1-277, pSG5-1-386, pSG5-1-523, pSG5-1-826, or pSG5-125-944 from pSG5 into SalI-digested pCEP4 vector (Invitrogen). oriP plasmid for expression of FLAG-EBNA3A aa 1 to 240 (1–240) was constructed by cloning PCR-amplified DNA, using Pfu polymerase (Gibco), oriP plasmid SV40 promoter, and FLAGEBNA3A, a forward primer containing an SfiI site (5⬘-GGCCGAGGCCGCCT CGGCCT-3⬘), and a reverse primer containing an NotI site (5⬘-GGATTAGCG GCCGCTTATACAATGTTACCCACGGAGC-3⬘). The amplified fragment was digested with SfiI and NotI and inserted into SfiI-NotI-digested oriP plasmid in the place of wild-type fE3A. Internal deletion mutants of FLAG-EBNA3A

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(⌬170–240, ⌬240–300, ⌬300–386, ⌬386–523, ⌬523–612, ⌬620–820, ⌬386–410, ⌬440–470, ⌬470–500, and ⌬500–523) were constructed from the oriP plasmid for the expression of fE3A. Fragments upstream and downstream to the deletion were PCR amplified using Pfu polymerase and an oriP plasmid containing FLAG-EBNA3A (fE3A) as the template. The upstream fragment contains part of the SV40 promoter and the N-terminal part of FLAG-EBNA3A before the deletion, and the downstream fragment contains the C-terminal part of EBNA3A after the deletion. Amplification of the upstream fragments used forward primers containing an SfiI site and reverse primers with numbers indicating the last EBNA3A amino acid encoded by the resulting amplified fragment, as follows:: forward primer 5⬘-GGCCGAGGCCGCCTCGGCCT-3⬘and reverse primers C169 (5⬘-TCCGGCGGCCAGGGTTTGCA-3⬘), C239 (5⬘-AATGTTAC CCACGGAGCTCTG-3⬘), C299 (5⬘-CAGGGCATCGCTGACAAAGCT-3⬘), C385 (5⬘-TCTTATAAATATAGGGGGTC-3⬘), C439 (5⬘-CCCGTGACTGGTA GCTGTCT-3⬘), C469 (5⬘-ACACGGGGCCATGCCGTGTTG-3⬘), C499 (5⬘-AC ACGCCACTCGCCCGTCGC-3⬘), C522 (5⬘-CCCCGCAGCCTGTGTCAGG G-3⬘), and C619 (5⬘-GGGCTGCACCTCAACACTAG-3⬘). Amplification of the downstream fragments used 5⬘ phosphorylated forward primers with numbers indicating the first EBNA3A amino acid encoded by the resulting amplified fragment and reverse primers with an NotI site as follows: the forward primers were N241 (5⬘-CAGAGCTGTAATCCCCGCTAC-3⬘), N301 (5⬘-ACCACTAG TATCCAAACACCG-3⬘), N387 (5⬘-CTGCACAGGTTGCTGCTGAT-3⬘), N411 (5⬘-GGGAGCACTTATGGCACACC-3⬘), N471 (5⬘-GTAGCACAGGCC CCACCTAC-3⬘), N501 (5⬘-CCAGTACCCGCCCCGGCTGG-3⬘), N524 (5⬘-GC CTTTGCACCCGTTAGACC-3⬘), N613 (5⬘-GCTAGTGTTGAGGTGCAGCC3⬘), and N821 (5⬘-CCCGTGTCTCCTGCCGTTAAC-3⬘); the reverse primer was 5⬘-TTCTACGCGGCCGCTTAGGCCTCATCTGGAGGAT-3⬘. The upstream and downstream amplified fragments were digested with SfiI and NotI, respectively, and three-fragment ligated into the SfiI-NotI-digested oriP plasmid in the place of wild-type FLAG-EBNA3A to make the appropriate deletion mutants. oriP plasmid containing the FLAG-EBNA3A deletion mutant ⌬410–439 used upstream and downstream fragments that were PCR amplified using Pfu polymerase with the primer pair 5⬘-GGCCGAGGCCGCCTCGGCCT-3⬘ and 5⬘-G CTCCCGCTAGCCTTTTCCAGTACCTCCTT-3⬘ and the pair 5⬘-AGTCACG CTAGCGCGCAAGTCCCAGAACCC-3⬘ and 5⬘-TTCTACGCGGCCGCTTA GGCCTCATCTGGAGGAT-3⬘, respectively; an oriP plasmid containing FLAG-EBNA3A was used as the template. The upstream and downstream amplified fragments were digested with SfiI plus NheI and NheI plus NotI, respectively, and were ligated into SfiI-NotI-digested oriP plasmid by threefragment ligation. The deletion mutants were verified by sequencing. Complementation assay. EBNA3AHT-infected LCLs (107) were transfected with 30 ␮g of oriP plasmid DNA expressing fE3A, fE3C, fE3A mutants, or control oriP plasmid. LCLs were harvested during log-phase growth, washed once with complete medium, resuspended in 400 ␮l of complete medium with DNA in a cuvette (0.4-cm gap; Bio-Rad), incubated for 10 min at 25°C, pulsed with 220 V at 960 ␮F, and cultured in 14 ml of LCL-conditioned medium with 4HT for 3 to 4 days. Cells were then washed, and 0.5 ⫻ 106 to 1 ⫻ 106 cells were cultured in 10 ml of complete medium with or without 4HT in a 25-cm2 culture flask. Every 4 to 8 days, cell numbers were counted, cultures were split, and the total numbers of viable cells relative to those of the initial culture were calculated. Western blot analysis. Total cell lysates or immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membrane (Bio-Rad), and reacted with EBV-immune human sera, rabbit polyclonal antiserum to RBP-J␬ (41), or anti-FLAG monoclonal antibodies M2 and M5 (Sigma). Membranes were reacted with horseradish peroxidase-conjugated species-specific secondary antibodies (Santa Cruz) and developed with chemiluminescence reagent (NEN). Immunoprecipitation. IB4 cells (107) were transfected with 30 ␮g of the oriP plasmid expressing FLAG-tagged EBNA3A (fE3A), FLAG-tagged EBNA3C (fE3C), indicated FLAG-tagged EBNA3A mutants, or a control oriP plasmid. For electroporation, log-phase IB4 cells were resuspended in 400 ␮l of complete medium with DNA in a cuvette. Following a 10-min incubation at 25°C, the culture was pulsed with 220 V at 960 ␮F. After 48 h, cells were lysed by vortexing in immunoprecipitation buffer (150 mM NaCl, 1% NP-40, 50 mM Tris [pH 7.4], 2 mM EDTA) containing protease inhibitors (10 ␮g of aprotinin per ml and 0.5 ␮M phenylmethylsulfonyl fluoride), incubated on ice for 1 h, and centrifuged to remove insoluble debris. The supernatant was incubated overnight with M2conjugated Sepharose beads (Sigma) at 4°C. The beads were washed with immunoprecipitation buffer four times. Proteins were eluted with sodium dodecyl sulfate sample buffer and were subjected to Western blotting with FLAG- or RBP-J␬-specific antibodies.

Reporter assays. BJAB cells (107) in log-phase growth were electroporated with 0.5 ␮g of pGK-␤-gal, 10 ␮g of pLuc-Cp reporter construct, and 1 ␮g of pSG5-EBNA 2 alone or in combination with 10 ␮g of oriP plasmid expressing fE3A wt or mutant constructs (5, 27, 28). After 24 h, cells were lysed in reporter lysis buffer (Promega). Clarified lysate luciferase (Luciferase assay system; Promega) and ␤-galactosidase (Galacto-Light; Tropix) activities were measured using an Optocomp I luminometer (MGM Instruments). Luciferase assays were corrected for transfection efficiency based on ␤-galactosidase activity.

RESULTS EBNA3A aa 170 to 240, 300 to 386, and 386 to 523 are critical for LCL growth. To identify EBNA3A domains critical for maintaining LCL growth, a series of FLAG-tagged EBNA3A mutants was cloned into an oriP-based vector and tested for transcomplementation of conditionally expressed EBNA3AHT in LCLs that had been established following infection with EBV genomes in which the first codon of a mutant estrogen hormone binding domain is fused in frame to the last codon of EBNA3A (Fig. 1) (29). Deletion mutations were designed to test the importance of EBNA3A residues associated with biochemical or functional activities as well as residues of unknown significance. Almost all parts of EBNA3A were evaluated except aa 125 to 169, which includes the essential nuclear localization sequence (25). The EBNA3A triple point mutant, EBNA3A AAGA, has A199AGA202 substituted for T199LGC202, within the core RBP-J␬ binding domain and is a null mutant for EBNA3A repression of EBNA2 in transient assays and in LCLs (5, 7). The LCL transfection efficiency in these experiments was at least 20 to 40%, as estimated from the number of enhanced green fluorescent protein-positive cells at day 3 with a control oriP plasmid that expresses enhanced green fluorescent protein (data not shown). As previously described (29), EBNA3AHT-infected LCLs grew in medium with 4HT but stopped growing over 7 to 10 days in medium without 4HT. Transfection with the vector control oriP plasmid had no effect on cell growth arrest in medium without 4HT (Fig. 2A). In contrast, EBNA3AHTinfected LCLs, which had been transfected with an oriP plasmid expressing wild-type FLAG-tagged EBNA3A (fE3A) continued to grow at similar rates in medium with or without 4HT (Fig. 2A). These data confirm that wild-type EBNA3A expression is required to sustain EBNA3AHT-infected LCL growth following 4HT withdrawal. EBNA3AHT levels in EBV/EBNA3AHT-infected LCLs growing in 4HT is ⬃50% of wt EBNA3A levels in wt LCLs, probably due to a destabilizing effect of the 4HT-activated estrogen receptor fusion on the unusually stable EBNA3A protein (29). After transfection with an oriP plasmid expressing wild-type EBNA3A and growth for 7 weeks in medium with 4HT, but without drug selection, wt EBNA3A expression stabilized at a level similar to that of EBNA3AHT, consistent with the notion that full wt EBNA3A expression has a selective advantage for cell growth (Fig. 2B and C) (29). Most importantly, after transfection with an oriP plasmid expressing wildtype EBNA3A and growth for 7 weeks in nonselective medium without 4HT, EBNA3AHT was not detected and wt EBNA3A was expressed at higher levels than in cells growing in the presence of 4HT (Fig. 2B and C). This is further evidence that wt EBNA3A transcomplements and sustains EBV/ EBNA3AHT-infected LCL growth in the absence of 4HT.

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FIG. 2. Transcomplementation assays of wt or mutant EBNA3As or wt EBNA3C in sustaining growth of EBV/EBNA3AHT-infected LCLs under nonpermissive conditions. (A) EBNA3AHT-infected LCL cells were transfected with 30 ␮g of the oriP plasmid expressing fE3A, fE3C, AAGA, ⌬2–124, ⌬170–240, ⌬240–300, ⌬300–386, ⌬386–523, ⌬523–612, ⌬620–820, or ⌬827–944, or a control oriP plasmid (Cont) and were cultured in conditioned medium with 4HT for 3 days. The cells were washed and resuspended at 1 ⫻ 106 cells/10 ml of complete medium with (⫹) or without (⫺) 4HT in 25-cm2 culture flasks (day 0). Every 5 to 7 days, cells were counted and cultures were fed with similar amounts of fresh medium. Total numbers of viable cells derived from the initial cultures were calculated and plotted at each time point. The number 10E5 along the y axis indicates that the cell number plotted should be multiplied by 100,000. (B) Protein lysates made from these cells on day 0 (3 days after transfection) were subjected to Western blotting with EBV-immune human serum to detect EBNA3A, EBNA3C, and EBNA3A mutants. (C) After 50 days of continuous

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During a 50-day assay with at least two different EBNA3AHT-infected LCLs (Fig. 1), some FLAG-EBNA3A mutants fully enabled LCL growth in the absence of 4HT, while other mutants were deficient or null for LCL growth. EBNA3A lacking aa 2 to 124 (EBNA3A ⌬2–124), EBNA3A ⌬523–612, or EBNA3A ⌬620–820 transcomplemented as well as wild-type LCLs, in five, three, or three independent experiments, respectively (Fig. 1 and 2A). In contrast, EBNA3A AAGA202, EBNA3A ⌬170–240, EBNA3A ⌬300–386 or EBNA3A ⌬386–523 failed to transcomplement, and transfected LCLs did not grow in medium without 4HT, in three independent experiments (Fig. 1 and 2A). EBNA3A ⌬240– 300, and EBNA3A ⌬827–944 partially transcomplemented and transfected LCLs grew at a slower rate in medium without 4HT than cells transfected with wild-type EBNA3A or failed to grow in one of three or one of five experiments, respectively (Fig. 1). Thus, EBNA3A aa 2 to 124, 523 to 612, and 620 to 820 are unimportant for LCL growth; aa 170 to 240, 300 to 386, and 386 to 523 are critical; and aa 240 to 300 and 827 to 944 are of intermediate significance. Western blot analyses using EBV-immune human serum and FLAG antibody showed that mutant EBNA3As were expressed in LCLs at day 3 after transfection at levels similar to wt EBNA3A (Fig. 2B). EBNA3A ⌬620–820 and ⌬827–944 were less evident in blots with human serum but were comparable to fE3A, using FLAG antibody (data not shown). We interpret these results as indicating that the human serum is particularly reactive with epitopes that are absent in EBNA3A ⌬620–820 or ⌬827–944 and that wt and mutant EBNA3A expression levels are similar. Small differences in wt and mutant EBNA3A levels in individual experiments are unlikely to affect complementation assays. Further, expression of FLAG-tagged EBNA3A wt, ⌬170–240, ⌬300–386, or ⌬386–523 in BJAB cells resulted in similar nuclear localization using Flag antibody (data not shown). EBNA3A protein levels at 50 days in EBNA3AHT LCLs transcomplemented by EBNA3A ⌬2–124, ⌬523–612, or ⌬620– 820 or partially transcomplemented by EBNA3A ⌬240–300 or ⌬827–944 stabilized at much higher levels in the absence of 4HT than in the presence of 4HT (Fig. 2C). EBNA3A ⌬240– 300 and ⌬523–612 stabilized at particularly high levels (Fig. 2C). Since EBNA3A expression was the only selective pressure over the 50-day period, this is evidence that EBNA3As deleted of aa 2 to 124, 240 to 300, 523 to 612, 620 to 820, or 827 to 944 significantly enhance EBV/EBNA3AHT-infected LCL growth in the absence of 4HT. Given their higher level of expression after LCL outgrowth, EBNA3A ⌬240–300 and ⌬523–612 may be less efficient than wt EBNA3A. EBNA3A aa 1 to 523 are not sufficient for LCL growth. Since all EBNA3A residues critical for LCL proliferation are within

culture with (⫹) or without (⫺) 4HT, protein lysates were again prepared from the transfected cells and subjected to Western blotting with EBV-immune human serum. In panels B and C, endogenous expression of EBNA3AHT (E3AHT) and EBNA3B (E3B) and expression of fE3A, fE3C, and fE3A mutants (fE3A, C, mutants) from transfected plasmids is indicated. Endogenous EBNA3C expression from the recombinant genome is not evident in blots of extracts from these LCLs because the human serum does not detect type II EBNA3C. HS, human serum.

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FIG. 3. EBNA3A aa 1 to 523 are not sufficient for maintaining LCL growth when EBNA3AHT-infected LCLs are grown under nonpermissive conditions. (A) EBNA3AHT clone 41-13 LCLs were transfected with 30 ␮g of the oriP plasmid expressing fE3A or FLAG-tagged EBNA3A deletion mutant (1–277, 1–386, 1–523, or ⌬827–944), or a control oriP plasmid (Cont) and cultured in conditioned medium with 4HT for 3 days. The cells were washed and resuspended at 5 ⫻ 105 cells/10 ml of complete medium with (⫹4) or without (⫺) 4HT in 25-cm2 culture flasks (day 0). Every 4 to 7 days, cells were counted and cultures were fed with similar amounts of fresh medium. Total numbers of viable cells derived from the initial cultures were calculated and plotted at each time. The number 10E5 along the y axis indicates that the cell number plotted should be multiplied by 100,000. (B) Protein lysates from cells on day 0 (3 days after transfection) were subjected to Western blotting with EBV-immune human serum to detect EBNA3A and EBNA3A deletion mutants (indicated with diamonds).

FIG. 4. EBNA3A aa 170 to 240 are required for RBP-J␬ association. (A and B) IB4 LCLs were transfected with 30 ␮g of the oriP plasmid expressing fE3A, fE3C, or indicated FLAG-tagged EBNA3A mutants or with a control oriP plasmid (Cont). At 48 h after transfection, protein complexes were immunoprecipitated with antibodies to FLAG and were subjected to Western blotting with FLAG- or RBPJ␬-specific antibodies. Diamonds indicate fE3A or fE3A deletion mutants.

the EBNA3A region of aa 125 to 523, we tested whether EBNA3A 1–523 or other C-terminally deleted EBNA3A mutants were able to sustain cell growth after removal from media with 4HT (Fig. 1). Neither EBNA3A 1–277, EBNA3A 1–386, nor EBNA3A 1–523 supported EBV/EBNA3AHT-infected LCL growth in the absence of 4HT, and ⌬827–944 only partially supported growth (Fig. 1 and 3A and data not shown). At day 3 after transfection, these EBNA3A deletion mutants were

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expressed similarly to wild-type EBNA3A (Fig. 3B). Thus, aa 524 to 944 of EBNA3A are critical for LCL growth, even though the region of aa 827 to 944 was the only deletion that alone was important for LCL growth and was not critical for LCL growth. EBNA3A association with RBP-J␬ is required for LCL growth. To evaluate the significance of RBP-J␬ association in LCL growth, EBNA3A mutants were assayed for association with RBP-J␬ using the oriP-based expression system in LCLs. LCLs were transfected, lysed, immunoprecipitated for EBNA3A wt or mutant proteins with FLAG antibody-Sepharose, and immunoblotted for EBNA3A and RBP-J␬ (Fig. 4).

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Endogenous RBP-J␬ in LCLs substantially associated with FLAG-EBNA3A or FLAG-EBNA3C and was not detected in control immunoprecipitations (Fig. 4). RBP-J␬ associated at similar levels with FLAG-EBNA3A ⌬240–300, ⌬300–386, ⌬386–523, ⌬523–612, ⌬620–820, and ⌬827–944, whereas less RBP-J␬ associated with FLAG-EBNA3A AAGA202 or ⌬2– 124, and RBP-J␬ did not immunoprecipitate with FLAGEBNA3A ⌬170–240 (Fig. 4A). These differences were not attributable to differential FLAG-EBNA3A protein expression or precipitation except for EBNA3A ⌬2–124, which was less well expressed than EBNA3A wild type or AAGA and is therefore closer to wild type than appears from the reduced amount of bound RBP-J␬ (Fig. 4A and data not shown). In sum, aa 170 to 240 of EBNA3A are required for RBP-J␬ association, and the EBNA3A AAGA202 mutation negatively affects association, whereas the EBNA3A ⌬240–300, ⌬300–386, ⌬386–523, and more C-terminal deletions have no discernible effect on RBP-J␬ association. Thus, partial and complete disruptions of RBP-J␬ association likely account for the null phenotype of EBNA3A AAGA202 and ⌬170–240 in EBV/EBNA3AHT-infected LCL growth under nonpermissive conditions but do not account for the null phenotype of EBNA3A ⌬300–386 and ⌬386–523. EBNA3A aa 1 to 523 had previously been shown to associate with RBP-J␬ in LCLs, whereas EBNA3A aa 1 to 277 did not associate with RBP-J␬, consistent with the idea that residues 277 to 523 are important for RBP-J␬ association (5). However, EBNA3A ⌬240–300, ⌬300–386, and ⌬386–523 were wt in RBP-J␬ binding in LCLs (Fig. 4A), indicating that aa 240 to 300, 300 to 386, or 386 to 523 cannot be uniquely important for RBP-J␬ association. Further investigation confirmed that EBNA3A 1–523 was wt in RBP-J␬ association, EBNA3A 1–277 was null, and EBNA3A 1–386 or EBNA3A 1–240 were only slightly less than wt, particularly after correction for lower EBNA3A 1–240 expression (Fig. 4B). Thus, EBNA3A 1–240 is near wt for association with RBP-J␬ and aa 241 to 277 negatively affect RBP-J␬ association. The strong negative effect of aa 241 to 277 on association of aa 1 to 240 with RBP-J␬ is likely due to a folding artifact of C-terminal truncation at aa 277, but aa 241 to 277 may have a destabilizing effect on RBP-J␬ association under physiologically appropriate conditions. EBNA3A aa 300 to 386 are required for repression of EBNA2-mediated Cp promoter activation. Although EBNA3A ⌬240–300, EBNA3A ⌬300–386, or EBNA3A ⌬386–523 were near wt in RBP-J␬ association, these residues could still be important for EBNA3A repression of EBNA2 activation of the Cp promoter through RPB-J␬ (3–5, 26, 36). In transient transfection assays in non-EBV-infected BJAB lymphoblasts (Fig. 5), EBNA2 transactivated the EBNA Cp promoter and coexpression of wt EBNA3A or EBNA3A ⌬2–124, ⌬240–300, ⌬386–523, ⌬523–612, ⌬620–820, and ⌬827–944 repressed transactivation (Fig. 5). EBNA3A ⌬170–240 and AAGA202, which are deficient in RBP-J␬ association, were also deficient in repression of EBNA2 activation of the Cp promoter (Fig. 4A and 5). More interestingly, EBNA3A ⌬300–386, which associated with RBP-J␬ as well as wild-type EBNA3A, did not repress EBNA2 activation (Fig. 4A and 5). In sum, all three mutants that were unable to repress EBNA2 activation of the Cp promoter failed to transcomplement EBNA3AHT-infected LCL growth, and all five EBNA3A mutants that transcomple-

J. VIROL.

FIG. 5. EBNA3A ⌬170–240, AAGA202, and ⌬300–386 are deficient in inhibition of EBNA2 activation of a multimerized Cp promoter. BJAB cells were transfected with the pLuc-Cp reporter construct containing eight copies of the RBP-J␬ binding site along with EBNA2 (E2⫹) alone or with the indicated fE3A construct. Relative luciferase activity was normalized to the ␤-galactosidase activity from cotransfected pGK-␤gal. The results are averages from two independent experiments and are representative of three independent repetitions. Bars indicate standard errors.

mented LCL growth repressed EBNA2-RBP-J␬-mediated transactivation, indicating that transcriptional regulation is critical for EBNA3A effects on LCL growth. EBNA3A aa 386 to 410 are critical for LCL growth, but not for association with RBP-J␬ or repression. To further investigate EBNA3A ⌬386–523, which was the only mutant that associated with RBP-J␬ and repressed EBNA2 activation of Cp promoter but did not sustain EBV/EBNA3AHT-infected LCL growth under nonpermissive conditions (Fig. 1, 2, 4A, and 5), EBNA3A mutants containing smaller deletions within aa 386 to 523 were constructed and assayed for RBP-J␬ association, repression, and growth affects (Fig. 6). EBNA3A ⌬410– 439, ⌬440–470, ⌬470–500, and ⌬500–523 were wt in sustaining LCL growth, whereas EBNA3A ⌬386–410 had a null phenotype (Fig. 6A). All five mutants were similarly expressed in LCLs at day 3 after transfection and were wt in RBP-J␬ association and repression of EBNA2 activation of the Cp promoter (Fig. 6B to D). Thus, the EBNA3A ⌬386–523 defect in LCL growth maps to aa 386 to 410, adjacent to aa 300 to 386, which are critical for repression, but deletion of EBNA3A aa 386 to 410 does not affect repression. These data are most consistent with the idea that the role of EBNA3A aa 386 to 410 is different from that of aa 300 to 386. DISCUSSION These EBNA3A reverse genetic analyses are the first strong evidence that EBNA3A association and transcriptional regulation with RBP-J␬ are critical for wild-type EBNA3A maintenance of LCL growth. EBNA3A mutants ⌬170–240 and AAGA202, which were deficient in RBP-J␬ association and in repression of EBNA2 activation of the EBV Cp promoter (5), did not sustain LCL growth after endogenous EBNA3AHT

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inactivation. Further, EBNA3A ⌬300–386, which was wt in RBP-J␬ association but was also unable to repress EBNA2 activation of the Cp promoter, could not sustain LCL growth. Also, deletion of aa 240 to 300 or aa 386 to 410, which are between or adjacent to the residues that affect transcriptional regulation, were also intermediate or null in sustaining cell growth, consistent with the possibility that they could have significant, undefined effects on RBP-J␬-mediated transcriptional regulation. Moreover, EBNA3A mutants deleted of aa 2 to 124, 410 to 523, 523 to 612, or 620 to 820 were wt in RBP-J␬ association and repression and maintained LCL growth. Hence, all EBNA3A mutations deficient in RBP-J␬ binding or repression of EBNA2 activation of the RBP-J␬-dependent Cp promoter, including the AAGA202 triple point mutant, were unable to sustain LCL growth, and almost all EBNA3A residues that were unimportant for transcriptional regulation through RBP-J␬ were unimportant for cell growth. This is compelling evidence that transcriptional regulation through RBP-J␬ is central to EBNA3A maintenance of LCL growth. Putative EBNA3A-RBP-J␬-regulated genes that would be critical for LCL growth have not been identified. In EBV/ EBNA3AHT-infected LCLs, EBNA3AHT translocates to the cytoplasm within 24 h of shift to medium without 4HT, and cell growth ceases gradually over the ensuing 4 to 7 days. During these 7 days, EBNA1, EBNA2, EBNA3B, EBNA3C, LMP1, c-myc, and CD23 protein levels remain unchanged, and CD21 expression is only slightly decreased (29). Transcript profiling can identify candidate EBNA3A-regulated cell genes likely to affect cell growth. Current studies are evaluating cellular RNA levels in EBV/EBNA3AHT-infected LCLs that are still growing after shift to medium without 4HT, so as to identify EBNA3A-regulated RNAs that are critical for LCL growth. This should enable more precise studies of the biochemical mechanisms critical for EBNA3A transcriptional affects on LCL growth. Within the EBNA3A N-terminal 523 aa, aa 240 to 300, 300 to 386, or 386 to 410 are likely to have significant unique biochemical interactions since EBNA3A ⌬240–300, ⌬300–386, and ⌬386–410 were wt in RBP-J␬ association; wt, null, and wt, respectively, in repression of EBNA2 activation of the Cp promoter; and intermediate, null, and null, respectively, in sustaining LCL growth (as summarized in Fig. 1). EBNA3A aa 300 to 386 could specifically enable repression of EBNA2-

FIG. 6. EBNA3A aa 386 to 410 are essential for EBNA3A effects on LCL growth but not for RBP-J␬ association or repression of EBNA2 activation of a multimerized Cp promoter RBP-J␬ site with a luciferase reporter. (A) EBNA3AHT clone 163 LCLs were transfected with 30 ␮g of the oriP plasmid expressing fE3A or indicated FLAGtagged EBNA3A mutants or with a control oriP plasmid (Cont) and cultured in conditioned medium with 4HT for 4 days. The cells were washed and suspended at 1 ⫻ 106 cells/10 ml of complete medium with (⫹) or without (⫺) 4HT in 25-cm2 culture flasks (day 0). Every 7 to 8 days, cells were counted and cultures were fed with similar amounts of fresh medium. Total numbers of viable cells derived from the initial cultures were calculated and plotted at each time point. The number 10E5 along the y axis indicates that the cell number plotted should be

multiplied by 100,000. (B) Protein lysates made from these cells on day 0 (3 days after transfection) were subjected to Western blotting with EBV-immune human serum (HS) to detect EBNA3A and EBNA3A deletion mutants. Endogenous expression of EBNA3AHT (E3AHT) and EBNA3B (E3B) and expression of fE3A and fE3A mutants (fE3A, mutants) from transfected plasmids are indicated. (C) IB4 cells were transfected with 30 ␮g of the oriP plasmid expressing fE3A or indicated FLAG-tagged EBNA3A deletion mutants or with a control oriP plasmid (Cont). At 48 h after transfection, protein complexes were immunoprecipitated with antibodies to FLAG and were subjected to Western blotting with FLAG- or RBP-J␬-specific antibodies. (D) BJAB cells were transfected with the pLuc-Cp reporter construct along with EBNA2 (E2⫹) alone or with the indicated fE3A construct, and activation of transcription by EBNA2 was evaluated by relative luciferase activity. The results are averages of duplicate samples and are representative of two experiments with similar results. Bars indicate standard errors.

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mediated Cp promoter activation and LCL cell growth by recruiting a repressor, inhibiting an EBNA2-associated activator, or by altering the interaction of RBP-J␬ with DNA. Although EBNA3A aa 240 to 300 and 386 to 410 do not affect EBNA3A association with RBP-J␬ or repression of EBNA2 through RBP-J␬ in transient assays, these residues may alter interactions through RBP-J␬ at specific promoters or be a scaffold for a new regulatory factor. Transcriptional regulation through RBP-J␬ can be determined by other transcription factors that regulate individual promoters. In contrast to the Cp promoter, EBNA3A coactivates with EBNA2 at the EBV LMP1 promoter (27). EBNA3A sequences responsible for this effect have not been identified. Further, neither EBNA nor LMP1 expression was altered following EBNA3A inactivation in the EBNA3AHT LCLs. Thus, the biochemical roles of aa 240 to 300, 300 to 386, and 386 to 410 are uncertain. The gradual cell growth arrest following EBNA3AHT inactivation contrasts with the rapid growth inhibiting effects of EBNA3A or EBNA3A 1–523 overexpression in LCLs (5). EBNA3A overexpression in LCLs also has no immediate effect on EBNA or LMP1 expression, but EBNA2 dissociated from RBP-J␬, c-myc, and CD21 protein levels fell, and cell growth ceased until wild-type levels of EBNA3A expression were restored or c-myc was activated through other mechanisms. In contrast, EBNA3A at physiologic levels in LCLs is critical for maintaining LCL growth but through mechanisms that are independent of EBNA2 regulation of c-myc. EBNA3A aa 524 to 944 were also important for LCL growth. EBNA3A 1–523 could not sustain EBNA3A-dependent LCL growth despite being wt for RBP-J␬ association and Cp promoter regulation. EBNA3A aa 827 to 944 are probably a major component of the EBNA3A 524–944 requirement, since EBNA3A ⌬827–944 was intermediate for sustaining LCL growth, whereas almost all of the remaining sequence of aa 524 to 826 could be deleted in segments, without any unique effect on LCL growth. EBNA3A aa 827 to 944 includes two (A/V) LDLS motifs within the sequence of aa 857 to 890 that can bind CtBP and contribute to rodent fibroblast cell growth (14). Further point mutational analyses of these motifs can more accurately evaluate the significance of EBNA3A association with CtBP for EBNA3AHT-infected LCL growth in the absence of 4HT. In contrast, almost all EBNA3A residues from aa 410 to 820 are individually relatively unimportant for maintaining LCL growth. Nevertheless, EBNA3A aa 524 to 666 can repress and aa 627 to 805 can activate transcription in Gal4-DNA binding domain-dependent reporter assays when fused to the Gal4DNA biding domain (3, 4, 7, 14). In initial B-lymphocyte infection, these activation, repression, or CtBP binding domains may have more critical roles in regulating virus or cell gene expression or for affecting cell cycle entry and progression.

ACKNOWLEDGMENTS We thank Frederick Wang, Ellen Cahir-McFarland, Teruhito Yasui, and Chih-Wen Peng for their helpful suggestions and discussions. This research was supported by grants from the National Cancer Institute of the USPHS (CA47006 and CA87661). E.J. received support from grant 1 K08 AI49943-03 from the National Institute of Allergy and Infectious Diseases of the USPHS.

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