JOURNAL OF VIROLOGY, May 2003, p. 5639–5648 0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.10.5639–5648.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 10
Transcriptional Regulatory Properties of Epstein-Barr Virus Nuclear Antigen 3C Are Conserved in Simian Lymphocryptoviruses Bo Zhao,1,2† Rozenn Dalbie`s-Tran,1‡ Hua Jiang,3 Ingrid K. Ruf,1 Jeffery T. Sample,1,2 Fred Wang,3 and Clare E. Sample1,2* Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 381051; Department of Pathology, University of Tennessee College of Medicine, Memphis, Tennessee 381632; and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 021153 Received 10 December 2002/Accepted 21 February 2003
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is a large transcriptional regulator essential for EBV-mediated immortalization of B lymphocytes. We previously identified interactions between EBNA-3C and two cellular transcription factors, J and Spi proteins, through which EBNA-3C regulates transcription. To better understand the contribution of these interactions to EBNA-3C function and EBV latency, we examined whether they are conserved in the homologous proteins of nonhuman primate lymphocryptoviruses (LCVs), which bear a strong genetic and biological similarity to EBV. The homologue of EBNA-3C encoded by the LCV that infects baboons (BaLCV) was found to be only 35% identical in sequence to its EBV counterpart. Of particular significance, this homology localized predominantly to the N-terminal half of the molecule, which encompasses the domains in EBNA-3C that interact with J and Spi proteins. Like EBNA-3C, both BaLCV and rhesus macaque LCV (RhLCV) 3C proteins bound to J and repressed transcription mediated by EBNA-2 through its interaction with J. Both nonhuman primate 3C proteins were also able to activate transcription mediated by the Spi proteins in the presence of EBNA-2. Like EBNA-3C, a domain encompassing the putative basic leucine zipper motif of the BaLCV-3C protein directly interacted with both Spi-1 and Spi-B. Surprisingly, a recently identified motif in EBNA-3C that mediates repression was not identifiable in the BaLCV-3C protein. Finally, although the C terminus of BaLCV-3C bears minimal homology to EBNA-3C, it nonetheless contains a C-terminal domain rich in glutamine and proline that was able to function as a potent transcriptional activation domain, as does the C terminus of EBNA-3C. The conservation of these functional motifs despite poor overall homology among the LCV 3C proteins strongly suggests that the interactions of EBNA-3C with J and Spi do indeed play significant roles in the life cycle of EBV. actions with cellular sequence-specific DNA-binding proteins, rather than by binding directly to DNA (26, 45, 50, 57, 58). In addition to interactions with sequence-specific transcription factors, EBNA-3C binds to Nm23-H1, a protein associated with metastatic suppression and which may have some transcriptional properties, as well as to a variety of transcription factors that are likely to function as accessory or modulatory proteins, such as prothymosin alpha, CtBP, histone deacetylase, and DP103 (6, 16, 52, 54). One interaction common to all EBNA-3 proteins, mediated through a conserved motif in the N terminus, is with J (46, 57) (also known as CBF-1), which mediates Notch signaling. J also plays a central role in EBV latent infection, where the EBV protein EBNA-2 functions as a mimic of constitutively activated Notch by binding to J and increasing transcription from promoters that contain J-binding sites (21, 22, 24, 31). J-responsive promoters include not only promoters for cellular genes, but also the promoters for many of the EBV latent infection proteins, such as the EBNAs (transcribed from a common promoter, Cp), LMP-1, and LMP-2 (15, 21, 36, 59). By contrast with EBNA-2, the interaction of the EBNA-3 proteins with J prevents the interaction of J with DNA (as well as with EBNA-2 [25]), thereby repressing EBNA-2/J-mediated transcription (45, 55, 57). In this respect, the EBNA-3 proteins function similarly to the Drosophila melanogaster protein Hairless, which represses the function of the Drosophila homologue of J (4). Since the expression of the EBNA-3
Studies using recombinant Epstein-Barr virus (EBV) genomes have identified six latent infection proteins that are essential for EBV-mediated immortalization of B lymphocytes in vitro: EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-LP, EBNA-3A, EBNA-3C, and latent membrane protein (LMP) 1 (5, 17, 27, 29, 34, 53). Two of these viral proteins, and perhaps the least well understood, are members of the EBNA-3 family of transcriptional regulators. Although the three EBNA-3 genes (3A, 3B, and 3C) share a similar gene structure and are likely to have evolved from a common ancestral gene, the encoded proteins are highly divergent in amino acid sequence. The EBNA-3 proteins are also likely to have diverged functionally, since EBNA-3A and EBNA-3C, but not EBNA-3B, are essential for EBV-mediated immortalization of B lymphocytes. Despite their differences, all EBNA-3 proteins function as transcriptional regulatory proteins whose transcriptional effects are mediated through inter* Corresponding author. Mailing address: Department of Biochemistry, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105. Phone: (901) 495-3416. Fax: (901) 525-8025. E-mail:
[email protected]. † Present address: Department of Microbiology and Molecular Genetics, Channing Laboratory, Harvard Medical School, Boston, MA 02115. ‡ Present address: UMR 6073, Institut Nationale de La Recherche Agronomique, Centre National de la Recherche Scientifique, Universite´ de Tours, Nouzilly F-37380, France. 5639
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proteins themselves is increased by EBNA-2 (through its activation of the EBNA promoter Cp [21]), the interaction of the EBNA-3 proteins with J provides a mechanism for feedback repression. Thus, at least one function of the EBNA-3 proteins is to regulate EBNA-2-mediated transcription, thereby regulating the expression of many of the EBV proteins expressed during latent infection. Although the ability to bind J and regulate EBNA-2/Jmediated transcription is clearly an important function of the EBNA-3 proteins, the J-binding motif constitutes only about 50 of their approximately 1,000 amino acids (aa). Moreover, the fact that both EBNA-3A and EBNA-3C are required for immortalization suggests that each protein also has a unique contribution to this hallmark biological property of EBV. In addition to the J-interaction domain that mediates transcriptional repression, EBNA-3C has a potential bZIP domain characteristic of transcriptional activators, and its C terminus alone is capable of activating transcription when tethered to a heterologous DNA-binding domain (35). Two lines of evidence support a role for EBNA-3C as a transcriptional activator as well as a transcriptional repressor in the context of an EBV infection. First, in the EBV-positive Burkitt lymphoma (BL) cell line Raji, in which the EBNA-3C open reading frame (ORF) is deleted from the EBV genome, LMP-1 levels are very low but can be restored to levels similar to those in lymphoblastoid cell lines by exogenous expression of EBNA3C (1, 2). Second, in cotransfection experiments in the presence of EBNA-2, EBNA-3C can activate expression of the LMP-1 promoter, through the Spi binding site (35, 58). Spi-1, which frequently binds DNA in conjunction with other proteins, binds to both EBNA-2 and EBNA-3C in vitro, suggesting the possibility that EBNA-2 and EBNA-3C could form a complex on the DNA with Spi-1 (24, 28, 58). Despite the fact that the exact mechanism of this activation is unknown, the Spibinding site within the LMP-1 promoter provides a second example of a DNA element through which EBNA-3 proteins regulate EBNA-2-mediated transcription. These two functions of EBNA-3C are distinct because (i) the J DNA-binding site is both required and sufficient for repression of transcription, (ii) the Spi DNA-binding site is both required and sufficient for activation, and (iii) mutations in EBNA-3C that effect repression do not affect activation (57, 58). Although the significant contributions of both J and Spi proteins to cellular differentiation and proliferation suggest that their interactions with EBNA-3 proteins are likely to be relevant to EBV-mediated immortalization, this has yet to be demonstrated. Curiously, sites for both Spi and J (through which EBNA-3C exerts opposing effects on transcription) are present within the LMP-1 promoter. To evaluate the significance of these functions, we sought to determine whether they are evolutionarily conserved. While EBV infection is restricted to humans, highly related LCVs exist that naturally infect nonhuman Old World primates and which may be exploited to address parameters of EBV biology that, due to species restriction, cannot be addressed directly. Importantly, many of the defining properties of EBV are conserved in these related LCVs, including immortalization of host B lymphocytes in vitro, establishment of lifelong infection, and induction of diseases similar to those commonly associated with EBV, namely, infectious mononucleosis, lymphoproliferative disease, and lymphomas (8, 9, 13,
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37, 38). Not surprisingly, the genomes of EBV and the nonhuman primate LCVs characterized to date are colinear and highly homologous with some ORFs displaying striking homology to their EBV counterparts, e.g., the latency-associated proteins EBNA-1 and EBNA-LP and the lytic cycle protein BHRF-1 (56, 61 and 64% amino acid identity, respectively) (18–20, 40, 56). By contrast, LCV homologues of EBNA-2 share only 37 to 38% amino acid identity with their EBV counterpart (32, 39). Discrete regions of EBNA-2, however, are more highly conserved, allowing the identification of the domain that binds J (32). Due to the important roles that Notch plays in cell growth and differentiation, the mimicry by EBNA-2, a protein essential for EBV-induced immortalization, is believed to play a significant role in EBV biology and pathogenesis, an importance that is underscored by the conservation of the EBNA-2–J interaction among all characterized LCVs (30, 39). A comparative analysis of LCV proteins, therefore, is a useful approach to define EBV protein structure and function, particularly for large multifunctional proteins such as the EBNA-3s. One region of the EBV genome that early DNA hybridization studies suggested might be distinct from other LCVs is that region subsequently shown to encode the EBNA-3 family of transcriptional regulatory proteins (20). The EBNA-3 proteins encoded by the LCV of rhesus macaques (RhLCV) have recently been shown to associate with J in vitro (23). However, recombinant EBV in which the EBNA-3 genes had been replaced with their RhLCV counterparts was only able to induce limited growth of infected human B lymphocytes and was ultimately unable to immortalize these cells (23). This suggests that the RhLCV-encoded counterpart of at least one of the EBNA-3 proteins might not provide a function essential for immortalization of human B lymphocytes. Further characterization of the LCV homologues of the EBNA-3 proteins, therefore, may be able to provide better insight into the contribution of the EBNA-3 proteins to EBV-mediated immortalization of B lymphocytes. Here we report the characterization of the EBNA-3C homologue from baboon LCV (BaLCV) and the comparative evaluation of the transcriptional regulatory properties of the EBNA-3C homologues encoded by BaLCV and RhLCV. Our results demonstrate that the EBNA-3C homologue encoded by BaLCV also associates with J and that repression of EBNA2/J-mediated transcription is conserved among all the LCV 3C proteins. Additionally, the region of the BaLCV-3C protein encompassing the bZIP domain bound to Spi proteins, and both RhLCV and BaLCV homologues activated transcription through Spi-binding sites in the presence of EBNA-2. Finally, we demonstrate that the activation domain in the C terminus of EBNA-3C, previously demonstrated to be conserved in RhLCV-3C (23), is functionally conserved in BaLCV-3C, despite a lack of sequence homology. These findings strongly suggest that these functions and domains of EBNA-3C play important roles in the immortalization of B lymphocytes by LCVs. MATERIALS AND METHODS Cell lines. BL2 is an EBV-negative human BL cell line. S594 is a baboon lymphoblastoid cell line infected with BaLCV (cercopithicine herpesvirus 12) that was obtained by spontaneous outgrowth from baboon peripheral blood (41).
VOL. 77, 2003 Both cell lines were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (HyClone). Cloning and sequence analysis of the BaLCV-3C gene. Genomic DNA libraries enriched for BaLCV DNA were constructed as previously described (7, 48). Briefly, BaLCV-enriched DNA was isolated from S594 cells by using a modified Hirt procedure (42), digested with restriction enzymes, and used to generate a genomic library in the lambda phage vector ZAP Express (Stratagene). Initial screening was performed on a library of HindIII fragments by using the EBV BamHI-E fragment (containing all of the EBNA-3B gene as well as the majority of the EBNA-3A and EBNA-3C genes) as a probe under conditions of low stringency (hybridization at 72°C in 5⫻ SSC [1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0], 5⫻ Denhardt’s solution, 1% sodium dodecyl sulfate [SDS], and 200 g of salmon sperm DNA/ml, followed by washes at 50°C in 1⫻ SSC plus 0.5% SDS and 0.5⫻ SSC plus 0.5% SDS). This strategy led to the isolation of a genomic fragment containing the a homologue of the EBNA-3B gene and the 5⬘ half of the homologue of the EBNA-3C gene. To obtain the DNA fragment encoding the complete ORF of the EBNA-3C homologue, this HindIII fragment was used to screen a genomic library of XhoI and EcoRI fragments. This screen resulted in the cloning of an approximately 7-kbp XhoI/ EcoRI fragment. Following plaque purification, the pBK-CMV phagemid within the ZAP Express vector containing this genomic fragment was excised according to the manufacturer’s protocol. Sequence analysis of this clone revealed that it contained the entire ORF for the EBNA-3C homologue. Smaller fragments of this papio DNA clone were generated by restriction endonuclease digestion and subcloned into pBluescript II KS(⫹) (Stratagene) for DNA sequence analysis with both T7 and T3 sequencing primers. The entire BaLCV-3C ORF was isolated as a KpnI-to-EcoRI subfragment that was cloned into the mammalian expression vector pSG5 (Stratagene) in frame with a c-myc epitope tag. Plasmid construction. Reporter plasmids ⫺2350LMP-CAT (containing an LMP-1 promoter fragment from ⫺2350 to ⫹40 relative to the site of transcription initiation linked to the chloramphenicol acetyltransferase [CAT] reporter gene) and ⫺215/-144 LMP-1BLCAT2 (containing nucleotides ⫺215 to ⫺144 of the LMP-1 promoter, encompassing the Spi site, in the plasmid pBLCAT2 which contains the herpesvirus tk promoter [33]) have been previously described (35, 57, 58). C10BLCAT contains 10 tandem copies of a J-binding site in pBLCAT2 (57). To create a BaLCV-3C–glutathione S-transferase (GST) fusion protein, a SnaBI-to-NsiI (blunted) restriction fragment from the BaLCV-3C ORF, encoding aa 135 to 317, was inserted into the SmaI site of pGEX-3X (Pharmacia). To generate the expression vector Gal4-BaLCV-3C QP, an EagI-to-BamHI restriction fragment, encoding aa 638 to 783, was inserted into pM2 (49) in frame with the Gal4 DNA-binding domain. An expression vector for RhLCV-3C (from cercopithicine herpesvirus 15) was constructed by cloning the viral genomic DNA fragment containing the entire coding sequence beginning with the initiator methionine in frame with an N-terminal FLAG epitope in plasmid 3910 (kindly provided by E. Hatzivassiliou and G. Mosialos), in which expression is driven by the cytomegalovirus (CMV) immediate-early promoter. DNA transfections. Transfection of cells with plasmid DNA was accomplished by electroporation as previously described (58). Briefly, 8 ⫻ 106 EBV-negative B cells were electroporated with 5 g of reporter plasmid and 2 to 10 g of expression vector. As a control for transfection efficiency, 1 g of pCMV-human growth hormone (hGH) was included. At 36 h posttransfection, cells were harvested and extracts were prepared and assayed for CAT activity as previously described (35). hGH present in the cell medium was quantified using a radioimmunoassay kit according to the manufacturer’s recommendations (Nichols Institute). Promoter activity was calculated as CAT activity and was normalized to hGH levels to correct for differences in transfection efficiency. In vitro binding assays. GST fusion proteins were expressed in Escherichia coli from pGEX vectors (Pharmacia) following induction with isopropyl--D-thiogalactopyranoside, and purified on glutathione-Sepharose beads (Pharmacia). These proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE) followed by staining with Coomassie brilliant blue to evaluate the levels of full-length fusion proteins that were included in each binding assay. RNA transcripts encoding J, Spi-1, and Spi-B were generated from pSG5-derived plasmids by in vitro transcription using T7 polymerase (mCAP kit; Stratagene). The mRNAs were translated in the presence of [35S]methionine (DuPont) by using rabbit reticulocyte lysate (Promega). GST fusion proteins bound to glutathione-Sepharose beads were incubated with [35S]methionine-labeled proteins for 30 min at 4°C. Glutathione-Sepharose beads with bound GST fusion proteins and any associated proteins were collected by centrifugation. Unbound proteins were removed by five washes with Tris-buffered saline (pH 8.0) containing 0.5% Triton X-100. Bound proteins were eluted by boiling in SDS-PAGE sample buffer, subjected to SDS-PAGE, and visualized by autoradiography.
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Nucleotide sequence accession number. We deposited the nucleotide sequence of BaLCV-3C under GenBank accession no. AY260938.
RESULTS Sequence analysis of the BaLCV-3C gene. To enable us to better assess the biological significance of the functions that we and others have identified for EBNA-3C, we cloned and determined the nucleotide sequence of the corresponding BaLCV gene, which we designate BaLCV-3C. Like the EBV EBNA-3C gene, the BaLCV-3C gene contained both a short and a long coding exon (predicted) separated by an intron of 85 nucleotides. Although the predicted BaLCV-3C intron was 11 nucleotides longer than the corresponding intron in the EBNA-3C gene, it was 70% identical to its EBV counterpart, with the greatest identity located at the splice junctions (Fig. 1A). A schematic diagram of EBNA-3C showing functional domains or motifs as well as regions that bind the various associated cellular proteins is shown in Fig. 1B. The BaLCV3C gene encodes a predicted protein of 975 aa with 35% overall identity to EBNA-3C from the B95-8 EBV strain (Fig. 1C). Identity was largely restricted to the N-terminal half of the molecule (aa 1 to 513), which had an identity of 47%; this portion encompassed the domain (aa 182 to 231) that we have identified as interacting with the J protein (57). C terminal to this region, the EBV and BaLCV proteins diverged significantly, with an identity of only 17% between aa 514 and 881, but were more homologous (45% identity) over the terminal 94 aa. The EBNA-3 proteins are three of the four proteins (the other being EBNA-2) that exhibit a difference in sequence between the type I and type II strains of EBV (alternatively referred to as types A and B, respectively), such that human antiserum often does not cross-react between the EBNA-3 proteins of the different viral strains (47). The type II EBNA3C gene contains a diagnostic sequence that is absent from the type I sequence located between nucleotides 1691 and 1779 of the AG876 EBNA-3C gene (51). This type-specific sequence was present, although of limited homology, within the BaLCV3C gene, suggesting that the isolate of BaLCV may be closer to the type II isolate of EBV than to type I, similar to our conclusions derived from the study of BaLCV-3A (alternatively named HVP-3A [7]). Since the sequence of the EBNA-3C homologue encoded by RhLCV had been recently reported (23), we compared its sequence to that of BaLCV-3C (Fig. 1C). The identity between these two LCV 3C proteins is slightly higher than that between EBNA-3C and BaLCV-3C, reflecting a greater homology within the C-terminal half of the protein. Additionally, BaLCV-3C contains two small deletions not found within either EBV EBNA-3C proteins or the RhLCV-3C protein: a 10-aa deletion at the extreme amino terminus and an 82-aa deletion (aa 894 to 976 of the AG876 sequence); presumably, therefore, these amino acids are dispensable for LCV-mediated transformation. One characteristic of the EBV EBNA-3 protein sequences is the presence of repeat elements within the C-terminal half of the molecule. A GPPAA motif is repeated 10 times in the type I EBV isolate B95-8 (aa 560 to 594) (Fig. 1C), and a related but less-well-conserved repeat, APPST, has been reported in the same location within type II EBNA-3C (AG876) (51), though
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FIG. 1. Conservation of the 3C gene and protein in BaLCV. (A) The BaLCV-3C intron is conserved both in location and in sequence. Nucleotides that are identical between BaLCV-3C (upper line) and EBNA-3C (lower line) are indicated by vertical bars. Diamonds indicate the junctions between coding and noncoding regions (splice junctions). The amino acids encoded and their locations within the respective 3C protein are indicated. (B) A schematic diagram of EBNA-3C shows functional domains, amino acid motifs, and regions demonstrated to associate with the cellular proteins that are listed below. (C) The BaLCV-3C amino acid sequence (Ba) compared to EBNA-3C type I (B95-8 EBV isolate sequence [E1]), type II (AG876 EBV isolate [EII]), or the RhLCV-3C (Rh) sequence. A consensus sequence is listed below, with identical residues shown and similar residues indicated by an asterisk. The residue numbers are shown at the end of each line. The J-binding motifs are boxed. The wavy line indicates the GPPAA repeat motif, and the dotted underlines indicate the repeat element in the EBNA-3C type I activation domain or similar motifs in BaLCV-3C and type II 3C proteins. A putative bZIP motif is shaded.
no function has been attributed to this motif. A similar motif was not present in BaLCV-3C. Three exact repeats of a second motif, PQAPYQGYQEPPA, are found in the C-terminal domain of EBNA-3C that functions as a transcriptional activation domain (aa 744 to 781) (Fig. 1C). Although an exact repeat of this motif is not present in type II EBNA-3C, similar sequences are found in the corresponding location of the protein (Fig. 1C) and have been previously reported in the region of RhLCV-3C that functions as an activation domain (23). Similarly, loosely related but not perfect repeats were present in
the C terminus of BaLCV-3C (Fig. 1C). Another functional motif in EBNA-3C is the PLDLS motif (located at aa 728) that associates with the cellular protein CtBP. Although this motif is highly conserved throughout evolution in proteins that bind CtBP, neither BaLCV-3C nor RhLCV-3C contains a PLDLS motif. BaLCV-3C represses transcription mediated through EBNA-2/J. Our laboratory has reported that the domains of EBNA-3A and EBNA-3C that bind J contain three blocks of sequence conserved between all EBNA-3 proteins (57). The
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FIG. 2. BaLCV-3C associates with the cellular protein J. (A) The putative J-binding site in BaLCV-3C aligned with the homologous regions of type 1 EBV EBNA-3C and RhLCV-3C. Shaded residues indicating amino acids that are conserved between EBNA-3A, -3B, and -3C proteins fall into three conserved blocks: left (L), middle (M), and right (R). Mutations within these blocks that affect binding are indicated with an asterisk. (B) 35S-labeled J generated by in vitro translation was incubated with GST or with GST fusion proteins containing either a fragment of EBNA3C encompassing the J-binding domain (aa 184 to 365), an irrelevant EBNA-3C C-terminal domain (aa 715 to 992), or the putative J-binding domain of BaLCV-3C (aa 131 to 317). In this experiment, the amount of GST–EBNA-3C conserved domain fusion protein was slightly less than the amount of the other fusion proteins. Bound complexes were collected by centrifugation and analyzed by SDS-PAGE and autoradiography. The first lane contained 10% of the amount of 35S-labeled J added to the binding reaction mixtures (lanes 2 to 5). Molecular mass markers (in kilodaltons) are indicated.
corresponding region of BaLCV-3C has 72% amino acid similarity to the domain in EBNA-3C (Fig. 2A), with a particular conservation of the middle block of sequence that our group has shown by mutational analysis to play an important role in binding to J (57). RhLCV-3C is more divergent in this middle block, though the substitutions are relatively conservative, but it is more highly conserved within the left and right blocks that we have demonstrated also contribute to the interaction with J (D. R. Marshall, R. M. Stiffin, and C. E. Sample, unpublished data). RhLCV-3C has been previously reported to bind J in vitro (23). To determine whether this domain of BaLCV-3C could interact directly with J, we generated a GST fusion protein containing the region of BaLCV-3C encompassing this putative J-binding motif and incubated it with J generated by in vitro translation. As shown in Fig. 2B, this domain of BaLCV-3C was sufficient to mediate an interaction with J in vitro. To determine whether the interactions of BaLCV-3C and RhLCV-3C with J are sufficient to repress EBNA-2/J-mediated transcription, we generated mammalian expression vectors encoding full-length BaLCV-3C and RhLCV-3C proteins, which were then cotransfected into the human EBV-negative B-lymphoma cell line BJAB together with an EBNA-2 expression vector and a CAT reporter gene controlled by a promoter containing 10 tandem copies of the J-binding site. When expressed in the absence of EBNA-2, none of the LCV 3C proteins had any effect on expression of the reporter gene (data not shown). However, as shown in Fig. 3, BaLCV-3C and RhLCV-3C proteins efficiently downregulated transcription activated by EBNA-2 through J, as previously reported for EBNA-3C (57). Thus, the ability to downregulate transcription mediated by EBNA-2 through J is a conserved property of LCV 3C proteins. BaLCV-3C activates transcription of the LMP-1 promoter
through interactions with Spi proteins. In addition to its effect on J-regulated transcription, EBV EBNA-3C serves as a coactivator with EBNA-2 of transcription from the LMP-1 promoter (58). To determine whether the ability of EBNA-3C to function as a coactivator of LMP-1 expression is also conserved in BaLCV-3C and RhLCV-3C, we transfected cells with ⫺2350 LMP-1 CAT (containing ⫺2350 to ⫹40 relative to the site of transcription initiation), which we have previously shown to be activated by EBNA-3C (58), together with expression vectors encoding EBNA-2 and either the EBNA-3C, BaLCV-3C, or RhLCV-3C protein. As shown in Fig. 4A, BaLCV-3C and RhLCV-3C proteins, like EBNA-3C, activated expression from the LMP-1 promoter in the presence of EBNA-2.
FIG. 3. LCV 3C proteins universally repress transcription mediated through J. EBV-negative B cells were cotransfected with a CAT reporter gene controlled by 10 copies of a J-binding site plus either empty pSG5 expression vector (control) or expression vectors for EBNA-2 alone or in the presence of EBV-3C, BaLCV-3C, or RhLCV3C. Each data point is the mean from four separate transfections corrected for transfection efficiency. Error bars indicate standard deviations.
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FIG. 4. LCV 3C proteins activate transcription mediated through Spi-binding sites. EBV-negative B cells were transfected with a CAT reporter gene controlled by the LMP-1 promoter (⫺2350 to ⫹40) (A) or a smaller fragment of the LMP-1 promoter (⫺215 to ⫺144) (B), which either encompasses the Spi-binding site (black bars) or mutated Spi site (grey bars), in the presence of empty vector (control), or expression vectors encoding EBNA-2 alone, or EBNA-2 in the presence of either EBV-3C, BaLCV-3C,or RhLCV-3C. Data are from four separate transfections corrected for transfection efficiency. Error bars indicate standard deviations.
The DNA element that mediates coactivation by EBNA-3C has been localized to a binding site for members of the Spi family of proteins (58). Moreover, we have shown that EBNA3C, like EBNA-2, binds to Spi proteins in vitro and contains sequences within its C terminus that function as an activation domain (35, 58). To determine whether the LCV 3C proteins could activate transcription mediated through a fragment of the LMP-1 promoter encompassing the Spi-binding site, we used ⫺215/⫺144 LMP-1BLCAT2, which we demonstrated to be responsive to EBNA-3C (58). The activity of this LMP-1 promoter construct is much less than that obtained with larger constructs, likely due to binding of fewer transcription factors to this small fragment. Indeed, only two cellular DNA-binding proteins, Spi-1 and an unidentified protein, have been shown to bind to this small fragment (24). Nonetheless, as shown in Fig. 4B, both LCV 3C proteins were as active as EBNA-3C in increasing transcription in conjunction with EBNA-2 through this truncated LMP-1 promoter. None of the LCV 3C proteins increased transcription when expressed in the absence of EBNA-2 (data not shown). To determine whether the Spi site was essential for this activation, we mutated the GGA core of the Spi-binding site, which inhibits binding of Spi proteins to this DNA fragment (58). Neither EBNA-3C nor its LCV homologues were able to activate transcription from the mutant LMP-1 promoter, demonstrating that the Spi binding site is essential for LCV-3C-mediated activation. The interaction that members of our laboratory previously demonstrated between EBNA-3C and Spi proteins is mediated through a region of EBNA-3C encompassing the bZIP
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motif (58). In BaLCV-3C, a potential leucine zipper was identified between aa 263 and 291, analogous to the position of the bZIP motif within EBNA-3C (the putative bZIP motif is highlighted in Fig. 1C), with three of the four leucine residues conserved. In addition, many of the amino acids between the repeated leucine residues are conserved between all three LCV 3C proteins. To determine whether this domain of BaLCV-3C is capable of binding Spi proteins, which would be consistent with its ability to activate transcription through Spi sites (Fig. 4B), we generated a fusion protein between the region encompassing the putative bZIP domain of BaLCV-3C and GST and tested the ability of this fusion protein to bind to the two Spi family members, Spi-1 and Spi-B, which were previously demonstrated to interact with EBNA-3C (58). As shown in Fig. 5, the region of BaLCV-3C encompassing the basic leucine zipper motif bound to both Spi-1 and Spi-B in vitro, although in this experiment the EBNA-3C bZIP fusion protein was expressed at much lower levels than the BaLCV-3C bZIP fusion protein, enabling it to pull down less Spi-1. Nevertheless, this interaction is consistent with the observed ability of BaLCV-3C to activate transcription via Spibinding sites, as demonstrated above (Fig. 4B). Thus, the ability to activate transcription mediated through the Spi family of proteins is a second example of EBNA-2-mediated transcription regulated by EBNA-3C that is conserved among LCV 3C proteins. BaLCV-3C contains a C-terminal transactivation domain. The transactivation domain that members of our laboratory previously described in the C terminus of EBNA-3C (aa 724 to 826) (35) is rich in glutamine and proline residues (34%), as are several known transactivation domains. The amino acid
FIG. 5. BaLCV-3C interacts with Spi proteins in vitro. In vitrotranslated Spi-1 (left panel) was incubated with GST, GST fused to an irrelevant domain of EBNA-3C (GST–EBNA-3C 715-992), or a region encompassing the putative bZIP motif of either EBNA-3C (GST-3C bZIP) or BaLCV-3C (GST-BaLCV-3C bZIP). In vitro-translated Spi-B (right panel) was incubated with GST or GST–BaLCV-3C bZIP. In this experiment, GST-3C bZIP was expressed at lower levels than GST–BaLCV-3C bZIP. Bound complexes were collected by centrifugation and analyzed by SDS-PAGE and autoradiography.
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FIG. 6. BaLCV-3C contains a potent C-terminal activation domain. A C-terminal region of BaLCV-3C, encoding aa 638 to 880, was fused to the Gal4 DNA-binding domain in the expression vector pM2. This construct was cotransfected into EBV-negative B cells with a CAT reporter gene controlled by Gal4-binding sites (Gal4CAT) or a control promoter in which the Gal4 sites were absent (E1bCAT). Empty vector, encoding only the Gal4 DNA-binding domain (Gal4), was used as a negative control, and Gal4 fused to the activation domain of VP16 (Gal4-VP16) was included as a positive control.
sequence of the corresponding domain of BaLCV-3C is quite divergent from that of EBNA-3C; however, it too is rich in glutamines and prolines (31%) (Fig. 1C). Moreover, although it does not contain perfect amino acid repeats as does the C-terminal domain of EBNA-3C, there is a similar motif that is loosely repeated within the C terminus of BaLCV-3C (Fig. 1C). To determine whether this domain could function as a transactivation domain, we generated an expression vector that encodes aa 639 to 784 of BaLCV-3C fused to the GAL4 DNAbinding domain. This expression vector was cotransfected into EBV-negative BL cells with a reporter gene under the control of multiple Gal4 binding sites (Gal4CAT), or one lacking Gal4 binding sites (E1bCAT) as a negative control. As shown in Fig. 6, this domain of BaLCV-3C had no effect on activation of a basal promoter lacking Gal4 binding sites (E1bCAT). However, when targeted to the promoter by the presence of Gal4binding sites (Gal4CAT), the C-terminal domain of BaLCV3C functioned as a very efficient activation domain, stimulating transcription to a similar degree to the prototypic activation domain of the herpes simplex virus transactivator VP16. The activation domain of EBNA-3C activated transcription to a lesser extent, approximately fivefold (data not shown). DISCUSSION LCV proteins that are dissimilar to their EBV counterparts have been very useful in identifying functional domains essential for EBV-mediated immortalization of B lymphocytes, such as the J-binding domain in EBNA-2 and the TRAF and TRADD binding domains in LMP-1 (11, 30). EBNA-3C is a large, multifunctional protein that binds a variety of general transcriptional modulatory proteins but only two known sequence-specific DNA-binding proteins, Spi-1 and J. Although the BaLCV-3C protein was only 35% identical to EBNA-3C, much of this identity was localized within the amino-terminal half of the protein containing the binding sites for both J and Spi. The J-binding domain that we have previously identified was conserved, and both the RhLCV-3C (23) and BaLCV-3C proteins bound to J in vitro. More importantly, both LCV 3C proteins were able to efficiently downregulate transcription
mediated by EBNA-2 through J. Though the interacting domain has not been closely delineated, EBNA-3C binds to Spi proteins through a region encompassing the putative bZIP motif. The corresponding region of BaLCV-3C also bound both Spi-1 and Spi-B, and both BaLCV-3C and RhLCV-3C proteins were able to activate the LMP-1 promoter in cooperation with EBV EBNA-2. Thus, two distinct transcriptional regulatory properties of EBNA-3C (repression through Jbinding sites and activation through Spi-binding sites) are conserved in the simian 3C proteins. By contrast to the motifs mediating interactions with these sequence-specific transcription factors, the binding site for the accessory protein CtBP (PLDLS), which is contained within the highly divergent C-terminal residues (54), was not conserved in BaLCV-3C or in RhLCV-3C. Although we have not investigated directly the ability of the LCV proteins to bind CtBP, it seems unlikely that they bind to CtBP via a divergent sequence, since the PXDLS motif is used in organisms as distant as Drosophila. For Hairless, the Drosophila protein that EBNA-3C mimics (by binding J and repressing transcription), interaction with CtBP has been proposed to play a role in the repression of J-mediated transcription (3). However, studies with EBNA-3C have clearly demonstrated that the CtBP-binding motif in EBNA-3C can be mutated without affecting the ability of EBNA-3C to repress J-mediated transcription (54). Since CtBP is generally regarded as a transcriptional repressor, it is surprising that the PLDLS motif of EBNA-3C is located within its activation domain. However, CtBP can also function as a context-specific coactivator. Thus, the functional significance of the interaction with CtBP is unclear. The sites in EBNA-3C that interact with the other transcription modulatory factors are not as well delineated, but all reside within the C-terminal region of EBNA-3C that is poorly conserved among the 3C proteins. Surprisingly, despite its divergent sequence, the C-terminal domain retained the ability to function as an activation domain. Previously, Gill et al. noted that the sequence of this activation domain was similar to that of Sp1, rich in glutamine and proline residues and containing bulky hydrophobic residues (14). Although there is little sequence conservation per se, these features are retained within the C terminus of BaLCV-3C. Both the BaLCV-3C (Fig. 6) and the RhLCV-3C (23) sequences functioned as more-potent activation domains than the EBNA-3C sequences. This greater activity might reflect the fact that similar but not identical sequences were used and that the surrounding sequences also contribute to the activation, or it might reflect the fact that the BaLCV and RhLCV sequences do not contain a binding motif for the repressor protein CtBP. Alternatively, the simian 3C proteins might have activation domains that are intrinsically more active for unknown reasons. Regardless, the conservation of function in a particularly diverse region of BaLCV-3C suggests that the activation domain may indeed play a role in transcriptional activation by EBNA-3C. In EBV, J plays a central role in regulating expression of all the latent promoters: for the six EBNAs (Cp), LMP-1, and LMP-2, and the J-binding sites within these promoters are conserved in the corresponding promoters within the BaLCV and RhLCV genomes (10, 12, 43). Furthermore, both RhLCV and BaLCV EBNA-2 homologues bind to J (32, 39). One question that has remained unanswered is whether each
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EBNA-3 protein serves the same purpose in binding to J, or whether these interactions differ in some respect. We have demonstrated that BaLCV-3A (7), RhLCV-3A (B. Zhao and C.E. Sample, unpublished observations), and LCV 3C proteins from both RhLCV and BaLCV (this study) are all able to modulate transcription through J despite a significant divergence among these LCV proteins. These findings confirm the central role that J plays in the control of LCV gene transcription and, furthermore, suggest that modulation of this transcription by both LCV 3A and 3C proteins is an essential element in EBV-mediated immortalization. Whether these interactions serve additive or unique functions remains to be elucidated. The conservation of the ability of LCV 3C proteins to bind to Spi-1 and Spi-B, and to activate transcription from the LMP-1 promoter in a Spi-dependent manner, certainly suggests that activation of transcription through Spi proteins also plays an important role in the biology of LCVs. Furthermore, the Spi-binding site is conserved in the RhLCV LMP-1 promoter (44). Although the only gene thus far demonstrated to be activated by EBNA-3C is the EBV LMP-1 gene, Spi proteins regulate a variety of genes and play important roles in the development of the B-cell lineage, and it is therefore highly likely that cellular genes are also activated by EBNA-3C in association with EBNA-2. Thus, the interaction of EBNA-3C with Spi transcription factors may represent a unique function of EBNA-3C that contributes to EBV-mediated immortalization by activating cellular gene expression, as well as activating expression of the viral oncoprotein LMP-1. These new findings, which demonstrate conservation of EBNA-3C interactions with two highly important and conserved transcription regulatory pathways, now set the stage for future genetic experiments to explore the role of each of these functions of EBNA-3C in EBV-mediated immortalization of B lymphocytes. ACKNOWLEDGMENTS We thank Evelyn Stigger-Rosser and Jennifer Moore for excellent technical assistance, Andrew Brooks for the EBNA-3C schematic shown in Fig. 1B, and the Hartwell Center for Bioinformatics and Biotechnology at SJCRH for oligonucleotide synthesis, DNA sequencing, and assistance with computer analysis. This research was supported by Public Health Service grants CA56645 and CA73561 (to C.E.S.), CA68051 (to F.W.), CA56639 (to J.T.S.), Cancer Center Support (CORE) grant CA21765, and the American Lebanese Syrian Associated Charities (ALSAC). REFERENCES 1. Allday, M. J., D. H. Crawford, and J. A. Thomas. 1993. Epstein-Barr virus (EBV) nuclear antigen 6 induces expression of the EBV latent membrane protein and an activated phenotype in Raji cells. J. Gen. Virol. 74:361–369. 2. Allday, M. J., and P. J. Farrell. 1994. Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells. J. Virol. 68:3491–3498. 3. Barolo, S., T. Stone, A. G. Bang, and J. W. Posakony. 2002. Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless. Genes Dev. 16: 1964–1976. 4. Brou, C., F. Logeat, M. Lecourtois, J. Vandekerckhove, P. Kourilsky, F. Schweisguth, and A. Israel. 1994. Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J, by direct protein-protein interaction with Drosophila hairless. Genes Dev. 8:2491–2503. 5. Cohen, J. I., F. Wang, J. Mannick, and E. Kieff. 1989. Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc. Natl. Acad. Sci. USA 86:9558–9562.
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