The BRRF1 Early Gene of Epstein-Barr Virus Encodes a Transcription Factor That Enhances Induction of Lytic Infection by BRLF1

The Na expression vector pRC-FLAG-BRRF1 and the Z expression vector pSG5-Z were described previously (51, 53). pSG5-R (a gift from Diane Hayward) contains the genomic R sequence beginning with the second exon (including the ATG start codon for R) but lacking the first intron (containing the Na gene) (24). Appropriate control vectors, pRC (Invitrogen) and pSG5 (Stratagene), were used in all experiments. The pRK5-BALF4 expression vector encodes the EBV gp110 protein and was previously shown to enhance the infection efficiency of EBV virions derived from the B95-8 strain of EBV (42). Zp-CAT, Zp-CAT ΔZIA/ZIB, Zp-CAT ΔZII, and −65Zp-CAT (all gifts from E. Flemington) were described previously (17). Briefly, Zp-CAT contains the region of the BZLF1 promoter from −221 to +12 (relative to the transcription start site) linked upstream of the chloramphenicol acetyltransferase (CAT) gene. Zp-CAT ΔZIA/ZIB and Zp-CAT ΔZII represent mutants of Zp in which the ZIA and ZIB sites and the ZII site, respectively, have been mutated via site-directed mutagenesis. The plasmid −65Zp-CAT contains nucleotides −65 to +12 of Zp upstream of CAT. EApBS-CAT (also called BMRF1-CAT) contains the BMRF1 promoter sequence from −331 to +1 upstream of CAT and was described previously (44). CRE-CAT contains three consensus CRE sites upstream of CAT; CRE(m)-CAT is identical to CRE-CAT except for specific mutations in the CRE site. The SG424 vector encodes the Gal4 DNA binding domain alone, whereas Gal4-CREB, Gal4-ATF1, Gal4-ATF2 (all gifts from M. Green), and Gal4-c-Jun (a gift from A. Baldwin) contain the respective proteins linked in-frame to the Gal4 DNA binding domain. Gal4-EIB-CAT (a gift from M. Green) consists of five copies of the Gal4 DNA binding site linked upstream of the minimal EIB TATA box and CAT gene. The CR2/puro expression vector was a gift from L. Hutt-Fletcher; CR2 is the primary cellular receptor for EBV (15). CMV-c-Jun expresses full-length c-Jun under the control of the cytomegalovirus (CMV) promoter. Plasmids were purified by use of Qiagen Maxiprep kits (Qiagen) according to the manufacturer's instructions.

293 cells infected with the R-KO virus (293 R-KO), Z-KO virus (293 Z-KO), and wild-type virus (293 WT) have been described previously (11, 14). In the R-KO virus, nucleotides 103,638 to 105,083 (B95.8 coordinates [accession no. V01555 ]) within the BRLF1 gene were removed via the insertion of a tetracycline resistance cassette into the virus (14). In the Z-KO virus, nucleotides 102,389 to 103,388 (B95.8 coordinates) within the BZLF1 gene were removed via the insertion of a kanamycin resistance cassette. The R-KO, Z-KO, and WT viruses also encode enhanced green fluorescent protein (GFP) and a hygromycin B resistance gene (both cloned into the B95.8 deletion of EBV, where the second copy of oriLyt normally resides). 293 R-KO, 293 Z-KO, and 293 WT cells were maintained in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS), penicillin-streptomycin, and hygromycin B (100 μg/ml; Roche). HeLa cells are a cervical adenocarcinoma line and were maintained in Dulbecco's modified Eagle medium with 10% FBS and penicillin-streptomycin. AGS R-KO cells (see below) were maintained in F-12 medium containing 10% FBS, penicillin-streptomycin, and 100 μg of hygromycin B/ml. BL30 R-KO cells and LCL R-KO cells (see below) were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin-streptomycin, and 400 μg of hygromycin B/ml. Raji cells, an EBV-positive Burkitt lymphoma cell line, were maintained in RPMI 1640 with 10% FBS and penicillin-streptomycin.

Transfections of 293 R-KO and 293 WT cells were done by use of Lipofectamine 2000 (Invitrogen). Cells (6 × 10⁵) were seeded the day prior to transfection in 2 ml of medium in a six-well plate. Transfections were performed according to the manufacturer's instructions, except that the reagent/DNA ratio was scaled down to 2 μl/1 μg. Transfections of HeLa cells for reporter assays were performed by use of Fugene 6 (Roche) according to the manufacturer's instructions. AGS R-KO cells were electroporated with 5 μg of total DNA in a Zapper electroporation unit as described previously (60). BL30 R-KO and LCL R-KO cells were transfected in buffer V (Amaxa Biosystems) with an Amaxa Nucleofector (Amaxa Biosystems). Cells were cultured for 24 h in antibiotic-free medium, followed by two washes in 1× phosphate-buffered saline (PBS). Cells (10⁷) were resuspended in 100 μl of buffer V (per condition) and 5 μg of total DNA was added. Settings G-16 and T-20 were used for BL30 R-KO and LCL R-KO cells, respectively. After being transfected, cells were incubated overnight in 1.5 ml of antibiotic-free medium at 37°C in a 12-well plate; 2.5 ml of complete medium was then added.

For the production of R-KO viral stocks, 5 × 10⁶ 293 R-KO cells were plated in 10 ml of RPMI 1640-10% FBS-penicillin-streptomycin in a 100-mm-diameter dish the day prior to transfection. The cells were transfected with 1.5 μg each of pSG5-R, pSG5-Z, pRC-FLAG-BRRF1, and pRK5-BALF4 expression plasmids by use of Lipofectamine 2000 (12 μl of reagent per condition) according to the manufacturer's instructions. At 72 h posttransfection, supernatants were filtered through 0.45-μm-pore-size filters and viral stocks were stored at 4°C. AGS cells (a gift from L. Hutt-Fletcher) are an EBV-negative gastric adenocarcinoma cell line. In order to create an AGS line infected with the R-KO virus, we first transfected AGS cells with 1 μg of an expression vector encoding CD21-puromycin resistance by using Fugene 6 (Roche) according to the manufacturer's instructions. At 48 h posttransfection, cells were selected with 0.5 μg of puromycin HCl (Roche)/ml; puromycin-resistant colonies were pooled. For infections with R-KO virus, puromycin-resistant cells were seeded at 50% confluence in a 60-mm-diameter dish and were incubated with R-KO viral stocks. Four days after infection, cells were selected with hygromycin B (100 μg/ml); one hygromycin B-resistant colony representing an AGS cell infected with the R-KO virus was isolated. BL30 cells are an EBV-negative Burkitt lymphoma line. For the generation of BL30 R-KO cells, approximately 5 × 10⁶ BL30 cells were pelleted and resuspended in 5 ml of R-KO viral stock for 3 h at 37°C. After the incubation, the cells were diluted into 50 ml of medium. Selection with 400 μg of hygromycin B/ml was begun at 5 days postinfection. For the generation of LCL R-KO cells, 3 × 10⁶ peripheral blood mononuclear cells (provided by the laboratory of Susan Fiscus, University of North Carolina at Chapel Hill) were resuspended in 3 ml of R-KO virus and incubated overnight at 37°C. Seven milliliters of RPMI 1640-20% FBS-penicillin-streptomycin was then added, and cyclosporine A (Sigma) was added to achieve a final concentration of 500 ng/ml. Fifty percent of the medium was changed every 7 days until transformation into LCLs was apparent (at ∼3 to 4 weeks postinfection). LCLs were then maintained in RPMI 1640-10% FBS-penicillin-streptomycin.

Total RNAs were prepared by use of RNAeasy kits (Qiagen), including DNase treatment, according to the manufacturer's instructions. Ten micrograms of total RNA was subjected to denaturing agarose gel electrophoresis, and fractionated RNAs were transferred to a membrane by use of the Turboblotter system (Schleicher and Schuell) according to the manufacturer's instructions. After being transferred, RNAs were cross-linked to the membrane via UV irradiation (1,200 J). The following double-stranded DNA fragments were used as probes for Z, Na, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively: a PstI/HindIII fragment from pSG5-Z, a BglII/XhoI fragment of pRC-FLAG-BRRF1, and a mouse GAPDH DECAtemplate (Ambion). Double-stranded DNA fragments were gel purified with a Qiagen gel extraction kit (Qiagen) and radiolabeled with a Prime-A-Gene kit (Promega), and unincorporated radioactivity was removed by the use of Sephadex G-50 columns (Amersham); all steps were performed according to the manufacturer's instructions. Prehybridization and hybridization (2 × 10⁶ cpm of probe) were performed in Quikhyb solution (Stratagene) according to the manufacturer's instructions. Membranes were exposed to film overnight at −80°C.

Immunoblotting was performed as described previously (1). Briefly, cells were lysed in NP-40 lysis buffer supplemented with protease and phosphatase inhibitors; equivalent amounts of protein were then separated in sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis gels. For the examination of c-Jun phosphorylation status, cells were kept at 4°C at all times during the harvesting procedure. After being transferred and blocked, membranes were incubated overnight at 4°C with the appropriate primary antibodies diluted in 5% milk in 1× PBS and 0.1% Tween 20 (PBS-T). Primary antibody dilutions were as follows: 1:100 anti-BRLF1 (Argene), 1:100 anti-BZLF1 (Argene), 1:100 anti-BMRF1 (Capricorn), 1 μg of M2 anti-FLAG (Sigma)/ml, and 1:5,000 anti-β-actin (clone AC-15; Sigma). Anti-c-Jun (Cell Signaling) and anti-phosphoSer73-c-Jun (Cell Signaling) antibodies were diluted 1:1,000 in 5% bovine serum albumin in 1× PBS-T. The appropriate horseradish peroxidase-conjugated secondary antibodies (Promega) were used at a dilution of 1:10,000 in 5% milk-1× PBS-T for 1 h at room temperature. After being washed, bound antibodies were visualized by use of ECL reagent (Amersham) according to the manufacturer's instructions.

CAT assays were performed as described previously (19) with cell extracts harvested at 48 h posttransfection. Chloramphenicol acetylation was determined by thin-layer chromatography followed by PhosphorImager (Molecular Dynamics) quantification.

Supernatants from 293 R-KO cells were harvested at 48 h posttransfection and filtered through a 0.45-μm-pore-size filter. Raji cells (2 × 10⁵) were then incubated in 0.5 ml of virus for 3 h at 37°C in a 12-well plate. One and one-half milliliters of medium was then added to each well and the cells were incubated for an additional 48 h at 37°C. Phorbol-12-myristate-3-acetate (PMA; Sigma) and sodium butyrate (Sigma) were then added to achieve a final concentration of 50 ng of PMA/ml and 3 mM sodium butyrate. The number of GFP-expressing Raji cells was quantitated 24 h later by fluorescence microscopy.

The phenotype of a recombinant EBV in which the majority of the R gene has been disrupted through insertional mutagenesis (293 R-KO) (Fig. 1) was previously described for 293 cells as well as primary B cells immortalized by this virus (14). The R-KO virus is similar to the wild-type virus in terms of its ability to immortalize primary B cells, but 293 cells carrying the R-KO virus could not be induced into the lytic form of EBV infection unless a vector expressing the BRLF1 gene product was supplied in trans (14). These results were interpreted as showing that the lytic defect of the R-KO virus is completely rescued by the BRLF1 IE protein in trans (14).

Notably, however, the EBV sequences deleted from the R-KO virus (nucleotides 103,638 to 105,083 in the B95.8 strain coordinates) not only include the BRLF1 gene, but simultaneously include essentially all of the Na promoter, except for the sequences located within 36 bp of the Na transcriptional start site (Fig. 1). Thus, we hypothesized that the R-KO virus cannot efficiently transcribe the Na message. Furthermore, since the BRLF1 expression vector which was previously used to rescue the lytic defect of the R-KO virus in 293 cells also contained the EBV sequences encoding the Na gene product as well as the Na promoter (14), this vector could potentially express both BRLF1 and Na.

To determine if the R-KO virus is indeed defective for Na expression, we performed a Northern blot analysis of total RNAs from 293 WT, 293 Z-KO, and 293 R-KO cells transfected with an empty vector, a vector expressing Z (which induces the expression of Na [53]), and a vector expressing R that does not contain the intronic sequence in which the Na gene lies. As shown in Fig. 2, as expected, the expression of either Z or R in the 293 WT line induced transcription of the 1.3-kb Na message. R induction of the BZLF1 message, as well as Z induction of the BRLF1 message, in the 293 WT line could be detected upon a longer exposure (data not shown). In the 293 Z-KO line, Z induced the transcription of Na, whereas R did not, indicating that R cannot induce Na expression without the help of Z. In 293 R-KO cells, neither Z nor R expression induced the transcription of Na, confirming that 293 cells infected with the R-KO virus cannot transcribe Na under the conditions in which it is normally expressed. Thus, 293 R-KO cells provide an environment to study the effects of Na during the viral life cycle.

To determine if Na affects the ability of R to induce a lytic EBV infection in 293 R-KO cells, we transfected these cells with a vector control, a vector that expresses Na (FLAG-tagged), a vector that expresses R but not Na, or a combination of the R and Na vectors. The efficiency of early lytic gene induction was assessed by quantitating the expression of IE and early lytic proteins 48 h after transfection. As shown in Fig. 3A, the R vector alone induced only a low-level expression of the early BMRF1 protein in 293 R-KO cells, whereas 293 WT cells transfected with the R vector expressed considerably more BMRF1. Furthermore, although Na alone did not activate Z or BMRF1 expression in 293 R-KO cells, the combination of Na and R was much more effective than R alone in inducing both Z and BMRF1 expression (Fig. 3B).

As shown in Fig. 1, the R-KO deletion also removes a portion of the R-derived N terminus of RAZ (Fig. 1) as well as the first RAZ splice site, suggesting that the R-KO virus cannot express RAZ. RAZ is a chimeric protein consisting of the N terminus of R fused to the C terminus of Z that may function to repress Z activity (18, 52). To determine whether the absence of RAZ expression in 293 R-KO cells had an impact on the lytic infection induced by R, we transfected 293 R-KO cells with R alone, R and RAZ, R and Na, or R, Na, and RAZ in combination. The transfection of RAZ did not enhance the capacity of R alone or R in conjunction with Na to induce BMRF1 expression (data not shown). Thus, the inability of R to efficiently activate early lytic gene transcription in 293 R-KO cells likely reflects the loss of the Na, rather than RAZ, function in the mutant R-KO virus.

To determine if the ability of Na to enhance R-induced lytic infection is a universal phenomenon among various cell lines, we derived several other cell lines containing the R-KO virus, including a gastric carcinoma line (AGS R-KO), a lymphoblastoid cell line (LCL R-KO), and a Burkitt lymphoma line (BL30 R-KO), and examined whether the ability of transfected R to induce early viral gene expression was likewise enhanced by Na expression (Fig. 4). The results indicated that the synergy between Na and R in inducing BMRF1 expression is cell line dependent. AGS cells infected with wild-type EBV have a relatively high proportion of lytically infected cells (39). Interestingly, AGS cells infected with the R-KO virus demonstrated the expression of Z even in the absence of R (Fig. 4A), suggesting that cellular factors are sufficient to activate Z transcription in this cell line. Nevertheless, the ability of transfected R to induce BMRF1 transcription was enhanced 2.2-fold by the presence of Na in AGS R-KO cells (Fig. 4A). In contrast, in the BL30 R-KO line, although R alone induced some BMRF1 expression, the addition of Na did not enhance BMRF1 expression (Fig. 4B). Finally, in the LCL R-KO line, although transfected R was clearly visible, no significant BMRF1 expression was observed in the presence of R alone or with the combination of R and Na (Fig. 4C). Thus, the ability of R to induce a lytic EBV infection, as previously described (46, 68), is clearly cell line dependent. Furthermore, the synergistic effect of Na and R together also appears to be modified by either host-cell-dependent or virus-dependent conditions (such as epigenetic modifications of the viral genome) that are currently poorly defined.

The ability of Na to enhance R activation of lytic gene transcription in at least some cell types (particularly 293 R-KO cells) suggests that the combination of Na and R may likewise increase the amount of lytic viral replication. To determine if this increase in early gene transcription results in an increase in the amount of infectious virus, we harvested the supernatants of 293 R-KO cells transfected with either vector DNA, R alone, Na alone, or a combination of R and Na. The filtered supernatants were than used to infect Raji cells, which express the EBV receptor at a high level, and the amount of infectious virus in each 293 cell supernatant was quantitated by determining the number of GFP-positive Raji cells. As shown in Fig. 5, 293 R-KO cells transfected with the combination of R and Na produced fivefold more infectious EBV than cells transfected with R alone. Thus, the ability of Na to enhance R-mediated lytic gene expression in this cell type also correlates with increased lytic viral replication.

The previous results suggested that Na is important for the efficient induction of lytic infection by R in some cell types. In addition, the ability of Na to enhance R activation of lytic viral genes in some cell types suggests that either Na enhances BRLF1 transcriptional function or Na itself is a transcriptional activator. To determine if Na itself is a transcriptional activator, we performed reporter gene assays with HeLa cells, using constructs in which either the BZLF1 (Zp) or BMRF1 (EAp) promoter was positioned upstream of the CAT gene. The Zp-CAT construct contains the BZLF1 promoter sequence from −221 to +12, which has been shown to be sufficient for the induction of Zp activity by a variety of stimuli (4, 17). Although the expression of Na had no effect on the activity of the construct driven by the BMRF1 (EAp) promoter, it increased the activity of the construct driven by Zp sevenfold (Fig. 6). In contrast, a Zp construct with a deletion of the Zp sequence 5′ of −65 was not activated by Na. These results indicate that Na specifically activates Zp and that sequences in Zp located between −65 and −221 are required for this effect.

Zp has been extensively characterized, and a number of different positively acting regulatory elements have been mapped within the Zp sequences located between −65 and −221 relative to the transcription start site (Fig. 7A). Sequences previously shown to be important for Zp activation by a variety of different stimuli include a CRE motif (ZII) (17), which binds CREB, ATF1, ATF2, and c-Jun (1, 34, 37, 63); a series of ZI sites (5, 16, 17), which bind MEF2D, Sp1, and Sp3 (22, 35, 36); and ZIII sites, which bind Z and may mediate positive autoregulation (16), although this point is controversial (29, 31, 68). In addition, several SMAD binding elements, including one which overlaps the ZII site, have been implicated in the activation of Zp in response to transforming growth factor beta (33). To determine if a ZI or ZII site is required for Na transactivation of Zp, we examined the ability of Na to activate transcription from wild-type Zp-CAT versus a Zp-CAT plasmid containing specific mutations in two ZI sites (Zp-CAT ΔZIA/ZIB) or a Zp-CAT construct containing a mutant CRE (ZII) site (Zp-CAT ΔZII) (Fig. 7A). Mutation of the ZIA and ZIB sites had no effect on the Na induction of Zp, whereas mutation of the ZII CRE site completely eliminated Na's ability to activate Zp (Fig. 7B). These results suggest that Na activates Zp through the ZII CRE site.

To determine if a consensus CRE motif is sufficient to mediate Na transactivation, we compared the ability of Na to transactivate a reporter plasmid containing three consensus CRE sites upstream of a minimal adenovirus E1b promoter versus a construct containing three copies of a mutant CRE motif (Fig. 7C). These results indicated that the CRE motif is sufficient to mediate the Na transactivator effect.

The ZII CRE site of Zp binds the transcription factors CREB, ATF-1, ATF-2, and c-Jun (1, 34, 37, 63), all of which have been shown to activate Zp in reporter assays (1, 34, 63). Since Na activates Zp through the ZII CRE site, we examined whether Na can activate CREB, ATF-1, ATF-2, or c-Jun fusion proteins linked to the Gal4 DNA binding domain. The abilities of ATF-1, ATF-2, CREB-1, and c-JUN Gal4 fusion proteins to activate a reporter plasmid containing five copies of the Gal4 DNA binding site upstream of the CAT gene, in the presence or absence of cotransfected Na, were examined in HeLa cells. As seen in Fig. 8, Na specifically activated the c-Jun transcriptional function, while it did not significantly affect the function of ATF-1, ATF-2, or CREB-1. Since the DNA binding activity in these experiments was mediated through the Gal4 DNA binding domain of the fusion proteins, these results suggest that Na activates promoters by enhancing the c-Jun transcriptional function.

Efficient c-Jun transcriptional functioning requires the phosphorylation of serines 63 and 73 in c-Jun by JNK (12, 25, 30). c-Jun phosphorylated at Ser63 and Ser73 is more stable (41) and has an enhanced ability to transactivate target promoters (43). We therefore determined if Na expression affects c-Jun phosphorylation. The c-Jun phosphorylation status was monitored by using an antibody specific for c-Jun phosphorylated at Ser73 versus an antibody that recognizes total c-Jun. Although HeLa cells transfected with c-Jun alone displayed a low level of basal c-Jun phosphorylation (Fig. 9, P-c-Jun, lower arrow), the presence of Na significantly increased the amount of heavily phosphorylated c-Jun (P-c-Jun, upper arrow). The specificity of the phospho-c-Jun antibody for phosphorylated c-Jun was verified by using blocking peptides specific for phospho-c-Jun (data not shown). The slightly increased level of total c-Jun in the presence of Na possibly reflects the increased stability of c-Jun in response to phosphorylation (41).