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Epstein-Barr virus (EBV) is associated with three lymphoproliferative diseases of B-cell origin: acute infectious mononucleosis, endemic Burkitt's lymphoma (BL), and lymphoma occurring in immunocompromised individuals. In addition, it has been shown that there is a likely etiologic role for EBV in the development of undifferentiated nasopharyngeal carcinoma (NPC) (14). Studies have shown the presence of rightward transcripts from the BamHI A region of EBV in NPC tumor tissue. Sequence analyses of cDNA clones from a transcription library of a tumor xenograft (C15) suggested that primary transcripts were subjected to differential splicing, giving rise to the existence of a family of related transcripts in NPC tissues (4, 5, 7, 9, 18). All NPC tissues tested contained either four or five rightward transcripts of BamHI A which have a common 3' terminal open reading frame (ORF), termed BARF0. PCR analyses and in situ hybridization, using a riboprobe specific for the BARF0 ORF, also detected BamHI A transcripts in EBV-positive BL cell lines as well as in all EBV-transformed lymphoblastoid cell lines (LCLs) studied (4).

In a recent study (15), cDNA cloning, reverse transcriptase-PCR (RT-PCR), and Northern blotting were used to further define the structures of the BamHI A rightward transcripts. Three BamHI A cDNAs were isolated and found to be previously unidentified mRNAs that contained the BARF0 ORF and additional ORFs encoded by multiple exons, including one, termed RK-BARF0, which extended the size of the BARF0 ORF from 174 to 279 amino acids (aa). Fries et al. (6) raised a rabbit antiserum to a synthetic peptide containing an amino acid sequence encoded within the BARF0 ORF. This antiserum detected a glutathione S-transferase-BARF0 fusion protein and both BARF0 and RK-BARF0 proteins expressed from transfected constructs in H1299 epithelial cells. The serum also immunoprecipitated the 20-kDa protein BARF0 and the 30-kDa protein RK-BARF0 translated in vitro and identified a membrane-associated doublet of 30- and 35-kDa proteins in all of the EBV-infected cell lines tested.

In further work the BARF0 ORF was shown to encode an HLA class I-restricted cytotoxic T lymphocyte (CTL) epitope which was recognized by CTLs from EBV-seropositive, but not -seronegative, individuals (11). In EBV-positive cells this CTL epitope was found to be effectively removed by differential splicing of the BARF0 ORF. These splicing events also resulted in the generation of novel RK-BARF0 protein derivatives of 16 to 20 kDa (10).

Cellular localization of RK-BARF0. In order to further characterize the biological functions of RK-BARF0 and BARF0 the cellular localization of these proteins was analyzed. The Flag-RK-BARF0 and BARF0 cDNAs (6) were cloned into the KpnI-BamHI and BamHI sites, respectively, of the vector pEGFP-C1 (Clontech) and were transiently expressed in the EBV-negative BL cell line DG75 (3). Localization of the green fluorescent protein (GFP) fusion proteins (GFP-Flag-RK-BARF0 and GFP-BARF0) and GFP alone was determined by fluorescence microscopy on living cells (Fig. 1A). As expected, GFP alone, being present in both the nucleus and cytoplasm, was distributed throughout the cell. In contrast, both the GFP-Flag-RK-BARF0 and GFP-BARF0 cell transfectants showed prominent nuclear staining that formed bright condensed structures. This result was surprising, as RK-BARF0 was thought to be membrane associated (6). As the cytoplasm of DG75 cells is fairly small it was difficult to determine the proportions of GFP-Flag-RK-BARF0 and GFP-BARF0 that were present in the cytoplasm. To confirm that RK-BARF0 was indeed associated with the nucleus, Flag-tagged RK-BARF0 cDNA, cloned into the expression vector EBO-pLPP (10), was transiently introduced into HeLa cells or stably expressed in DG75 cells. The transfected cell lines were treated with propidium iodide (2 µg per ml) to stain nucleic acid and with anti-Flag monoclonal antibody (MAb) (diluted 1:60 in phosphate-buffered saline) (Eastman Kodak) to identify Flag-tagged proteins. Indirect immunofluorescence showed that the Flag-tagged proteins were present throughout the cell but were predominantly localized in the parts of the nucleus other than the nucleoli (Fig. 1B). These results confirmed the data for localization of the GFP fusion proteins and indicated that a sequence within the BARF0 ORF was sufficient for targeting the proteins to the nucleus.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Fluorescence microscopy in transfected EBV-negative cells. The tagged RK-BARF0 and BARF0 gene constructs are illustrated and include the RK exon (open box), the BARF0 ORF (grey box), and the in-frame sequence (dark grey box) located 5' of the BARF0 ORF (according to references 6 and 15) as well as the Flag epitope (Flag) and GFP (cross-hatched box). The location of the 20-mer peptide used to raise the polyclonal anti-RK-BARF0 rabbit serum (6) is shown by a black oval. (A) DG75 cells were transiently transfected with plasmids encoding GFP, GFP-Flag-RK-BARF0, or GFP-BARF0, and GFP fluorescence was analyzed in living cells. (B) The Flag-RK-BARF0 gene was either transiently expressed in HeLa cells or stably expressed in DG75 cells. Cells were stained with an anti-Flag MAb (anti-Flag) and propidium iodide (PI stain) and analyzed by indirect immunofluorescence of fixed cells.

Fractionation of cells expressing Flag-RK-BARF0 and its derivatives. We have previously shown that the RK-BARF0 transcript undergoes differential splicing utilizing an identical 5' splice site and one of three different 3' splice sites. This results in the generation of three RK-BARF0 derivatives of 190 or 177 aa and a frameshifted chimeric protein product (which is totally unrelated to RK-BARF0 in its C-terminal half) of 150 aa (10) (Fig. 2A). To confirm the fluorescence study results and to determine whether the RK-BARF0 16- to 20-kDa protein derivatives were also localized to the nucleus, DG75 cells stably expressing Flag-RK-BARF0 were fractionated into nuclear, membrane, and cytoplasmic fractions as described recently (6). Protein was detected by immunoblotting by using an anti-Flag antibody, diluted 1:300 (12). The total lysate of transfected cells showed low amounts of full-length RK-BARF0 protein but high levels of 16- to 20-kDa Flag-tagged RK-BARF0 derivative proteins (Fig. 2B). The 32-kDa Flag-RK-BARF0 protein was found to be almost exclusively associated with the nuclear fraction, while the 16- to 20-kDa derivatives were present in all cellular fractions. These results indicated that the sequence responsible for the nuclear association of RK-BARF0 was encoded within the region which was removed by splicing of the RK-BARF0 transcript. Interestingly, the region removed was arginine rich and had been suggested to be involved in binding of nucleic acids (15).

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Fractionation of EBV-negative DG75 cells expressing Flag-RK-BARF0. (A) Summary of the differential splicing of RK-BARF0 as reported recently (10). The locations of the splice sites are shown, and the sizes (in amino acids) of the full-length Flag-tagged RK-BARF0 and its 16- to 20-kDa protein derivatives are given. For graphic details see the legend of Fig. 1. Note that the products F-S#1 and F-S#2 maintain the BARF0 frame, whereas F-S#3 undergoes a frameshift in its C terminus (cross-hatched). (B) Cells stably expressing Flag-RK-BARF0 were separated into nuclear (N), membrane (M), and cytoplasmic (C) fractions. For controls, total extracts of cells expressing either the control vector (lane 1) or Flag-RK-BARF0 (lane 2) were used. Total and fractionated proteins were separated by SDS-12% polyacrylamide gel electrophoresis, and their expression was analyzed by immunoblotting by using an anti-Flag MAb. Molecular size markers (expressed in kilodaltons) are shown, and the positions of Flag-RK-BARF0 and its 16- to 20-kDa derivatives are indicated by arrows. (C) Cells stably expressing Flag-RK-BARF0 were separated into cytoplasmic (C) and nuclear fractions. Samples of the nuclear extract were then incubated with buffers containing either low (150 mM) or high (500 mM) NaCl concentration, and the corresponding nuclear supernatant (w) and pellet (p) were collected and analyzed by immunoblotting.

Specificity of the polyclonal rabbit RK-BARF0 antiserum. The study generating the original data on the membrane localization of RK-BARF0 utilized cellular fractionation of an EBV-positive LCL and a polyclonal rabbit anti-RK-BARF0 serum (6). This anti-RK-BARF0 serum (diluted 1:1,000) was therefore utilized to probe the Flag-RK-BARF0-expressing DG75 cell fractions. As shown in Fig. 3A, the 30- and 35-kDa proteins, previously identified by Fries et al. (6), were as expected present primarily in the membrane fraction, suggesting that they did not represent RK-BARF0. More importantly, these proteins were also detected in the EBV-negative DG75 cells (Fig. 3C, lane 1), indicating that the anti-RK-BARF0 serum cross-reacted with cellular proteins.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Specificity of the RK-BARF0 antiserum. (A) DG75 cells stably expressing Flag-RK-BARF0 were separated into nuclear (N), membrane (M), and cytoplasmic (C) fractions and subjected to SDS-12% polyacrylamide gel electrophoresis. For controls, total extracts of cells expressing either the control vector (lane 1) or Flag-RK-BARF0 (lane 2) were used. (B) Total cell extracts of the EBV-negative B-cell lymphoma line BJAB (lane 1), of the EBV-positive LCL IS (lane 2), and of the EBV-negative BL cell lines BL41 (lane 3), BL30K (lane 4), Ramos (lane 5), DG75 (lane 6), and BL30A (lane 7) were separated by SDS-12% polyacrylamide gel electrophoresis. (C) Total cell extracts of DG75 cells transfected with plasmids encoding GFP (lane 1), GFP-BARF0 (lane 2), GFP-Flag-RK-BARF0 (lane 3), and Flag-RK-BARF0 (lane 4) were separated by SDS-10% polyacrylamide gel electrophoresis. Protein expression was analyzed by immunoblotting by using either the polyclonal anti-RK-BARF0 serum or an anti-Flag MAb (indicated at the bottom). Molecular size markers (expressed in kilodaltons) are shown, and the positions of the cross-reactive 30- to 35-kDa proteins and the tagged RK-BARF0 and BARF0 proteins are indicated at the left. The asterisks mark proteins which were detected by the anti-Flag MAb but not by the anti-RK-BARF0 serum.

Analyses of BARF0 transcripts in EBV-infected cells. In EBV-positive BL cells, LCLs, and NPC cells, transcripts encoding the BARF0 ORF were recently shown to utilize a 5' splice site and one of three different 3' splice sites (10). The expression pattern of the BamHI A rightward messages is very complex, and so far no major transcript has been described. To obtain a semiqualitative estimation of the frequency of the differentially spliced BARF0 transcripts, expressed relative to the amounts of all of the BamHI A transcripts, RT-PCR was performed as outlined recently (10). Briefly, total RNA from a panel of EBV-positive cells was reverse transcribed with oligo-dT primers. For negative controls, water and the RT sample missing the RT enzyme were used. A positive control consisted of plasmid DNA of Flag-RK-BARF0. The primers BARF0-F3 (5'-TCTGCCGTGAAGGGTTG; position 160789 within the B95.8 EBV genome [2]) and BARF0-R6 (5'-GTGTTTTATTGCATGTCTCACACC; position 160973 within the same genome) were specific for the 185-bp-long 3' end of the BARF0 ORF and should represent all BamHI A transcripts. The primers BARF0-F (5'-GCCCGAGGAGCTGTAGACC; position 160308 within the B95.8 EBV genome) and BARF0-R6 covered the complete BARF0 ORF and amplified full-length (666 bp) and spliced forms (314, 353, and 363 bp) (Fig. 4A). Comparison of the PCR-amplified products revealed that the differentially spliced BARF0 products (Fig. 4B) composed a small but significant proportion of the total viral BamHI A transcripts which were unspliced in their 3'-terminal ends (Fig. 4C).

View larger version (37K): [in this window] [in a new window]   FIG. 4.   RT-PCR of EBV-positive cells. (A) Schematic diagram of the genomic RK-BARF0 organization in EBV and the locations of the primers used. The numbering refers to the EBV sequence of strain B95.8 (2) and indicates start, stop, and splice sites as reported recently (10, 15). For graphical details see the legend of Fig. 1. (B) Total RNAs from the EBV-positive cell lines SB (an LCL) (lane 1), MutuI-BL (lane 2), and MutuIII-BL (lane 3), from the tumor xenograft C15-NPC (lane 4), and from EBV-negative DG75 cells stably expressing Flag-RK-BARF0 (lane 5) were analyzed by RT-PCR. The positive (+) and negative () RT samples, the water control (W), and the positive control DNA of the plasmid encoding Flag-RK-BARF0 (DNA) were amplified by using the primers BARF0-F and BARF0-R6. The PCR products were separated and visualized by electrophoresis on an ethidium bromide-containing 2.5% agarose gel. Some markers of the 1-kbp DNA ladder (M) are shown on the right, and the positions of the unspliced and spliced cDNAs are indicated by arrowheads. (C) RT-PCR performed by using the primers BARF0-F3 and BARF0-R6. Lanes 1 through 5 correspond to the positive RT samples used in the RT-PCR shown in panel B, and one negative RT sample is shown as a control. The molecular size of the unspliced cDNA is given.

Analysis of RK-BARF0 expression in EBV-infected cells. To further characterize the recombinant 16- to 20-kDa RK-BARF0 protein derivatives, two-dimensional (2D) gel electrophoresis was employed. Protein extracts of the EBV-positive cells of the NK LCL, which stably expressed either a control vector or Flag-RK-BARF0 (10), were subjected to 2D gel electrophoresis by using the Immobline Drystrip (pH range, 3 to 10.5) and an ExelGel sodium dodecyl sulfate (SDS) gradient (8 to 18%) according to the instructions of the manufacturer (Pharmacia), followed by immunoblotting by using an anti-Flag MAb. The anti-Flag MAb detected three sets of proteins, ranging from 16 to 20 kDa and with isoelectric point (pI) values ranging between 5 and 8, in the Flag-RK-BARF0-transfected cells but not in the control cells (Fig. 5A and B). These values correlated well with the predicted sizes (Fig. 2A) and pI values of the splice variants generated from the Flag-RK-BARF0 message. Each set of Flag-tagged proteins consisted of proteins with similar molecular weights but different pI values, suggesting that these proteins were posttranslationally modified. Since the pattern of differential splicing within the BARF0 mRNA also occurs in EBV-infected cells, as determined by RT-PCR, it is tempting to speculate that these truncated protein variants may be expressed in vivo and have some biological activity. Indeed, differential splicing is well known to regulate gene expression and to create protein isoforms with different functions (reviewed in reference 17).

View larger version (78K): [in this window] [in a new window]   FIG. 5.   2D gel electrophoresis. Total cell extracts of NK LCLs stably expressing a control vector (left) or Flag-RK-BARF0 (right) were separated according to their isoelectric points (first dimension) and molecular weights (second dimension). Protein expression was analyzed by immunoblotting by using an anti-Flag MAb (upper and lower) followed by the anti-RK-BARF0 serum (lower). Molecular size markers (expressed in kilodaltons) and the isoelectric point range are given. Arrows and arrowheads indicate the positions of proteins specifically detected by the anti-Flag MAb and by the anti-RK-BARF0 serum, respectively.