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Previous Article | Next Article 
Molecular and Cellular Biology, March 2003, p. 2192-2201, Vol. 23, No. 6
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.6.2192-2201.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Viral Oncology Unit, Division of Medicine, Wright-Fleming Institute, Imperial College of Science, Technology and Medicine at St. Mary's, London W2 1PG,1 Department of Infectious Diseases, Imperial College Faculty of Medicine,3 Clinical Sciences Centre, Medical Research Council, Hammersmith Hospital, London W12 0NN, United Kingdom,4 College of Life Sciences, Shaanxi Normal University, Xian 710062, People's Republic of China,2 Department of Pathology, Free University of Amsterdam, Amsterdam, The Netherlands5
Received 6 September 2002/ Returned for modification 7 October 2002/ Accepted 17 December 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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170 to >200 kbp) is characterized by unique DNA sequences interrupted by tandem repetitive sequences, internal repeats (IR) 1 to 4, and terminal repeats (TR). The repeats themselves are of various sizes, ranging from about 100 to >3,000 bp, but all reside within genes. Three of these repetitive elements (called IR 1, IR 3, and TR, respectively) are found in latent viral proteins, EBV nuclear antigens 5 and 1, and LMP2A and -B (membrane proteins). The other two repetitive elements, IR 2 (or NotI repeats) and IR 4 (PstI repeats), lie within genes that have been classified as expressing early (replication-associated) functions (23, 30). The corresponding transcripts, called BHLF1 (BamHI H leftward reading frame 1) and LF3 (leftward reading frame 3), are found in the polyribosomal fraction of EBV-infected cells and represent the most abundant viral transcripts synthesized during the EBV lytic cycle (5, 11, 15, 17, 30). Their open reading frames are almost entirely composed of the repetitive elements. Structurally, there are similarities between IR 2- and IR 4-containing genes, and both lie adjacent to viral origins of lytic replication (13). Their protein products, however, have different sequences, and antibodies to one do not cross-react with the other (28, 29). Although EBV IRs resemble in size the short interspersed elements found in eukaryotic genomes, whereas short interspersed elements are generally noncoding and can act as retrotransposons, there is no evidence for retrotransposition of the EBV tandem repeats. They appear to remain physically as stable components of their respective viral genes. Nonetheless, like chromosomal repetitive elements in general (21), by offering the possibility of template-primer misalignments or generation of template hairpin structures during replication, the viral repeats provide opportunities for replication infidelity or transcriptional variation and mutations. Such events are postulated as relevant to viral evolution (14, 24, 41).
A remarkable feature of any microbial or mammalian cell is the incredible accuracy achieved in maintaining the fidelity of its genetic information. However, sequence alterations can and do occur due to RNA/DNA polymerase slippages during transcription or replication. In the RNA virus field, products have been observed with genetic alterations associated either with RNA polymerase slippages at tandem repetitive sequences or mRNA editing that produces frameshifts within a single open reading frame. Such mechanisms lead both to nucleotide deletions and insertions of G residues in paramyxoviruses (4, 16) or, in bovine parainfluenza virus 3, to the expression of all three reading frames in its P gene (32). An RNA polymerase stuttering or slippage model has been proposed to explain such events (40).
In the retrovirus field, infidelity of the reverse transcriptase frequently results in mutation events, many of which appear nonrandom (2). Further, many retroviruses use translational frameshifting on the ribosome at stem-loop structures for generating their pol gene products (31). In both Escherichia coli and Thermus thermophilus, frameshifting events at runs of A residues allow two components of DNA polymerase III to be expressed from a single gene. In the latter case, this mechanism has been attributed to slippage on the ribosome during translation (46), whereas in E. coli, the data suggest that the frameshifting has occurred during transcription (22).
In the DNA virus field, the fidelity of individual DNA replication complexes, whether mainly of cellular or mixed viral and cellular origin, is sufficiently high that records of polymerase slippages and frameshift alterations are rare. To expand or alter their limited coding potential, DNA viruses mainly appear to rely on the use of multiple, alternative splicing sites for processing transcripts that then allow different proteins to be expressed from a single gene, as observed, for example, with papovaviruses and adenoviruses. A classical case of infidelity, however, in the DNA virus field involves the lysozyme gene of bacteriophage T4. This has been attributed to stuttering of a replication complex at short runs of A residues at two sites in the gene (38). Polymerase slippages that alter mini- and microsatellite sequences in DNA are also well documented (6) and, as seen with the RAD50 gene in colorectal cancer and gastric carcinomas, may be related to tumorigenesis (19). Elsewhere, sequence slippages have been reported in clinical isolates involving herpes simplex virus type 2 (25).
Our attention became focused on slippage/frameshifting phenomena while studying the EBV LF3 (IR 4-containing) major transcript in virally associated cell lines and tumors, where alterations possibly explainable by such mechanisms appeared to occur (43). The literature on LF3 already pointed to sequence variations which, if accurate, could best be explained by the occurrence of mutation events (5, 11, 23, 30). A cartoon based in part on literature data is shown (Fig. 1). Further evidence for genetic diversity came from studying cell lines derived from a Burkitt's lymphoma patient, Daudi, which differed in their responsiveness to interferon alpha (12) as well as in their abilities to express a protein from the LF3 gene (44).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor materials. North African NPC xenografts (C15, C17, and C18, from Pierre Busson) were maintained by propagation in nude mice (3). Primary Chinese NPC biopsies were obtained from Hong Kong, and Burkitt's lymphoma biopsies were obtained from Blantyre, Malawi. Southern blot data showed all tumors to be EBV positive.
RNA and DNA isolation for amplification protocols and sequencing. Total RNA was isolated as a pellet from xenografts, cell lines, and fresh biopsy specimens by the guanidinium-cesium chloride method, as described previously (43), and polyadenylated RNA was selected on oligo(dT) mRNA purification columns (Pharmacia). Polyadenylated RNA (1 µg) or total RNA (5 µg) was treated with 10 U of DNase I (Boehringer) to remove any DNA in the RNA samples. DNA isolated from the supernatant of the total RNA preparation by standard procedures was used as a control for DNase I digests or as a substrate for PCR amplifications.
DNA sequencing. PCR products were either sequenced directly from gel-isolated materials or cloned into a pBIISK vector (Stratagen) or M13mp19 and then sequenced with the ABI Prism dye terminator cycle sequencing kit as specified by the manufacturer (Applied Biosystems Inc.).
cDNA synthesis, RT-PCR, and PCR amplifications. To obtain the 5'-end sequence of the LF3 transcripts from different EBV strains and tumor biopsies, DNase I-treated RNAs (1 µg of polyadenylated RNA or 5 µg of total RNA) were used for first-strand cDNA synthesis with a gene-specific primer, 5'-CTGCAGCCGGGTCCGGGGTT-3' (EBV nucleotides 3470 to 3489) (30). Synthesized cDNA was diluted (to 100 µl) in l0 mM Tris-HCl (pH 8.0)-0.1 mM EDTA. PCR amplifications were carried out on 5 µl of cDNA over 36 cycles with 5'-GGTCCGGGGTTCCGGCCCTG-3' (3479 to 3498) and 5'-CGCTCGGGGGGTGCACACCT-3' (3684 to 3665) as primers, as described previously (43). Products were separated by electrophoresis on 1% agarose, and identities were verified by Southern blot hybridization and DNA sequencing. PCR amplification of DNA for sequencing purposes used the same primers and conditions as for cDNA.
RACE analysis. For 3' end sequence determination on LF3 transcripts, the rapid amplification of cDNA ends (RACE) protocol (9) was employed. mRNA (1 µg) was primed with 5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3' and reversibly transcribed with Moloney murine leukemia virus reverse transcriptase (Gibco-BRL). cDNA was amplified by PCR with a gene-specific primer, 5'-CCCGAGCTCCAGGGCCGGAA-3' (1161 to 1142), and an adapter primer, 5'-GACTCGAGTCGACATCG-3'. Positive bands, identified by Southern blotting, were subcloned and sequenced.
Determination of IR 4 repeat copy numbers from cell lines and tumor biopsies. Total chromosomal DNA, digested with BanI and DdeI, was separated by electrophoresis on a 1% agarose gel, transferred to nylon membranes, and hybridized with a 32P-labeled PstI fragment from Daudi cells (11). Product migration, relative to size markers, allowed copy numbers to be derived. Raji DNA, giving a fragment 2.5 kbp in size, with 24.7 copies of the repeat (30), served as an added control for this experiment; 0.7 of a repeat unit lacks a PstI site.
IR 4 repeat studies. To obtain the sequence of the internal 102-bp repeat region, high-fidelity PCR was performed with primers 5'-CCCGAGCTCGAGGGCCGGAA-3' and 5'-CTGCAGCCGGATCCGGGGTT-3' (italic sequences are restriction enzyme sites used in cloning of the DNA products). PCR utilized heat-denatured total cellular DNA (1 µg) from uninduced or chemically induced Daudi-ICRF cells, 400 ng of each primer, 0.1 mM each of the deoxynucleoside triphosphates, and 2 U of HiFidelity DNA polymerase mix (ABgene). Amplification involved an initial denaturation at 95°C for 5 min, followed by 40 cycles of 30 s at 95°C, 20 s at 60°C, and 40 s at 72°C. PCR products were digested with XhoI and BamHI restriction enzymes and cloned into M13mp19. Representative clones were sequenced with dye terminators.
Generation of antipolypeptide antisera. Peptides from the three coding frames in the IR 4 repeat region were used: PP, H2N-PPPERGSGPSGPGGPPGRPPSSRAGTPDPAAAGH-COOH; PR, H2N-PRRSGAADPADPVGHPAAPRAPGPEPRTRLQPAT-COOH; and PA, H2N-PAGAGQRTQRTRWATRPPPELQGRNPGPGGSRPP-COOH. They were synthesized with a 433A solid-phase synthesizer (Applied Biosystems Inc., Poster City, Calif.). Peptide-keyhole limpet hemocyanin conjugates were emulsified at l mg/ml in Freund's complete adjuvant, and 2 ml was injected subcutaneously into New Zealand White rabbits. At 4 weeks, a second immunization was given with peptide-keyhole limpet hemocyanin in Freund's incomplete adjuvant. Immunization was repeated until a strong antibody titer (>l:1,000, checked by peptide enzyme-linked immunosorbent assay [ELISA]) was reached, and then the rabbits were bled. The resulting serum samples and preimmunization serum samples were checked for specificity and reactivity by peptide ELISA and immunoblot staining with chemically induced HH514 (P3HR-1-derived) cell extracts as described previously (27). No cross-reaction by ELISA was observed with antipeptide sera on the individual (see above) peptides.
Protein extraction and Western blotting analyses.
Confluent cells (30 ml), treated or not treated with chemical inducing agents (tetradecanoyl phorbol acetate and n-butyrate) (11), were harvested and pelleted. After three washes with ice-cold phosphate-buffered saline, cell pellets were resuspended in 0.6 ml of cytolysis buffer [20 mM Tris-HCl (pH 7.6), 50 mM KCl, 400 mM NaCl, 150 mM 2-mercaptoethanol, 1% (vol/vol) Triton X-100, and 20% glycerol]. Lysates were incubated on ice (20 min) and spun at 4°C (12 min), and supernatant containing cytoplasmic protein was transferred to a clean 1.5-ml microfuge tube and stored at -70°C prior to use. In all cases, samples from 2 x 106 cells were loaded for electrophoresis purposes. Western blots were performed as described previously (29).
Immunofluorescence analyses. Healthy proliferating cells, with or without chemical induction, were pelleted, washed with ice-cold phosphate-buffered saline, and resuspended (in phosphate-buffered saline) to 2 x 106 cells/ml, and 20 µl was dropped onto glass slides coated with 3-aminopropyltriethoxysilane over an area of about 1 cm in diameter, dried at room temperature, and stored at -70°C (26). Cells were fixed with 4% paraformaldehyde (10 min) and blocked with 10% normal swine serum (20 min) and then incubated with rabbit anti-LF3 antibody (1:50 dilution) (29) or preimmune serum for 1 h. Cells were incubated with biotinylated swine anti-rabbit immunoglobulin (1:500 dilution; Dako) for 45 min, followed by Texas Red-conjugated streptavidin (1:200 dilution; Amersham) for 45 min. All antisera were diluted in 3% bovine serum albumin (Sigma) in phosphate-buffered saline, and intervening washes of slides were carried out twice with phosphate-buffered saline for 5 min. Cells were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) and examined under a fluorescent microscope (Zeiss Axiophot).
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RESULTS |
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We began our studies by examining in greater detail the apparent frameshift mutations at the 5' and 3' ends of the LF3 gene, asking whether the sequence variations observed in cell lines such as those described above are reflected in the gene from primary EBV-associated tumors, whether sequence differences are tumor type specific, whether mechanistically the frameshifts have occurred during DNA replication or transcription (within a particular cell line or tumor), and how much diversity is observed. DNA (by PCR) and RNA (by RT-PCR) sequences over both 5' and 3' apparent frameshift variable regions were determined. Sequence data from relevant regions of the LF3 gene were obtained from both virus producer (P3HR-1 and BL74) and nonproducer (Raji, Daudi-ATCC, Daudi-ICRF, and 100K) human cell lines, from North Africa NPC xenografts (C15, C17, and C18), and from primary biopsies, two from Asian NPC tumors (NPC 1 and 2) and nine (BL001 to BL007, SD, and IB) from Malawian African Burkitt's lymphomas. Similar data were also obtained from a marmoset cell line, M81.
DNA sequence studies showed the unique regions of LF3 to contain invariable sequence. On the other hand, as given in Table 1, runs of pyrimidines at the 5' end of the gene were variously found to be either C6, TC4, or TC3, and at the 3' end, either C6 or C5. From a single source, the sequence data obtained on the mRNAs were identical with the DNA data. There appear to be two discrete patterns among the lymphomas and two among the NPCs, which overlap but are not entirely identical. However, the data are too limited to attach any significance to these results at present.
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In every case examined (data not given), including Raji, with a ubiquitous internal sequence primer, the same-sized product was obtained, indicating that LF3 transcripts all terminate at the same position as that shown in Fig. 2. Sequencing of RACE products also showed that the first AAUAAA polyadenylation signal in the transcript, as noted, was used in every case and identified the poly(A) tail position shown. Thus, based on the two assumptions cited, were an LF3 protein identified in materials cited in patterns 2 to 5, as for example, in the case of BL74 cells (Table 1), mechanisms other than frameshifting events at the ends of the repetitive regions are necessary to permit protein expression.
Frameshifts in internal IR 4 repeats also occur to produce genetic variations.
It is generally assumed from sequence evidence that although point mutations may occur within the repeats in LF3 from different cell types, the size (102 bp) of the repetitive element is invariant (11, 23, 30, 43). There is, however, a single report of a 103-bp repeat (in AG876 B cells), involving expansion of a frequently observed C6 sequence to C7 (5), which alters the coding frame. The question of internal repeat variability could be addressed in detail were it possible to sequence across the repetitive elements, but this has to date defeated all attempts both by us and by others. This is probably due to the GC-rich (85%) repetitive element; in the LF3 transcript, this folds into a predictably highly stable secondary structure and behaves like a double-stranded RNA in enzymatic digests (12).
To explore this question, we adopted a cloning methodology previously used to search for DNA fidelity during replication (35). For this, we used cellular DNA from chemically induced or uninduced Daudi-ICRF cells as the work of others suggested that protein synthesis might require chemical inducing agents (29). Daudi-ICRF cells were chosen because preliminary data suggested that a protein could be produced in this cell line (44), although none had been predicted from simple sequence data (Table 1 and Fig. 2), nor was one detectable in another Daudi line, Daudi-ATCC (11). Of relevance, Daudi lines need not be identical. Historically, whereas their precise origins remain obscure, their natural history allows variation (20); four separate lines were produced over time from the tumor patient Daudi prior to and after chemotherapeutic response, followed by tumor relapse.
In our present study, PCR primers were chosen that should produce products which, when cloned into suitable M13 vectors, would yield blue plaques from a 102-bp repeat unit and white plaques from 101- or 103-bp repeats. For amplification, the Extensor Hi-Fidelity PCR enzyme (ABgene) was used because of its low error rate, in the range of 5 x 10-5 to 3 x 10-6 errors/base, depending on the nature of the template (information from the manufacturers). We obtained both blue and white plaques. Cloned DNA products were sequenced. Difficulties in designing suitable primers for PCR amplification of a complete repeat unit meant that amplification products were 90 and 91 bp, respectively. Only one variation was seen in the DNAs (Fig. 3). That is, whereas blue plaque isolates gave a product containing a C6 sequence from the repeat units, white plaque-derived materials had a C7 sequence. The ratio of C6 to C7 found in the Daudi 90- and 91-bp products was 7:1 in uninduced and 9:1 in chemically induced cells. This difference, probably not significant, implies that the frameshifts generally observed in LF3 are independent of chemical induction.
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Frameshifting is independent of the number of IR 4 repeats in the LF3 gene in different cells. For eukaryotic cells, it has been observed that when replication frameshifts involve reiterated sequences, the number of potential misaligned intermediates and correct base pairs that can stabilize such misalignments increases as the length of the run increases (21). To ask whether repeat copy numbers might correlate with the observed slippage patterns of LF3, the numbers of IR 4 repeats from different EBV strains was determined, with a technique described previously (30). In each case examined (see Table 1), except for uncloned P3HR-1, where two copy number variants (n = 21.7 and n = 25.7) were found, each cell line had its own repeat copy number. These ranged from 21.7 to 33.7, indicating a potential difference of up to 408 amino acids in LF3 proteins from different sources. (The 0.7 represents the 3'-most incomplete repeat, shown in Fig. 2.) Unlike reports from studies on microsatellites (reviewed in reference 39), where replication errors resulted in copy number variations, we saw no correlation between repeat numbers and sequence specified, as best illustrated by the African Burkitt's lymphoma biopsies. Thus, whereas repeat copy numbers may be of relevance, no biological significance was apparent in this study.
LF3 protein expression. Sequence variations might be of little consequence if LF3 were an untranslated gene whose function, if any, lay in its RNA (see reference 37 for discussion of this topic). However, as argument against this notion, not only is the primary transcript of LF3 polyadenylated, but proteins have been identified in several B-cell lines (29). Since determining frameshifts within all internal repeats is difficult by sequencing methodologies, protein structures of LF3 in individual cells or tumors cannot be predicted by such methods. Protein data may be highly relevant, however, to understanding the cellular selective pressures that lead to the frameshift mutational events. However, identification of proteins from the LF3 gene has also proved problematic. As noted earlier, for example, whereas in M-ABA cells one group (29) identified a protein, another (23) did not, and in our hands detection of proteins from a single cell type, such as M-ABA or P3HR-1, did not always give reproducible results (data not shown).
In the light of the gene sequence data (Table 1, Fig. 2 and 3), a polyclonal antibody generated from a recombinant clone that expressed 46 amino acids of the C-terminal part of the protein in M-ABA cells (N. Mueller-Lantzsch, personal communication), often used in experiments aimed at examining LF3 protein expression, may be suboptimal for protein assays in other cells. This antibody did, however, allow detection of a protein where none is predicted (Fig. 2) in the Burkitt's lymphoma-derived line BL74 (29) and in Daudi-ICRF cells (44), supporting further the notion of frameshifts within internal repeats.
We used the antibody to probe Western immunoblots of proteins extracted from several other cell lines and controls, with and without chemical induction, and from the C15 NPC xenograft. Full-length proteins of expected sizes (Fig. 4A) were identified in Daudi-ICRF and its mutant variant Daudi-100K, in M-ABA cells, and in the EBV producer lines P3HR-1 and BL74. For C15, although the repeat copy number of LF3 is at the lower end of the range (22.7 copies, Table 1), the discrete protein band observed was only half its expected size. No proteins of expected sizes were observed for Raji and M81 cells. Since chemical induction was an important factor in allowing a protein to be identified (29; our data) but appeared to have little effect on frameshifting, this finding may merely reflect the increased mRNA levels found in induced cells (11).
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To evaluate protein expression further, antibodies were raised to peptides (designated PP, PR, and PA, from the first unique dipeptide in different reading frames, see Fig. 2) from the three internal repeat elements. With these antibodies and induced Daudi-ICRF cells, specific immunoreactive polypeptides were observed with PR (Fig. 4B) or PP but not with PA (not shown), induction being required (as in Fig. 4A) to observe the protein. With P3HR-1 cells, the PP antibody recognized a full-length discrete protein and smaller species in cells with or without induction (Fig. 4B). The data are consistent with findings given in Fig. 2 and 3. From both cells the polypeptide bands were notably strong. That the degraded bands were not so sharp for P3HR-1 as they were for Daudi-ICRF may reflect the fact that with regard to numbers of repetitive sequences, P3HR-1 is a mixed population of cells (Table 1). Again, the data support the hypothesis of internal frameshifts within the LF3 gene. The polypeptide degeneracy in products from some sources, which cannot be explained by frameshift mutations, raises the possibility that in the highly structured RNA from LF3 (12) either slippages are taking place on the ribosome during translation or that, in some cases, specific protein degradation is occurring.
Immunostaining of cells for LF3 protein give data consistent with genetic diversity. To examine on a single cell basis whether chemical induction leads to large amounts of protein being generated in a small number of cells, or vice versa, immunostaining experiments were carried out. Such studies were also relevant for confirming the subcellular location of the LF3 translation product and whether it resides largely in the cytoplasm, as suggested by Western blot results, or might also be localized in the nucleus, in accordance with a role in DNA replication (13). For these studies, EBV-positive cell lines M-ABA, M81, and Burkitt's lymphoma-derived cell lines Raji, Daudi-ICRF, BL74, and P3HR-1 were used, both with and without induction, with Ramos (EBV negative) and B95-8 cells (with an LF3 deletion) as controls.
Data given in Fig. 5A show that whereas the low-virus-producer cell line M81 had appeared negative for expression of the LF3 protein on Western blots (Fig. 4A), on immunostaining following induction, small numbers of cells with low levels of fluorescence were observed. No expression was seen in Raji (or B95-8) cells. From immunostaining data, the LF3 protein appears in detectable amounts in a subpopulation of cells only (at most 10%), expression being generally enhanced upon chemical induction and more prominent in the cytoplasm than in the nucleus. An example of cytoplasmic (panel a) and nuclear (panel b) staining patterns in P3HR-1 cells is shown in Fig. 5B, where in the latter the stained cells appear to be in mitosis. Overall, the protein data accord with the genetic data.
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DISCUSSION |
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LF3 contains only a small amount of unique sequence, the bulk of its information residing in multiple copies (Table 1) of a mainly 102-bp GC-rich repeat, designed IR 4. It is found transcriptionally expressed in a variety of virally infected cell lines and tumors (8, 11, 12, 43, 45). Its protein product has been postulated to be associated with productive expression of EBV (29), and in our studies, without the use of chemical inducing agents, protein was detected only in a cell line (P3HR-1) that can spontaneously generate virus. The frameshifts observed occur at short runs of pyrimidines (mainly cytidines) found throughout the gene. Sequence data, as illustrated in Fig. 2, support the need for frameshifting events to occur in order to generate a protein in some cells, and molecular (Fig. 3) and immunofluorescence (Fig. 5) data suggest that this occurs in a minority of cells only (10% or fewer) in most isolates. Notably, most EBV-infected cells do not spontaneously produce virus. LF3 is probably not unique to EBV in that a gene similar to it has been predicted to exist in the recently sequenced rhesus lymphocryptovirus (34), and it is worth keeping an open mind with regard to the possibility that among the many repetitive sequences in the human genome, one or more might well have properties similar to those found for LF3.
In normal eukaryotic cells, although DNA replication fidelity is exceedingly high, not all of the enzyme components involved in replication have proofreading-associated functions. Structural elements that promote stable frameshifts have been defined in part as those, like LF3, which contain a high frequency of reiterated (including homopolymer) sequences and tandem repeats (6). For LF3 as well, its high GC content (85%) could make it vulnerable and subject during replication to aberrant mispairing or replication complex stuttering mechanisms that contribute to slippage events (21, 24, 41). Coupling these data with the fact that malfunctions of mismatch repair processes occur in tumors (18), with hindsight it is probably not surprising that, consistent with molecular hybridization and translational data (11, 43, 45), variation in the sequence of this viral gene in different cell isolates occurs. We propose that sequence frameshifts in LF3, having occurred initially at the DNA level in short runs of pyrimidines, reflect DNA polymerase slippage and its failure of correction, rather than internal recombination events - since it seems unlikely that the latter would be sufficiently promiscuous to allow for all the variables in sequence observed or predicted (Table 1 and Fig. 2) and account for the apparent genetic diversity observed. The occurrence of frameshifting events in a gene consisting of repeats usually divisible by three theoretically allows expression of a unique protein in virally infected cells from any particular source, depending upon the repeat copy numbers in the gene and the sites at which mutations occur.
There are other unusual aspects to this gene. Western blots earlier identified proteins from LF3 that varied between 115 to 140 kDa, depending (presumably) on its repeat copy number in an individual cell (29). Two other things were noted in this cited work that are relevant to our findings and support protein diversity. One was that only a small fraction of human serum samples (4 of 57) that were positive for EBV early and late antigens also recognized the recombinant LF3 protein from M-ABA cells. The other was that protein derived from a recombinant construction containing the whole LF3 gene, expressed in E. coli, was rapidly degraded; only a protein considerably reduced in size (by removal of most of the repetitive sequences), as seen in the C15 NPC tumor (Fig. 4A), proved stable.
Our Western blot data (Fig. 4) with a single antibody (29) point to another aspect of diversity, epitomized by specific degradation of the protein, with integrals of about 34 amino acids in some (but not all) cells. In M-ABA, P3HR-1, and Daudi-ICRF cells, ladders extending from a predicted full-sized peptide to much smaller peptides (<40 kDa) were observed, whereas the chemically modified Daudi-100K and BL74 cells produced mainly a full-sized protein and the NPC xenograft C15 gave a truncated (about half the predicted size) stable protein; with polypeptide antibodies made from different repeat sequences (see Materials and Methods and Fig. 2), cytoplasmic extracts from Daudi-ICRF and P3HR-1 cells gave similar ladders. Notably here, in line with sequence data, one polypeptide antibody proved more responsive in the first case and another in the second (Fig. 4B).
Although slippages on the ribosome during translation, such as postulated for some RNA and RNA viruses (31, 42, 46) but not reported for transcripts from DNA viruses, might account for the protein diversity, it seems an unlikely mechanism to explain the degradation products observed. From different cell sources, the transcription products of LF3 generally represent point mutations only, and their sequences are not apparently influenced by the repeat copy number in a particular cell. An alternative explanation for the polypeptide ladders seen may be suggested by sequences within the repeats themselves, in that two of the three LF3 coding frames contain rare aspartic acid-proline (DP) dipeptides (Fig. 2), known to be susceptible to acid degradation. In vitro, the conditions for this specific degradation are quite drastic, low pH and high temperature (33), and not commensurate with the data shown in Fig. 4. Thus, the observed degradations do not seem wholly explainable by pH changes during cell culture or isolate work-up. They might occur, however, if the proteins were targeted to lysosomes, where they would encounter not only sustained low-pH conditions but also numerous acid hydrolases, such as those suggested to be used for specific enzymatic degradation of a salivary gland protein (10).
There is no evidence of a lysosome-targeting signal in LF3 proteins, and at this stage in our work, although we have evidence for the cytosolic location of the majority of the LF3 protein expressed (Fig. 5), we cannot comment on its specific localization. Notably the degradative process has specificity (as predicted, Fig. 2) and is not equally effective on all LF3 proteins (see those from the chemically modified 100K protein from Daudi-ICRF cells or the tumor-derived line BL74). The protein in the C15 xenograft may also have largely evaded the degradative pathway by losing most of its repeats. In the rhesus lymphoproliferative virus (34), the LF3 homologue identified should also have DP sequences in two out of its three coding frames, raising interesting questions about an apparently conserved protein function between these viruses.
In the work presented here, we have examined LF3 not because of its putative role in lytic DNA replication, but with regard to its notable genetic diversity. There may be a correlation between these topics. LF3 is strategically placed to play an important, possibly initiating role in lytic replication (8, 11, 13), being located adjacent to one of the two EBV lytic origins of replication, both of which contain AT-rich palindromic elements and a large homopurine-homopyrimidine tract that could adopt a classical triple-helical hinge and single-stranded DNA regions (11). Such structures conform to those found in replication origins of other DNA viruses (7), where susceptibility to mutations, above background levels, has been reported to occur during replication (35). As elsewhere (41), they allow polymerase pauses to occur during DNA replication, with dissociation of the enzyme and ultimate induction of slippage.
Our findings raise questions about selective pressures that give rise to the diversities that define LF3, both with regard to the gene and its protein, where a 'tug-of-war' between the virus and its host may take place. Chemical inducing agents, well known to support lytic replication of EBV, can influence the outcome. The frameshifting events themselves may be a survival stratagem of the virus, allowing it to produce a potentially immunogenic protein in some hosts but to evade the immune system and proliferate in others, that is, generate a viral defense mechanism. Destroying the viral protein in the absence of an effective immune mechanism by targeting it to a pathway where it can be degraded may reflect a host defense mechanism (1). The phenomena observed with LF3 merit further investigation not just with regard to virus-host interactions, but also for the light they might shed on cellular repetitive elements, often relegated to the status of junk DNA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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