Affiliations of authors: M. I. Gutiérrez, M. M. Ibrahim, King Fahad National Centre for Childrens Cancer and Research (KFNCCC&R), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; J. K. Dale, S. E. Straus, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; T. C. Greiner, Department of Pathology, University of Nebraska, Omaha; K. Bhatia, KFNCCC&R, King Faisal Specialist Hospital and Research Centre, and Department of Pathology, University of Nebraska.
Correspondence to: Kishor Bhatia, Ph.D., P.O. Box 3354, MBC 98-16, Riyadh, 11211 Saudi Arabia (e-mail: Kishor_Bhatia{at}kfshrc.edu.sa).
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ABSTRACT |
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INTRODUCTION |
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In the latent cycle, the replication of the viral DNA is coupled to the replication of the host genome without production of infective viral particles. Reactivation (i.e., a switch from latency to a productive lytic cycle of EBV) is frequently seen in immunocompromised individuals (5), suggesting that the tight control over latency is, at least in part, dictated by host factors, possibly including immune surveillance mechanisms. However, because latently infected B cells grown in vitro do not spontaneously undergo lytic replication, it is likely that the virus itself may exert some control over the switch from latency to lytic replication. Indeed, one potent regulator of the switch from latency to productive infection (i.e., lytic replication) is the protein encoded by the EBV BamHI fragment Z (BZLF1, ZEBRA, or Zta) (6,7). Although the specific in vivo signals that induce the switch from latency to lytic infection are unclear, BZLF1 expression is stringently regulated by several regulatory domains within its promoter (8,9). A wide variety of signals, including phorbol esters (e.g., 12-O-tetradecanoylphorbol 13-acetate [TPA]), calcium ionophores, transforming growth factor (TGF)-1, and anti-immunoglobulin, trigger expression of BZLF1 in vitro, leading to the lytic cascade of events (1013). Many of these signals regulate BZLF1 expression through the 220-base-pair (bp) fragment immediately upstream of the transcription initiation site.
An additional factor known to influence the efficiency of the shift between latency and lytic replication is the EBV type. EBV types, termed A and B, are distinguished by signatory nucleotide changes in specific EBV genes, including EBV nuclear antigens (EBNAs) 2, 3A, 3B, 3C, and Epstein-Barr-encoded small RNA (EBER) (14,15). Both EBV types have been detected in immunocompromised and immunocompetent hosts (16,17). Type B EBV enters the lytic cycle more readily than type A EBV (18). Moreover, different EBV isolates respond with different efficiencies to exogenous inducers of reactivation. For example, anti-immunoglobulin treatment more efficiently induces BZLF1 in the EBV strain Akata than in the EBV strain B95.8 (6,19). Although both of these strains are type A viruses, they differ in the DNA sequences of the promoter regulatory and coding regions of BZLF1 (2022). Because BZLF1 is necessary and sufficient to mediate the switch from latency to the lytic cycle, it is possible that differences in the lytic potential of different EBV isolates may reflect the presence of different BZLF1 regulatory and/or coding sequences. We therefore asked whether the sequences of the major promoter regulatory elements (Zp) of the BZLF1 gene differ among type A and B EBVs in tumors and nontumor tissues.
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MATERIALS AND METHODS |
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We obtained DNA from a total of 52 tumor samples. DNA was available from 15 EBV-associated Burkitts lymphomas that were previously characterized for the presence of EBV and c-myc translocations (23). DNA was also available from 17 non-Hodgkins lymphomas, five nasopharyngeal carcinomas, two T-cell lymphomas, and 13 specimens from post-transplant lymphoproliferative disorders. These specimens have been described in previous studies (2427). Thirty-seven of the 52 tumors originated from North and South America, seven from Africa, and eight from Far East Asia (Japan and Hong Kong).
DNA was isolated, by using a standard phenol/chloroform extraction technique after proteinase K digestion, from peripheral blood lymphocytes of healthy individuals from North and South America (n = 21), Japan (n = 6), and Saudi Arabia (n = 13) and from peripheral blood lymphocytes of patients with acute infectious mononucleosis (IM) (n = 6). DNA was also isolated from peripheral blood lymphocytes of seven patients with chronic active EBV infection. Seventeen samples from the seven patients with chronic active EBV were obtained at different times (16 years after diagnosis) during the course of the disease. Research protocols for studies involving the patients with chronic active EBV were approved by the National Institute of Allergy and Infectious Diseases (Bethesda, MD). All research subjects who provided peripheral blood lymphocytes for the study gave informed consent. Because the EBV strain B95.8 is often used as a reference, we included this as the EBV prototype control.
Polymerase Chain ReactionSingle-Strand Conformation Polymorphism and DNA Sequence Analyses
Five hundred nanograms of genomic DNA obtained from all tumor and nontumor samples was directly used as a template in polymerase chain reactions (PCRs). PCR was used to amplify the fragment from nucleotides 221 to +12 (with respect to the transcription start site) of the BZLF1 promoter (nucleotides 103420103182 of the EBV genome, Genbank accession no. NC_001345). The primers used were 5'-agcatgccatgcatatttc-3' and 5'-ttggcaaggtgcaatgttt-3'. PCR conditions consisted of 5 minutes at 95 °C, 30 cycles of 30 seconds at 95 °C, 30 seconds at 60 °C, and 1 minute at 72 °C, followed by a long extension of 10 minutes at 72 °C. [32P]dCTP was included in the PCR buffer. PCR products were separated by electrophoresis through 6% nondenaturing acrylamide gels (19 : 1, acrylamide to bis) containing 10% glycerol at 6W for 18 hours and visualized by autoradiography. Autoradiograms were exposed for 216 hours. Single-strand conformation polymorphisms (SSCPs) were apparent by differences in the patterns of migration of the PCR product. Amplified product obtained from the EBV strain B95.8 served as a reference control.
Five hundred nanograms of genomic DNA from samples that showed distinct SSCP patterns were then amplified in a separate, nonradioactive PCR. These PCR products were directly sequenced either manually (28) or with the automated MegaBACE 1000 Sequencer (Molecular Dynamics, Piscataway, NJ). At least two independent reactions with two different sequencing primers (sense and antisense) were carried out for each PCR product to confirm the sequences. Detected sequence variations are reported at their base position location relative to the transcription start site of the BZLF1 gene.
EBV Typing
Type A and B EBVs were determined from genomic DNA of tumor and chronic active EBV samples by PCR with primers for the EBNA-3A and EBNA-3C genes as described (29). PCR with EBNA-3A primers yields an amplification product of 276 bp for type A EBV or 237 bp for type B EBV. PCR with EBNA-3C primers yields an amplification product of 153 bp for type A EBV and 246 bp for type B EBV.
Statistical Analysis
The association of each polymorphic Zp (promoter element of BZLF1) with type A and B EBVs and the distribution of the variants in different cell types (i.e., malignant and nonmalignant) were analyzed by using the Fishers exact test (SPSS 11.0; Chicago, IL). All statistical tests were two-sided.
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RESULTS |
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The purpose of this study was to determine whether discriminatory alterations could be identified in the regulation of the lytic pathway in type A and B EBVs and/or EBV associated with tumor and nontumor tissues. PCRSSCP analyses were initially used to identify whether sequence variations existed within the major regulatory Zp domains (nucleotides 221 to +12, with respect to the initiation of transcription of BZLF1). We first analyzed 37 samples obtained from North and South America; 20 carried type A EBV and 17 carried type B EBV. Of the 37 samples, seven were obtained from cell lines derived from non-Hodgkins lymphomas, 17 were obtained from primary non-Hodgkins lymphoma biopsy specimens, and 13 were obtained from post-transplant lymphoproliferative disorder biopsy specimens.
PCRSSCP analyses demonstrated two distinct patterns of migration (Fig. 1, A). Amplified products of EBV Zp from several tumor samples migrated faster than the amplified Zp product from EBV strain B95.8 (Fig. 1, A
; compare lane 1 with lanes 2, 4, 6, and 8). Of the 37 samples analyzed, 20 demonstrated a pattern similar to that of EBV strain B95.8, and 17 showed an alternate pattern of migration. For further sequence analyses, we chose DNA from 12 samples with the EBV strain B95.8 migration pattern and from eight samples with the alternate pattern. Direct sequencing of independently amplified products from these 20 samples confirmed the presence of discrete sequence variations (Fig. 1, B
). The sequences of the amplified products with SSCP patterns similar to that of B95.8 were identical to that found in EBV strain B98.5 and, therefore, were designated as the prototype promoter (Zp-P). Sequences of the samples with an altered SSCP pattern differed from that of Zp-P at three positions: T to G at position 100 (T 100
G), A to G at position 106 (A106
G), and A to G at position 141 (A141
G). This sequence containing all three substitutions was designated promoter variant 3 (Zp-V3). It is interesting to note that two of the three substitutions occurred within known functional domains of the BZLF1 promoter. The A106
G substitution occurred in the ZIIIB domain, a region that strongly binds BZLF1 (9), and the A141
G substitution occurred in the ZIC domain, a region that binds the nuclear regulatory factor SP-1 (8,10) and contributes to the ability of Zp to respond to TPA.
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Because geographic variations in EBV genome sequences exist, we analyzed seven EBV-containing lymphoma specimens from Africa and eight lymphoma and carcinoma tissues from Far East Asia (Japan and Hong Kong) by PCRSSCP. Direct sequencing of five of the specimens identified the same variants as those observed in the specimens from American subjects (data not shown). All samples from Africa (n = 2) and Far East Asia (n = 1) containing type B EBV carried Zp-V3 (Table 1). All samples from Africa containing type A EBV (n = 5) carried the Zp-P sequence, whereas all samples from Far East Asia (n = 7) containing type A EBV carried Zp-P (n = 2) or Zp-V3 (n = 5) (Table 1
). The data suggest that, regardless of its geographic origin, type B EBV carries the BZLF1 promoter Zp-V3. However, there is some degree of geographic heterogeneity for type A EBV, which carries Zp-P or Zp-V3.
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We next determined whether differences in the BZLF1 promoter sequence could also be detected in EBV isolated from patients with nonmalignant disorders, such as infectious mononucleosis or chronic active EBV infection. All samples were from the United States. The PCRSSCP Zp migration patterns of EBV isolated from patients with nonmalignant disorders (Fig. 2, A) and of EBV isolated from tumor samples (Fig. 1, A
) differed. Twelve DNAs were analyzed by DNA sequencing, which identified a variant promoter with four single-base substitutions. This variant promoter was designated Zp-V4. Three substitutions were identical to those detected in Zp-V3, and the fourth was a T
C substitution at position 196 (Fig. 2, B
). This substitution was located in the ZIA domain, which contains SP1 binding elements and is also the domain necessary for anti-immunoglobulin-mediated response (8,10,13). Five samples (four from two patients with chronic active EBV infection and one from a patient with infectious mononucleosis) of the 23 samples analyzed contained both Zp-V4 and Zp-P as evidenced by the mixed pattern of migration in the SSCP gel (see Fig. 2, A
, lanes 9, 11, and 15) and confirmed by the presence of heterogeneous sequences (P+V4 in Fig. 2, B
).
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To further determine which of the Zp sequences is present in healthy seropositive individuals, we analyzed peripheral blood lymphocytes from 21 individuals from North and South America, 13 individuals from Saudi Arabia, and six individuals from Japan by PCRSSCP. Twenty-one of these samples were also sequenced (data not shown). All samples, regardless of their geographic origin, carried the Zp-V4 sequence; two samples (5%) also carried the Zp-P sequence.
Table 2 summarizes the distribution of Zp sequences in all of the samples examined in our study. All of the 53 non-tumor samples carried Zp-V4, but none of the 52 tumor samples from three diverse geographic regions carried the Zp-V4 sequence (P<.001), suggesting that Zp-V4 is not associated with EBV in malignant cells.
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DISCUSSION |
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Because the lytic transactivator protein BZLF1 is necessary and sufficient to induce the lytic cycle (6,7), differences in this protein could help modulate the responsiveness to auto-reactivation signals or to other inducers of the lytic cycle. However, the reported sequence variations in the BZLF1 gene occur independently of EBV type (2022) and, hence, did not seem sufficient to account for the efficiency of the lytic cycle in type B EBV compared with type A EBV. Thus, we hypothesized that additional sequence variations in the promoter of BZLF1 could exist. We focused our studies on the 221-bp region designated Zp that encompasses the binding sites for the transcription factors SP-1, AP-1, and ZRE and that is primarily responsible for regulating BZLF1 transcription (813). We determined the sequence of Zp in 52 EBV-positive tumors that were collected from North and South America, Africa, and Far East Asia, because the distribution of polymorphic variants of EBV is influenced by geography. Our results demonstrate that polymorphisms in Zp (Fig. 1 and Table 1
) are strongly associated with the EBV type (Table 1
). Thus, tumors from North and South America, Africa, and Far East Asia containing type B EBV genomes are associated with Zp-V3. Similarly, tumors from the same geographic regions containing type A EBV genomes are associated with Zp-P, with the exception of type A EBV genomes from Far East Asia. Although some geographic dependence of these polymorphisms is evident, the association between EBV type and promoter polymorphism was statistically significant (P<.001).
It was surprising to determine that none of the Zp polymorphisms detected in tumor samples were strongly associated with nonmalignant EBV-associated diseases (chronic active EBV infection and infectious mononucleosis) or with nonsymptomatic EBV carriers. In fact, the most common Zp sequence detected in samples from patients and subjects with no evidence of malignant disease was a distinct variant, Zp-V4 (Fig. 2). Zp-V4 was consistently present, irrespective of the origin (North or South American, Arab, or Asian) or the EBV type. It is important to note that, although we did not detect the Zp-V3 sequence in the nonmalignant samples, it is possible that Zp-V3 could be present in levels below our limit of detection. By contrast, the absence of Zp-V4 in malignant cells, which cannot be attributed to the assay sensitivity because of the abundance of EBV DNA, was statistically significant (P<.001). These data suggest that if EBV containing Zp-V4 were more responsive to some physiologic signals, e.g., TGF-
1 or engagement of B-cell immunoglobulin receptor, it would be more likely to lyse the host cell and less likely to be found in malignant cells. Indeed, preliminary functional studies suggest that different Zp sequences vary in their ability to respond to known activators (TPA, calcium ionophore, and anti-immunoglobulin) (data not shown). In addition to the regulation conferred by Zp, negative regulatory elements that bind the cellular transcription factor YY1 exist upstream of the 221-bp Zp region (30,31), and may also vary among the EBV types, potentially modifying the responsiveness of each promoter.
Irrespective of the responsiveness of different BZLF1 promoter variants, this study provides evidence that the polymorphic BZLF1 promoter may be a marker for a viral subtype that does not associate with malignant cells.
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NOTES |
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Manuscript received July 1, 2002; revised September 3, 2002; accepted September 13, 2002.
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