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Epstein-Barr virus (EBV) has the potential to undergo latent and lytic pathways during infection. However, expression of many of the viral genes during the lytic-latent transition remains unclear. In this study, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydroxyurea (HU), two commonly used modulators of EBV life cycle, on the expression profiles of the entire genome of EBV persistent infected in B95-8 cells. After treatment with TPA for 48 h, the copy number of EBV genome in the cells increased about 2.5 fold, whereas HU treatment resulted in a reduction to approximately two-thirds of the original level. Except a small set of genes, the amounts of EBV mRNA are generally less abundant than that of beta-actin. The expression of a large fraction of the 80 EBV genes was found to be activated after TPA treatment with a noticeable increase of 19 and 21 fold, respectively in BSLF1 and BBLF4. In contrast, treatment of the B95-8 cells with HU, a nucleotide synthesis inhibitor, dramatically suppressed the expression of EBV lytic genes. In summary, we have demonstrated that real-time quantitative PCR is a reliable method to monitor the influence of drug-treatment in EBV genes regulation. Our results also provide a basis for further investigation on how the virus coordinates its own gene expression during latent-lytic pathway transition.

Since previous published studies about second malignant tumors (SMTs) in nasopharyngeal carcinoma (NPC) patients usually included a limited sample size and did not attain consistent results, we conducted a large retrospective study in a cohort of 1,549 patients to assess the risk of SMT in NPC patients following radiotherapy (RT) in Taiwan. The follow-up period ranged from 2 to 16 years, with a median of 7 years. Thirty-nine patients developed SMTs during the 7,145 person-year follow-up [standardized incidence ratio (SIR): 2. 8; 95% confidence interval (CI): 2.0 to 3.9]. Increased risks of developing SMTs were observed for head and neck (H/N) cancer (SIR: 16.5; 95% CI: 10.0 to 26.8), gastric cancer (SIR: 5.5; 95% CI: 2.2 to 11.4) and leukemia (SIR: 9; 95% CI: 1.9 to 26.3). Paraffin-embedded specimens of secondary H/N cancer (11), secondary gastric cancer (6) and their corresponding NPC specimens were examined by EBER in situ hybridization to assess the association between Epstein-Barr virus (EBV) and these SMTs. Twenty-six primary H/N and 5 gastric cancer specimens were chosen as the control groups. In H/N cancer, EBV was detected in 3.8% of the primary cancers and 9.1% of the secondary cancers. All the positive specimens resulted from hypopharyngeal cancer. Of the secondary gastric cancers, only 1 case (16.6%) was associated with EBV. None of the primary gastric cancers was associated with EBV. Our results indicate an increased risk of developing SMTs, with a preference for head and neck cancer, gastric cancer and leukemia, in NPC patients after RT in Taiwan. Only a small proportion of the secondary H/N and gastric cancers was associated with EBV., (Copyright 2000 Wiley-Liss, Inc.)

The ED-L1E promoter of the LMP 1 gene is a GC box-containing promoter. To test if Sp1/Sp3 are important for modulating ED-L1E promoter activity through the GC box, site-specific mutation and deletion constructs carrying a reporter gene were transfected into NPCTW076 and C33A cells. Results showed that deletion or mutation of the GC box abolished ED-L1E activity. Association of Sp1/Sp3 with the GC box was confirmed by electrophoretic mobility shift and supershift assays using Sp1- and Sp3-specific antibodies. Transfection of Sp1- and Sp3-expressing vectors into NPCTW076 and Sp-deficient Drosophila SL2 cells activated ED-L1E in a dose-dependent fashion and showed an additive effect. Data suggest that both factors may function as transcriptional activators and regulate the ED-L1E promoter in human epithelial cells., (Copyright 1999 Academic Press.)

Background: Nasopharyngeal carcinoma (NPC) is a common head and neck cancer in Taiwan. Early detection is the best way to improve survival for this disease. A prospective study was designed to assess the feasibility of a new screening method for NPC by use of polymerase chain reaction (PCR) based on the close association of NPC and Epstein-Barr virus (EBV)., Methods: One hundred thirty-three different tissues from nasopharynx, nose, and sinus were investigated by use of PCR to check for the presence of EBV genome. Subsequently, from April 1996 to March 1997, 55 patients were enrolled in a prospective screening study. All patients underwent nasopharyngeal swabs before biopsy. Polymerase chain reaction detection of EBV genome was conducted on swab samples. Anti-EBV viral capsid antigen (VCA) in IgA and IgG class were checked at the same visit., Results: The EBV genome was present in 91.4% (85/93) of NPC tissues and in 25.0% (10/40) of non-NPC tissues (p < .001, chi2 test). Of the 55 swabs, 49 (89.1%) specimens obtained enough cells for PCR examination. Thirty of these 49 patients were pathologically proved NPC. The presence of EBV were 86.7% (26/30) in NPC group and 42.1% (8/19) in non-NPC group. The sensitivity and specificity were 86.7% and 57.9%, respectively, which were similar to those of serologic method (87.5% and 43.5%) when the cut-off point was set at anti-VCA IgG > or = 160 and IgA > or = 10. Combining both methods elevates the specificity to 84.2%., Conclusions: The sensitivity of this PCR screening method is similar to that of the serologic method. Combining both methods can greatly increase the specificity, indicating this new method is a good supplement to the serologic screening of this endemic disease.

The location of the BcLF1 promoter of Epstein-Barr virus (EBV) has been identified by primer extension, which indicates that the +1 site of the BcLF1 mRNA is located at nucleotide 137676 of the EBV genome. According to deletion analysis, the region upstream from nucleotide -38 is not essential for transcription of BcLF1. A 23 bp region in the promoter, from nucleotide -38 to -16, was identified as necessary for regulating the expression of BcLF1, i.e. the promoter activity is activated by 12-O-tetradecanoylphorbol 13-acetate but is repressed by phosphonoacetic acid. The results presented also demonstrate that the oriLyt sequence in cis is essential for enhancing the expression of BcLF1.

The BZLF1 gene of Epstein-Barr virus (EBV), which encodes a transcription factor, Zta, is transcribed into monocistronic and bicistronic mRNAs from two different promoters during the immediate-early stage of the EBV lytic cycle. It is generally accepted that the Zta protein translated from the monocistronic mRNA profoundly influences the activation of the EBV lytic cycle. In this study, we constructed a plasmid, pCMV-RZLUC, which can transcribe a bicistronic mRNA consisting of BRLF1 and a BZLF1-luc fusion gene under latent conditions. P3HR1 cells transfected with this plasmid produce a luciferase activity which is approximately 17-fold higher than the activity exhibited by pRZLUC, a plasmid incapable of transcribing the bicistronic mRNA. Genetic analyses indicated that mutations in BRLF1 not only can decrease the translation of the fusion gene from the bicistronic mRNA but can also be complemented by a functional BRLF1 gene in cis. This observation implies that the product of BRLF1, Rta, is involved in the translation of the downstream gene. Results presented herein also demonstrate that these mutations cannot be complemented in trans with a plasmid overexpressing Rta, suggesting that the amount of Rta in the vicinity of the intercistronic region may be crucial for the translation. Furthermore, our results correspond to those of previous investigations indicating that the Zta protein can be translated from the bicistronic mRNA and that, similar to the translation of bicistronic ZLUC, mutations in BRLF1 also hinder the translation of Zta from the BRLF1-BZLF1 bicistronic mRNA. Translation of Zta from the bicistronic mRNA may play an essential role in the activation of the EBV lytic cycle.

Previously, we reported that the LMP 1 gene of Epstein-Barr virus (EBV) derived from nasopharyngeal carcinoma (NPC) tissues (i.e., NLMP 1 gene) was able to transform BALB/c3T3 cells. On the other hand, LMP 1 gene of B95-8 strain (i.e., BLMP 1 gene) was not able to transform these cells (Chen et aL, 1992). Further studies indicated that a 10-amino-acid deletion in the carboxyl terminus of NLMP 1 played an important role in transformation (Li et al., 1996). In this study, we tested if this 10-amino-acid deletion affected the induction of NF-kappaB activity by LMP 1. The long terminal repeat of the human immunodeficiency virus type 1 (HIV-1 LTR) contained two copies of NF-kappaB sites and was used to construct the Luc gene-based reporter plasmid, p kappaB-Luc. Plasmid p kappaB-Luc was co-transfected with plasmids containing the NLMP 1 gene, BLMP 1 gene, and their chimeric or deletion constructs, respectively, into C-33A and BALB/c3T3 cells. The activation was then measured by the luciferase activity. Results showed that the full-length proteins induced a similar level of NF-kappaB activity, the two 3' mutants (R15delta and D4delta) still induced a relatively high level of activity, and the two 5' deletion mutants (delta3058 and delta3243) of NLMP 1 gene did not show any significant activation in C-33A cells. However, none of these LMP 1 proteins induced NF-kappaB activity in BALB/c3T3 cells. Using subcellular fractionation analysis and an immunocytostaining method, the truncated proteins of delta3058 and delta3243 were detected in the cytoplasm of the cells whereas the full-length NLMP 1 protein was located at the cytoplasmic membrane. Stable BALB/c3T3 cell clones that expressed both truncated proteins were established and then their ability to induce tumors in nude mice was examined. Data showed that both truncated NLMP 1 proteins still maintained partial transformation activity. Our results suggested that there was no direct correlation between NF-kappaB activation and transformation activity of LMP 1 in BALB/c3T3 cell transformation and that the amino-terminal membrane-spanning domain was important for maintaining both functions of LMP 1.

Latent membrane protein 1 (LMP 1) is one of two Epstein-Barr virus (EBV)-encoded proteins that expressed in nasopharyngeal carcinoma (NPC) cells. Previous studies showed that a 3.5-kb transcript of the LMP 1 gene, in addition to the 2.8-kb transcript, was detected in a B95-8-EBV-containing, nude mice-passaged NPC tumor, C15. This indicated that a transcript was initiated from a region 5' to the putative promoter, ED-L1. We have isolated an EBV variant from a NPC tissue, and this virus strain contained a more pathogenic LMP 1 gene. DNA sequence analysis of the 5'-upstream region showed distinct variations as compared to that of B95-8 strain. To test if the LMP 1 gene of the NPC strain also contained an upstream promoter, we generated a series of deletion plasmids encompassing positions -1,030 to +20 of the LMP 1 promoter and tested for their abilities to drive the expression of the reporter gene in human epithelial cell lines, C-33A and NPC-TW076. We found that the region between -643 and -496 contained a promoter activity that was approximately five-fold higher than the putative promoter, ED-L1. This region between -643 and -496 was designated as ED-L1E. C-33A cells containing the genomic clone pT7(E) or the clone that had deleted a 94-bp ED-L1 sequence (delta94) was used to determine the transcription initiation sites by RNase protection assay. Results showed that a transcription initiation site was located at nucleotide 170,099 ("A") of EBV genome. The transcript was expressed in NPC biopsies and in human primary normal epithelial cells transfected with pT7(E) and delta94, respectively, as examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Furthermore, the ED-L1E was not regulated by the EBV-encoded nuclear antigen 1-mediated transcriptional enhancer family of repeats (FR) in C-33A cells. Our results suggested that the ED-L1E was specifically activated in epithelial cells. The biological significance of the selective usage of the ED-L1E promoter was discussed.

A previous study has shown that the BNLF1 of Epstein-Barr virus (EBV), isolated from a nasopharyngeal carcinoma biopsy (BNLF1-1510), was able to transform Balb/3T3 cells. On the other hand, BNLF1 of a prototype virus B95-8 (BNLF1-958) was not transforming unless the gene was transcribed from a strong promoter. In this study, we have generated chimeric BNLF1 by exchanging the DNA fragments between BNLF1-1510 and BNLF1-958 and examined their expression and transformation ability in Balb/3T3 cells. Results showed that transformation of Balb/3T3 cells by BNLF1-1510 was not due to the excessive expression of the gene. Transfection of Balb/3T3 cells with chimeric BNLF1 showed that the genes with 3' 453 bp sequence of BNLF1-1510 were oncogenic to the cells. Study also revealed that changing the numbers of the 33 bp repeats in the 3' region of the two BNLF1s did not affect the transformation characteristics. On the other hand, deletion of a 30 bp sequence of BNLF1-958, which is absent in BNLF1-1510, changed the gene from non-oncogenic to oncogenic and insertion of this 30 bp sequence into BNLF1-1510 abolished the transformation ability. BNLF1 without this 30 bp sequence was also found in the tumours of other EBV-related neoplastic disease, suggesting that absence of this 30 bp sequence in BNLF1 may be associated with the oncogenesis of these diseases.

An EBV variant has been identified in NPC tissues in Taiwan. This EBV variant contains a point mutation in exon I of the LMP I gene. This mutation results in the loss of an XhoI site at nt 169,426, which is present in strain B95-8. In addition, this variant contains a 30-bp deletion in exon 3 of the gene. The recent demonstration of the prevalence of EBV-containing nasal and peripheral T-cell lymphoma in this region drove us to evaluate the presence of this NPC-EBV strain in 7 cases of T-cell lymphoma, as well as in 48 NPC tissues, 2 cases of Hodgkin's disease and I B-cell lymphoma. Four samples of normal lymph node tissue, 40 of normal nasopharynx tissue and 78 throat washings of healthy individuals were included for comparison. We used sequence-specific primers and the polymerase chain reaction (PCR) method to amplify LMP I gene fragments containing these variations. Mutations were then confirmed by restriction-enzyme digestion and the DNA sequencing analysis. Our results showed that 57 of 58 tumor-tissues samples were EBV-positive. Among them, 56, including 6 T-cell-lymphoma samples, belonged to the NPC strain. This strain of EBV was also present in 92% of EBV-positive normal nasopharynx tissues and in 84% of EBV-positive throat washings of the healthy individuals tested. These results suggest that the NPC-EBV strain is prominently present in Taiwan.

EBNA 1 is the only antigen expressed in both Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma cells. Previous studies showed that the mRNA of EBNA 1 in these two tumor tissues was initiated from a promoter located in the Bam HI F fragment (Fp) on the viral genome. Two regulatory elements located in the downstream Bam HI Q region include an EBNA 1 binding site and a positive regulatory region between the Fp and the EBNA1 binding site. This data strongly suggested that a cellular factor(s) may modulate the usage of the Fp. To locate the shortest responsible viral sequence, we constructed a series of luciferase gene and chloramphenicol acetyltransferase (CAT) gene plasmids that contained various portions of the Bam HI F/Q region. Plasmid DNA was then introduced into cells to examine the promoter activity of each construct. By this method, we identified a 186-bp fragment within the Bam HI Q region that possessed the highest activity. This promoter was designated as Qp and found to be orientation-dependent and down-regulated by EBNA 1 in both the type I BL cells and human epithelial cells. Furthermore, RNase protection assay showed that a transcription initiation site was located at nucleotide 62,416 of the EBV genome. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further confirmed that the transcript was initiated from the Qp, and not the Fp. Therefore, our data suggested that a novel promoter, Qp, located within the Bam HI Q existed for the EBNA 1 expression in the latently infected type 1 BL cells. The biological significance of the selection of the Qp needs further investigation.

Different mutations were generated in the diffused-form early antigen (EA-D) of Epstein-Barr virus (EBV) for study of the effects of these mutations in DNA binding, stimulating the activity of EBV-specific DNA polymerase (EBV-DP), and binding to monoclonal antibody R3. Results revealed that the N-terminal 303 amino acids were essential for DNA binding and were sufficient for the enhancement of the activity of EBV-specific DNA polymerase. Deletion study also showed that the region recognized by the R3 monoclonal antibody was located between aa 315 and aa 377. Our results failed to demonstrate the binding between EA-D and EBV-DP, using the proteins synthesized in vitro, suggesting that direct contact between the two proteins is not required for the EBV-DP activity in vitro. We have generated fusion between EA-D and DNA-binding domain of yeast GAL4 protein; however, this fusion protein was not able to transactivate the promoter containing UAS sequence in P3HR1 cells.

Sequence variations of the 5'-upstream region of latent membrane protein 1 (LMP-1) in two Epstein-Barr virus (EBV) strains have been reported before (Chen et al., 1992). To investigate the effect of these variations on gene expression, we constructed a series of deletion plasmids encompassing positions -950 to +20 of the LMP-1 promoter region and tested for the ability to drive chloramphenicol acetyltransferase (CAT) gene expression in C33A cells. Results showed that the promoter activities of constructs from NPC strain were 3-fold lower than the corresponding constructs from the B95-8 strain. In addition, the region between -54 and +20 contained the basic, constitutive promoter activity for both strains. Sequence analysis of this region indicated that an activating transcription factor (ATF) binding site, TGACGTAG, which is present in B95-8 strain was changed to TCTCGTAG in NPC strain. A chimeric plasmid study suggested that these sequence variations in the ATF binding site may contribute to the 3-fold increase of CAT activity observed for B95-8 strain. Furthermore, the activity of the promoter constructs was not activated by EBV-encoded nuclear antigen 2 (EBNA-2) in C33A cells. However, the promoter activities were upregulated in B-lymphocyte cells such as CG3 and CA46 cells. The biological significance of this difference in promoter activity of LMP-1 gene between two strains and the involvement of the cellular factors were discussed.

The latent membrane protein 1 (LMP 1) gene of an Epstein-Barr virus (EBV) variant derived from an nasopharyngeal carcinoma (NPC) biopsy in Taiwan was isolated and characterized (Chen et al., 1992). Transient expression of the genomic sequence containing this gene showed two proteins with molecular masses of 62 kD and 50 kD, respectively, recognized by LMP-specific antibody S12. To determine if these two proteins were derived from independent promoters, we generated a series of mutant plasmids from plasmid pT7(E) that contained the upstream and the coding regions of the LMP 1 gene. These mutants were introduced into a human epithelial cell line, C33A, and LMP 1 proteins were examined by Western blotting analysis with the S12 antibody. Data showed that plasmid with a fragment containing approximately 3 kb of upstream sequence of LMP 1 gene produced the 62-kD protein. Removal of 2.7 kb of the upstream sequence (plasmid delta 2710, deleted to nucleotide 169,571) resulted in the production of both 62-kD and 50-kD proteins. This suggested that the upstream sequence interfered with the production of the 50-kD protein. Plasmid DNA deleted to Acc I site (nucleotide 169,223) generated only the 50-kD protein, designated as tr-LMP. Further deletion to nucleotide 169,038 resulted in the expression of another smaller LMP1 (49 kD, named as str-LMP1). The region between nucleotides 168,789 and 169,038 was tested to see if it possessed a promoter activity by using the luciferase gene as a reporter. Data showed that this region contained promoter activity with a level compatible to the previously reported ED-L1A promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of Epstein-Barr virus (EBV) DNA and human papillomavirus (HPV) DNA in 74 head and neck tumor tissues was examined by the polymerase chain reaction and DNA sequencing analysis. EBV DNA sequence was detected in all 30 nasopharyngeal-carcinoma tissue samples and in 30 of 44 other head and neck tumor samples. HPV DNA sequence was detected in 14 of 30 nasopharyngeal-carcinoma tissue samples and in 11 of 44 other tumor samples. Coinfection of both viruses was observed in 14 nasopharyngeal-carcinoma tissue samples but only in 5 other head and neck tumor samples including 3 hypopharyngeal-carcinoma tissue samples. Our data indicate that EBV is closely associated with nasopharyngeal- carcinoma and may also be related to hypopharyngeal-carcinoma. In addition, a relatively high percentage of EBV-positive nasopharyngeal- and hypopharyngeal-carcinoma tissue specimens contained HPV sequence. The significance of the coexistence of EBV and HPV in these tumor tissues requires further study.

A DNA fragment containing Epstein-Barr virus (EBV) terminal fragment sequence was obtained from a genomic library of nasopharyngeal carcinoma (NPC). One of the clones (clone 1510) contained the gene encoding latent membrane protein (LMP). Sequence analysis revealed that this gene had 95% homology with the LMP sequence of the B95-8 strain. Among the sequence variations, there was a change from G to T at nucleotide position 169,426, resulting in the loss of an XhoI site in exon 1 of the LMP gene. A pair of primers bracketing the XhoI site were designed to synthesize the EBV DNA fragment from nucleotides 169,081-169,577 by using the polymerase chain reaction (PCR) method. The PCR products were then subject to XhoI digestion and to DNA sequencing analysis. This restriction enzyme site polymorphism along with the sequence variations were also observed in 50 biopsy tissues as well as in the throat washings of 6 out of 20 healthy individuals that we examined, indicating that the EBV strain predominantly existing in these biopsy tissues was different from strains of B95-8, Jijoye or nude mouse passaged cells (C15) with an African origin, but closely resembled other nude mouse passaged CAO cells which were originally derived from China. Balb/c 3T3 cells carrying this NPC-LMP gene showed a transformed cell morphology and were tumorigenic in nude mice. The relationship between this unique type of EBV and NPC has yet to be established.

The subtypes of Epstein-Barr virus (EBV) according to the EBNA 2 gene were investigated in Taiwan by the polymerase chain reaction (PCR) and by Southern blot hybridization. The materials included 53 nasopharyngeal carcinoma (NPC) biopsies, 49 other head and neck cancers and 32 throat washings of normal individuals. EBV DNA was found in all NPC biopsies, 27 of 49 other head and neck carcinomas and 81% of normal individuals. Type A EBV was the predominant type of EBV in both normal individuals and patients with head and neck carcinomas in Taiwan. Type B EBV or coexistence of the A and B types comprised a small number of samples in this study.

The subtypes of Epstein-Barr virus (EBV) according to EBNA 2 genes were investigated in 53 nasopharyngeal carcinoma (NPC) biopsies in Taiwan by polymerase chain reaction (PCR) and by Southern blot hybridization method. EBV DNA was found present in all NPC biopsies. Type A was the predominant type and comprised of 94.3% of the EBV of NPC. Type B or coinfection of type A and type B comprised of only 3.8% and 1.9% respectively.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.

Two spontaneous outgrowing Epstein-Barr virus (EBV)-carrying lymphoblastoid cell lines (LCLs), CG2 and CG3, have been established from bone marrow cells of myeloid leukemia patients. CG2 was derived from a patient with chronic myelomonocytic leukemia (CMMoL) and who has a 45 XO karyotype. CG3 was derived from a patient with juvenile chronic myeloid leukemia (CML) and who carries a hypotetraploid karyotype, 91XXYY. Both CG2 and CG3 cells carry the same type of translocation; t(1;19)(q23;p13). Both cell lines are of an early B cell lineage as shown by their reactivities with monoclonal antibodies OKIa, B1, B2 and B4. The combination of horizontal discontinuous agarose slab gel and Southern hybridization results show CG2 and CG3 cells are of monoclonal origin and harbor episomal EBV genomes. Approximately 50 EBV genome equivalents were contained in CG2 and CG3 cells. Immunofluorescence studies demonstrate the expression of EBV-encoded antigen (EBNA) in almost all cells of these two lines. The expression of EA and VCA is only observed in a small percentage of cells and cannot be induced by treatment with TPA and SB. Therefore, CG2 and CG3 cells are probably nonproducer cell lines for EBV. The serum samples from both patients have been shown to contain elevated IgG antibody titers to EBV antigens. Both cells are found to be nontumorigenic in nude mice. These cells may provide an important tool in analyzing molecular epidemiological aspects of EBV infections in diseases such as CMMoL and juvenile CML.