Amino acid changes in functional domains of latent membrane protein 1 of Epstein–Barr virus in nasopharyngeal carcinoma of southern China and Taiwan: prevalence of an HLA A2-restricted ‘epitope-loss variant’

Jung-Chung Lin1, Jaw-Ming Cherng2, Hsiung-Ju Lin3, Chi-Wai Tsang4, Yi-Xi Liu5 and Steven P. Lee4

1 Department of Microbiology, Tzu Chi University School of Medicine, 701 Section 3, Chung Yang Road, Hualien 970, Taiwan, ROC
2 Department of Internal Medicine, Tzu Chi General Hospital, Hualien, Taiwan, ROC
3 Department of Pathology, Kaohsiung Medical University, China
4 CRC Institute for Cancer Studies, University of Birmingham, Birmingham, UK
5 Department of Virology, Cancer Research Institute, Chinese Academy of Medical Sciences, Beijing, China

Correspondence
Jung-Chung Lin
jclin{at}mail.tcu.edu.tw


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Full-length sequences of the Epstein–Barr virus (EBV) gene for latent membrane protein (LMP)-1 from 22 nasopharyngeal carcinoma (NPC) biopsy specimens and 18 non-neoplastic counterparts (NPI) were determined. Relative to the B95-8 strain, the amino acid sequences of the toxic-signal and transformation domains were changed variably in NPC and NPI specimens; in contrast, no change was observed in the NF-{kappa}B (nuclear factor {kappa}B) activation domain. HLA typing revealed that 47 % of NPC and 31 % of NPI specimens were HLA A2-positive. A major A2-restricted epitope within LMP-1 (residues 125–133) was analysed. At residue 126, a change of L->F was detected in 91 % (20/22) of NPC and 67 % (12/18) of NPI specimens. In addition, a deletion at residue 126 was detected in one NPC sample from Taiwan. At residue 129, a change of M->I was observed in all samples, regardless of whether they were NPC or NPI. The changes in this peptide between NPC and NPI specimens, including mutation and deletion, are statistically significant (P<0·05). A recent report indicated that this variant sequence is recognized poorly by epitope-specific T cells. Genotyping results indicated that 96 % of NPC and 67 % of NPI samples carried a type A virus. By scanning the entire sequence of LMP-1, eight distinct patterns were identified. Detailed examination of these patterns revealed that type A strains are more prevalent in NPC than in NPI specimens and are marked by the loss of an XhoI site, the presence of a 30 bp deletion and the presence of a mutated, A2-restricted, T cell target epitope sequence. These results suggest that an EBV strain carrying an HLA A2-restricted ‘epitope-loss variant’ of LMP-1 is prevalent in NPC in southern China and Taiwan.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Nasopharyngeal carcinoma (NPC) has unusually variable incidence rates around the world. High-risk areas are restricted to southern China, Hong Kong and south-east Asian countries, including Taiwan, Singapore, Malaysia, Indonesia and Vietnam (de Thé, 1982). Intermediate risk areas are confined to Alaska and north Africa, including Algeria, Morocco and Tunisia. However, the rest of the world, including the United States and Europe, comprises a low-risk area. Regardless of geographical origin, Epstein–Barr virus (EBV) is detected in all cells of undifferentiated NPC biopsy specimens. Apart from ethnic factors, which may influence the prevalence of EBV-associated tumours in different countries, EBV strain variation may also contribute to this geographical distribution of disease.

Multiple factors, including genetic and environmental factors and EBV infection, are believed to account for the marked racial differences and geographical variations in NPC (Raab-Traub, 1992). However, it is not known whether EBV-associated tumours reflect a particular sensitivity of affected patients or the existence of highly tumorigenic viral strains. Analyses of strain variation, based on restriction enzyme polymorphisms, have identified a predominant strain in NPC from southern China (Lung et al., 1990). Sequence analysis of the EBV BNLF-1 gene, isolated from a nude mouse-passaged Chinese NPC, identified a strain (CAO) that was marked by the loss of an XhoI site at the 5' end and a 30 bp deletion at the 3' end of the gene (Hu et al., 1991). This deletion (del-LMP-1) has since been found in another NPC-associated strain (1510) and, subsequently, in >90 % of strains that infect NPC in Taiwan and Hong Kong (Chen et al., 1992; Cheung et al., 1996).

LMP-1 is of special interest as it is generally considered to be the main EBV oncogene (Wang et al., 1985; Baichwal & Sugden, 1988). The unique structure of the LMP-1 protein suggests that the amino- and carboxy-terminal domains may interact with cellular proteins and, through these interactions, could affect cellular growth. LMP-1 can induce a variety of cellular genes that enhance cell survival (Henderson et al., 1991; Fries et al., 1996) and adhesive (Wang et al., 1990), invasive and angiogenic potential (Yoshizaki et al., 1998; Murono et al., 2001). It also interacts with and engages the TRAF (TNF receptor-associated factor) signal-transduction pathway, resulting in induction of EGFR (epidermal growth factor receptor) expression and activation of nuclear factor {kappa}B (NF-{kappa}B) (Hammarskjöld & Simurda, 1992; Miller et al., 1995; Mosialos et al., 1995).

Several functional domains have been mapped within LMP-1. Two regions within the C-terminal domain have been shown to initiate signalling processes: C-terminal activator region (CTAR)-1 (aa 194–232) and CTAR-2 (aa 332–386) (Huen et al., 1995; Mitchell & Sugden, 1995). A putative CTAR-3 domain (aa 275–330) was reported to bind Janus kinase 3 (JAK-3) (Gires et al., 1999).

Several components of the immune system contribute to the highly efficient control of virus replication and proliferation of immortalized, EBV-infected cells in healthy individuals. The best-characterized, and probably the most important, components are HLA-restricted specific CTLs that are directed against viral gene products of the latent state. Recently, HLA A2-restricted LMP-1 epitopes that are recognized by CTLs from healthy, EBV-seropositive individuals have been identified (Khanna et al., 1998). Because of the central role of LMP-1 in the development of EBV-associated tumours and its effects on cell growth, delineation of sequence variations of the functional domains and the A2-restricted CTL epitopes are relevant to understanding the biology and genetics of naturally occurring isolates.

To evaluate specific sequence variation within these functional domains and HLA A2-restricted epitopes, the complete sequence of the LMP-1 gene from EBV isolates from NPC biopsy specimens and NPI (non-neoplastic counterparts) was determined. Our results show that, relative to the B95-8 strain, three distinct features of sequence variation occurred simultaneously within the LMP-1 gene and were detected at a higher rate in NPC than in NPI specimens. These were: (i) loss of an XhoI cut site at the N-terminus; (ii) a 30 bp deletion at the C-terminus; and (iii) mutation in the HLA A2-restricted epitope sequence. By scanning the entire sequence of LMP-1, eight distinct patterns were identified. Detailed examination of these patterns revealed that a type A variant of EBV is more prevalent in NPC than in NPI specimens; it is marked by loss of the XhoI site and the presence of the 30 bp deletion and it always harbours the mutation in the A2-restricted epitope at amino acid 126.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Biopsy specimens.
In total, 22 NPC biopsy specimens were collected, of which nine originated from southern China and 13 from Taiwan. A portion of each specimen was submitted for histopathological diagnosis of NPC and the remainder was snap-frozen and kept at –80 °C for subsequent DNA analysis. For controls, 23 NPI specimens from patients with no evidence of NPC, but with clinical symptoms that were compatible with NPC, were obtained from the same anatomical site. These biopsy samples were subsequently diagnosed as chronic inflammation or inflammation and necrosis. All NPC biopsy specimens harboured EBV DNA, whereas EBV DNA was detected in 18 of 23 (78 %) of NPI samples, of which 10 were from southern China and eight from Taiwan.

Isolation of DNA.
Genomic DNA was prepared from NPC and NPI biopsy samples as described previously (Lin et al., 1993). Briefly, NPC and NPI samples were digested for 16 h at 55 °C in a buffer that contained 10 mM Tris/HCl (pH 8·0), 100 mM NaCl, 25 mM EDTA, 0·5 % Sarkosyl and proteinase K (0·1 mg ml–1). Samples were extracted twice with an equal volume of phenol/chloroform and dialysed against 10 mM Tris/HCl (pH 8·0) and 1 mM EDTA. RNase A (100 µg ml–1) was added to the dialysed sample and incubated at 37 °C for 2 h, followed by phenol/chloroform extraction and dialysis.

EBV genotyping.
The primers that were selected to amplify strain-specific variations in the EBNA-2 (EBV nuclear antigen 2), EBNA-3C and EBER (EBV-encoded RNA) gene loci have been reported previously (Lin et al., 1993). Conditions and parameters for amplification of EBV DNA sequences by PCR were as described by Lin et al. (1993).

Serology.
Enzyme immunoassays for quantitative determination of IgA and IgG antibodies against EBV viral capsid antigen (VCA) were performed by using an ELISA kit (Immuno-Biological Laboratories). Results are expressed as U ml–1 with a cut-off standard of 10 U ml–1, which represents an OD450 of approximately 0·35 in the ELISA test.

HLA typing.
Molecular typing of HLA by PCR was performed by using genomic DNA, as described by Gorski (1990). PCR was employed for amplification of the HLA-A and HLA-B gene loci. Primers used for HLA-A typing were: 5'-TGGCCCCTGGTACCCGT-3' and 5'-GAAAC(G,C)GCCTCTG(T,C)GGGGAGAAGCAA-3'. Primers used for HLA-B typing were: 5'-GGGTCCCAGTTCTAAAGTCCCCACG-3' and 5'-CCATCCCCGGCGACCTAT-3'. All PCR amplifications were set up in a ‘clean room’, using a laminar-flow hood to avoid contamination. Genomic DNA prepared from selected homozygous cell lines that were used in the 10th International Histocompatibility Workshop was used as a specificity control for DNA amplification and hybridization. Open-tube and negative-reagent controls were used to check for PCR contamination.

Sequence-specific oligonucletide probe hybridization.
Dot-blots were prepared on Hybond-N membranes (Amersham Biosciences) by using a stamped format as a grid for the transfer of PCR products. Group-specific probes (28-HLA-A and 38-HLA-B) were used to differentiate between HLA-A and HLA-B alleles (Gorski, 1990). Probes were labelled at the 3' end with non-radioactive digoxigenin-11-ddUTP, according to Genius system protocols (Boehringer Mannheim) and hybridized to the blots. Treated blots were exposed to Kodak X-OMAT AR film for 10 min to record chemiluminescent signal. Data analysis and allele assignments were computer-assisted.

PCR amplification and DNA sequencing.
The full-length LMP-1 gene was amplified by PCR using primers that contained an internal NcoI site at the 5' end and an additional XhoI site at the 3' end. Sequences of the primers were 5'-CTGACCATGGAACGCGACCTTG-3' (nucleotide coordination relative to B95-8, 169679–169658) and 5'-CTCGAGATAGTAGCTTAGCTGAA-3' (168166–168182). Approximately 100–300 ng genomic DNA was used in each reaction. PCR-amplified products were sequenced directly with an ABI Prism 377 DNA sequencer by using an ABI Prism BigDye Terminator cycle sequencing kit (Perkin-Elmer).

Identification of HLA A2-binding peptides within LMP-1.
A computer program that was designed to predict HLA-binding peptides (Parker et al., 1994) was employed to identify potential HLA A2-restricted epitopes within LMP-1. This program involves analysing the amino acid sequence, based on an estimation of the half-time dissociation of the HLA–peptide complex. Values greater than 1000 are normally considered to indicate significant epitopes in this analysis.

Statistical analysis.
Data are presented as mean±SD. Analysis of variance was used to determine statistically significant differences. Non-parametric data were compared by the Kruskal–Wallis test. P<0·05 was considered to be statistically significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pathological characteristics and serology
Table 1 shows pathological data and serology results. NPC has been classified into three types by the World Health Organization (WHO): squamous cell carcinoma (SCC; WHO type 1), non-keratinizing carcinoma (NKC; WHO type 2) and undifferentiated carcinoma (UDC; WHO type 3). In most large series of NPC samples, SCC is the least common type of NPC and none was found in this study. NKC represented 77 % of NPC samples analysed and the remaining 23 % were UDC. Among 22 NPC samples collected, male patients represented 73 % and patient age ranged from 20 to 60 years, with a mean of 43 years. Female patients represented 27 %, with a mean age of 48 years.


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Table 1. Pathological characteristics and serological study

 
Earlier studies demonstrated elevated titres of IgG and IgA to VCA in the serum of patients with NPC of WHO types 2 and 3. The ELISA results shown in this study (Table 1) demonstrate that NPC patients had markedly elevated titres of IgA and IgG against VCA, compared to NPI patients. Differences in titres of IgA and IgG between NPC and NPI are statistically significant (P<0·05). These anti-EBV serological profiles are characteristic of this pathology and confirm the histological diagnosis of NPC.

General sequence characteristics of the LMP-1 gene
Complete sequences of the LMP-1 gene were determined and compared with the sequence from the prototype B95-8 (type A strain) (Baer et al., 1984) and an NPC CAO strain (also type A). In addition, a type B strain (AG876) was sequenced in parallel. Sequence analyses revealed that the LMP-1 gene from NPC and NPI samples was approximately 99 % similar at the nucleotide level and 97 % similar at the amino acid level to the LMP-1 gene of the B95-8 strain. Although LMP-1 is highly conserved, it does contain a significant number of consistent nucleotide alterations. Of 81 nucleotide changes that were found in the LMP-1 ORF, 74 % (60/81) resulted in amino acid changes. Sequence analysis of the amino-terminal domain (aa 1–180) of LMP-1 from NPC and NPI samples revealed several distinct features. A unique polymorphism in the XhoI restriction site in exon 1 and a mutation in the HLA A2-restricted epitope sequence (aa 125–133) were observed in NPC samples. In the non-coding sequence in intron a region, there were three nucleotide changes at positions 169165 (T->G, 96 % NPC and 60 % NPI), 169181 (T->A, 96 % NPC and 60 % NPI) and 169183 (A->C, 96 % NPC and 60 % NPI). In the intron b region, three locations had nucleotide changes: positions 168998 (T->C, 71 % NPC and 60 % NPI), 169002 (C->T, 96 % NPC and 60 % NPI) and 169018 (A->C, 96 % NPC and 60 % NPI). In the long, cytoplasmic, carboxy-terminal region (aa 181–386) of LMP-1, four types of sequence variation were detected in both NPC and NPI samples. The LMP-1 gene from NPC and NPI samples had variable copy numbers (from three to seven copies) of the 11 aa repeat, compared to four and a half copies in the B95-8 strain. A 5 aa insertion (HDPLP) in the third 11 aa repeat sequence that disrupts the perfect repeat sequence was detected in the B95-8 strain, but was not present in any NPC or NPI sequence. The third variation was a deletion of aa 343–352 of the B95-8 LMP-1. The fourth variation comprised alterations in CTAR-1 and CTAR-2. These distinct features are analysed in detail below. DNA and amino acid sequences of 22 NPC and 18 NPI samples have been deposited in GenBank under accession numbers (AY601307AY601346).

XhoI polymorphic site
A unique polymorphism in an XhoI restriction site in exon 1 of the LMP-1 coding region of the B95-8 strain was detected previously in EBV isolated from Chinese and Taiwanese NPC (Hu et al., 1991; Chen et al., 1992). To determine whether the same change (G->T at 169425, which results in the loss of the XhoI cut site), was present consistently in both NPC and NPI samples, we examined DNA sequencing data from 22 NPC and 18 NPI tissue specimens. Loss of the XhoI cut site was detected in 96 % (21/22) of NPC and 67 % (12/18) of NPI specimens. Loss of the XhoI cut site can also occur at position 169423 (G->C), resulting in an amino acid change from Gly to Arg at codon 18 (Miller et al., 1994; Lin et al., 1995). However, this change was not detected in specimens used in the present study.

Analysis of HLA A2-restricted epitopes
To assess whether HLA A2-restricted epitopes were altered, DNA sequences of 12 potential HLA A2-binding peptides (aa 32–181) within LMP-1, identified by a computer-based program (Parker et al., 1994), were analysed. Those peptides with an estimated half-time dissociation score of 26 000–360 were selected and numbered accordingly (Table 2). A number of epitopes have recently been reported (Duraiswamy et al., 2003). Nucleotide and deduced amino acid sequences of peptides 1–12 were compared with those of the reference EBV strain (B95-8). DNA sequence changes that resulted in amino acid changes were detected in peptides 1, 2, 5, 8 and 11. Of particular interest is peptide 1, which was identified previously as the major HLA A2-restricted CTL epitope in LMP-1 (Khanna et al., 1998). Fig. 1a shows the different sequence patterns of peptide 1, with changes identified relative to the B95-8 (type A virus) prototype sequence.


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Table 2. Sequence variation in the HLA A2-restricted peptides of LMP-1

 


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Fig. 1. (a) Sequences of A2-restricted LMP-1 epitopes (aa 125–133) in EBV isolated from NPC and NPI samples. Nucleotide and amino acid changes relative to the B95-8 (type A strain) sequence are indicated. Nucleotide and amino acid changes of a type B strain (AG876) were also analysed. (b) Eight distinct patterns of amino acid change of LMP-1 that were identified from NPC and NPI isolates. The top line shows the amino acid sequence of the prototype B95-8 (Baer et al., 1984). The second line is from an NPC CAO strain (GenBank accession no. AF304432). AG876 (type B) was sequenced in parallel with NPC and NPI specimens. The genotype of the B95-8 and CAO strains is type A, based on the EBNA-2, EBNA-3C and EBER genes. Note that the A2-restricted epitope with a deletion at aa 126, which was found in a Taiwanese NPC sample (Table 5, NPC case 20), is included in pattern 3.

 
For comparison, the sequence of a prototype AG876 strain (type B virus) was also analysed. NPC-I represents the major form that varies from the B95-8 sequence and is found commonly in the majority of NPC specimens from both southern Chinese and Taiwanese patients, whereas NPC-II carries a deletion of the anchor residue of the epitope, a sequence change that is detected infrequently in Taiwanese NPC specimens.

The DNA sequence of peptide 1 displayed amino acid changes at two positions, 126 and 129 (positions 2 and 5); note that position 2 is an anchor residue that is essential for peptide binding to HLA A2 (Table 2). At aa 126 (position 2), Leu was substituted by Phe in approximately 67 % (12/18) of EBV isolates from NPI and 91 % (20/22) of isolates from NPC specimens. In addition, deletion at residue 126 was detected in one NPC sample from Taiwan. Thus, changes (including mutation and deletion) in peptide 1 between NPC and NPI samples are statistically significant (P<0·05). At aa 129 (position 5), a consistent change from ATG to ATT, resulting in an amino acid change from Met to Ile, was detected in 100 % of EBV isolates from both NPC and NPI samples.

In peptide 2 at residue 33, CTC (Leu)->ATC (Ile) was detected in 27 % (6/22) of NPC and 6 % (1/18) of NPI samples. Peptide 5 had a point mutation at residue 178, where CTG (Leu) was changed to ATG (Met) in 59 % (13/22) of NPC and 50 % (9/18) of NPI samples. It should be noted that peptide 8 overlapped with peptide 5, in which residue 178 was included. At residue 57 in peptide 11, TCC (Ser) was changed to GCC (Ala) in 32 % (7/22) of NPC and 6 % (1/18) of NPI samples. In summary, it would appear that only one change in peptide 1 is significantly different between NPC and NPI specimens.

The effect of sequence variations within the given peptide on predicted HLA A2 binding scores is shown in parentheses in Table 2. It should be noted that some changes in amino acid residue within the peptide do not affect HLA A2 binding scores.

Copy numbers of the 33 bp repeat in LMP-1
Previous reports have shown the presence of a varying copy of an 11 aa repeat between aa 250 and 308 in the carboxy-terminal domain of LMP-1. Upon examining DNA sequencing data, we found that the number of repeated sequences varied from a minimum of three copies to a maximum of seven copies. There was no correlation between copy numbers of repeat elements and disease status.

Sequence variations in NF-{kappa}B-activating domains
Two NF-{kappa}B-activating regions have been identified within CTAR-1 (aa 187–231) and CTAR-2 (aa 332–386) (Huen et al., 1995; Floettmann & Rowe, 1997; Izumi & Kieff, 1997; Eliopoulos et al., 1999; Izumi et al., 1999; Kieser et al., 1999). Deletion-mapping analysis has localized the major NF-{kappa}B-activating region of LMP-1 to critical residues 379, 380, 381, 383 and 384 (Floettmann & Rowe, 1997). DNA sequences within the codons for these amino acids were conserved both in NPC and NPI samples.

Transformation and toxic-signal domains
LMP-1 has transforming effects in continuous rodent fibroblasts. However, overexpression of LMP-1 results in toxicity. Both transforming (aa 364–386) and toxic-signal (aa 306–334) domains have been identified (Baichwal & Sugden, 1989). Within the transforming domain at position 366, a base change from T to A, resulting in the amino acid change Ser->Thr, was detected in 100 % of NPC and NPI samples (Table 3). The toxic-signal domain was changed variably in NPC and NPI samples (Table 3). The frequency of amino acid changes at positions 309 (Ser->Asn) and 334 (Gln->Arg) were similar in NPC and NPI samples. At position 322, Gln was substituted by Glu in 5 % (1/22) of NPC and 39 % (7/18) of NPI samples. In addition, at position 309, where Ser was changed to Asp in 6 % (1/18) of NPI samples, no change was detected in NPC samples. At position 324, Thr->Met was detected in 5 % (1/22) of NPC samples. It should be noted that the CTAR-3 domain overlaps with the toxic-signal domain.


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Table 3. Sequence variation of transformation and toxic-signal domains of LMP-1

 
Identification of eight distinct patterns in LMP-1
CTAR-2 encompasses the 10 aa deletion (343–352) of the CAO EBV strain. Analysis of CTAR-2 (aa 332–386) revealed eight distinct patterns, as shown in Fig. 1b. Relative to the B95-8 sequence, these nucleotide changes correspond to amino acid changes at positions 334 Gln->Arg, 335 Gly->Asp, 338 Leu->Ser or Leu->Pro and the deletion of 10 aa (343–352). The change of Leu->Ser at aa 338 was consistently associated with the deletion of 10 aa. Interestingly, this deletion was not detected in samples where the change of Leu->Pro at position 338 occurred.

Previous studies on defining EBV strains by LMP-1 were based on sequencing analysis data that were derived from either the amino- or carboxyl-terminus (Sandvej et al., 1997; Sung et al., 1998; Walling et al., 1999; Henry et al., 2001). To gain a complete picture of sequence variation, we have examined the entire LMP-1 sequence in detail and revealed that these eight distinct patterns coexist with other changes at the amino-terminus (residues 1–90), in addition to the HLA A2-restricted epitope region (residues 120–140) (see Fig. 1a and b). The feature at the amino-terminus (residues 1–90) is marked by amino acid changes at positions 3 His->Arg (96 % NPC and 67 % NPI), 17 Arg->Leu (96 % NPC and 67 % NPI), 25 Leu->Ile (96 % NPC and 67 % NPI), 82 Ala->Gly (96 % NPC and 67 % NPI), 84 Cys->Gly (96 % NPC and 67 % NPI) and 85 Ile->Leu (96 % NPC and 67 % NPI).

Frequency of occurrence of these eight distinct patterns and their genotypes is summarized in Table 4. Pattern 1 was detected in 36 % (8/22) of NPC and 28 % (5/18) of NPI samples; pattern 2, 18 % (4/22) NPC and 39 % (7/18) NPI; pattern 3, 14 % (3/22) NPC and none in NPI; pattern 4, 14 % (3/22) NPC and none in NPI; patterns 5, 6 and 7, 4·5 % (1/22) each of NPC and none in NPI; pattern 8, 4·5 % (1/22) NPC and 33 % (6/18) NPI. Patterns 3, 4, 5, 6 and 7 were only found in NPC samples. Interestingly, patterns 1–7 were associated with type A strains, whereas pattern 8 was found predominantly in NPI samples and harboured only type B strains, regardless of whether the sample was NPC or NPI.


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Table 4. Frequency of occurrence of LMP-1 variants and genotype

 
EBV genotyping
Genotyping of EBV was carried out by simultaneous analysis of three divergent gene loci (EBNA-2, EBNA-3C and EBER) (Lin et al., 1993). Both NPC and NPI biopsy samples were analysed in parallel with B95-8 (type A strain) and AG876 (type B strain) cells. The primers that were chosen to amplify the EBNA-2 and EBNA-3C genes encompass strain-specific deletions (Lin et al., 1993). A representative result is shown in Fig. 2. Amplification with EBNA-2 primers resulted in the expected 168 bp fragment in samples that contained type A viral strains and a 184 bp fragment from samples that harboured type B virus (Fig. 2a). Similarly, amplification with EBNA-3C primers resulted in the expected 153 bp fragment from B95-8 and a 246 bp fragment from AG876. The primers that were chosen to amplify EBER DNA encompass strain-specific point mutations, but do not contain strain-specific deletions (Lin et al., 1993). Therefore, strain specificity was assessed by single-strand conformation polymorphism (SSCP) analysis. Two distinct patterns of shift in mobility that were specific for either B95-8 or AG876 EBER DNA were the basis of differentiation of viral strains (Lin et al., 1993) (Fig. 2b). Table 5 summarizes the frequency of occurrence of LMP-1 variants and EBV genotypes.



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Fig. 2. Representative results of EBV genotyping. (a) Left top panel (EBNA-3C) and right top panel (EBNA-2): lanes 1–9 were from NPI cases 1–9 (see Table 5), respectively. Samples in lanes 10, 11 and 12 were B95-8 (type A), AG876 (type B) and water control, respectively. M, Molecular size markers. Left bottom panel (EBNA-3C) and right bottom panel (EBNA-2): lanes 1–9 were from NPC cases 1–9 (see Table 5), respectively. Samples in lanes 10, 11 and 12 were B95-8, AG876 and water control, respectively. (b) Representative result of PCR-SSCP analysis of the EBER region. Lanes 1 and 2 were AG876 and B95-8; lanes 3–8 were from NPI cases 1–3 and 5–7, respectively.

 

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Table 5. Summary of EBV genetic variations in NPC and NPI

 
Distribution of HLA A2 haplotype
To determine whether there was a haplotype that was associated predominantly with NPC patients, molecular typing of HLA was carried out with DNA samples from 19 NPC and 16 NPI specimens. The distribution of HLA A2 haplotypes is shown in Table 5. The results show that the frequency of A2 alleles was 47 % in NPC and 31 % in NPI patients (see Table 5).

The sequence variation profile of LMP-1 that is characteristic for EBV isolated from NPC and NPI patients is summarized in Table 5. These results, taken together, indicate clearly that, relative to the B95-8 sequence, a predominant variant of LMP-1 that was marked by a type A genome with loss of an XhoI restriction site, a 30 bp deletion and a mutated HLA A2-restricted epitope sequence was more prevalent in NPC than in NPI samples.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Despite the fact that EBV latent infections are common in populations worldwide, only a very small percentage of individuals will develop an EBV-associated malignancy. It is not known whether this situation reflects the existence of more sensitive individuals or of particularly tumorigenic EBV strains. It has been suggested that genetic changes, including the loss of an XhoI restriction site and a 30 bp deletion within the LMP-1 gene, define an EBV strain that is associated with increased tumorigenicity or with disease among particular geographical populations (Trivedi et al., 1994; Li et al., 1996). However, this notion has recently been challenged by the fact that virus with a deleted version of LMP-1 is present in the general population in endemic regions, but can also be found in non-endemic areas within Asia (Itakura et al., 1996) and in western countries (Sandvej et al., 1997). Moreover, it has been shown that the LMP-1 gene from non-endemic Russian NPC harbours a non-deleted version of LMP-1, whereas the gene with a deletion can be found in healthy subjects from the same area (Hahn et al., 2001). Therefore, we postulate that if highly tumorigenic EBV strains do exist, they would be found preferentially in consistently EBV-associated tumours, such as NPC, and differ significantly from the strains present in other, non-neoplastic counterparts of the same anatomical sites.

Widespread prevalence of LMP-1 sequence variations, particularly the XhoI polymorphism and the 30 bp deletion, indicates that they cannot be used as simple markers for oncogenic viruses that are related to particular forms of EBV-associated tumours. These findings, taken together, argue strongly against the notion that loss of the XhoI site and presence of the 30 bp deletion of LMP-1 alone predispose for NPC. Sequence variations in other parts of LMP-1, especially in regions that are related to its biological functions, need to be analysed in parallel. However, several of the structural changes detected occur at sites where they may affect biological functions of LMP-1. It is not clear which sites of changes are associated with loss of the XhoI site and which with the 30 bp deletion, or with both. Therefore, we examined the full-length LMP-1 sequence, focusing on known functional domains in general and the HLA A2-restricted epitope in particular. These analyses revealed eight distinct patterns or strains (Fig. 1b). A previous study, based on sequence variation and signature sequences, identified seven distinct strains (Edwards et al., 1999): the China 1, 2 and 3, the Mediterranean, the Alaskan, the NC and the B95-8 strains. The China 1 strain, which predominates in east Asia where NPC is endemic, is characterized by amino acid changes (relative to B95-8) at positions 212, 309, 322, 334, 338 and 366 and the deletion of aa 343–352, with the asparagine at position 322 being unique to this strain. The China 2 strain is marked by amino acid changes at positions 212, 245, 252, 309, 331, 338, 344 and 366, with the histidine at position 245 and the aspartic acids at positions 252 and 344 being unique to this strain. This strain was detected from both endemic and non-endemic isolates and was marked by the retention of aa 343–352. The China 3 strain had amino acid changes at positions 212, 338 and 366 and the deletion of aa 343–352. These data do not fit perfectly into any pattern or strain that we have identified in the present study, as the amino acid changes that encompass the HLA A2-restricted epitope were not analysed by these authors (Sung et al., 1998; Edwards et al., 1999). However, it is likely that China 2 can be assigned to our patterns 3, 4 or 8, based on the retention of aa 343–352. It is of interest to note that this 10 aa deletion is always preceded by a signature amino acid change at position 338 from leucine to serine, whereas that of leucine to proline results in retention of the 10 aa fragment.

Several of the samples analysed had amino acid changes that were characteristic of more than one strain. The 33 bp repeats in the carboxyl-terminus have been suggested as a site for recombination (Miller et al., 1994). Pattern 8 shares many of the AG876 cell line changes that are characteristic of type B strains in the amino-terminus (aa 1–140), but differs in the carboxyl-terminus by the retention of aa 343–352 (Fig. 1b), suggesting that it may arise from recombination. Similarly, patterns 3 and 4 (both type A) have amino acid changes in the carboxyl-terminus that resemble pattern 8 and are marked by retention of the 10 aa fragment, but differ in amino acid changes in the amino-terminus (Fig. 1b). Patterns 1, 2, 5, 6 and 7 are all characterized by identical changes in the HLA A2-restricted epitope and the 10 aa deletion, but differ slightly in their preceding signature amino acids.

An extensive study by Walling et al. (1999) arrived at 22 sequence patterns by analysis of the DNA sequence that encodes aa 196–378 of LMP-1. Mutations at positions 334 (Gln->Arg) and 338 (Leu->Ser or Leu->Pro), which were found frequently in our sequences, were also common in their isolates. The other isolates had some, but not all, of the amino acid changes of the patterns that were described in that publication. Incidentally, their pattern 2 (2a, 2b and 2c) was comparable with our pattern 1 in that, for example, where leucine was changed to serine at position 338, it was always followed by deletion of aa 343–352. Likewise, when leucine was changed to proline at position 338, it was always followed by retention of the 10 aa fragment.

Sequence analysis of the LMP-1 gene and promoter in EBV isolates from European individuals has defined four groups of LMP-1 variants, designated groups A, B, C and D (Sandvej et al., 1997). Each of these four groups is characterized by a specific pattern of amino acid mutations, relative to B95-8 LMP-1. However, none of these groups fits perfectly into the patterns of the CAO strain and our isolates. For instance, group C viruses are defined by the 30 bp deletion with an intact XhoI restriction site. Group D isolates are marked by loss of the XhoI site with retention of aa 343–352. Fielding et al. (2001) systematically analysed the signalling functions of LMP-1 by using a panel of well-defined LMP-1 variants, including representatives of the four defined groups (A–D) of European LMP-1 variants (Sandvej et al., 1997) and Chinese NPC-derived LMP-1. They reported that Chinese and group D variants activated the transcription factor NF-{kappa}B two- to threefold more efficiently than B95-8 LMP-1, whereas Chinese, group B and group D variants similarly activated activator protein 1 (AP-1) transcription more efficiently than did B95-8 LMP-1. However, there were no amino acid substitutions in the core binding regions for TNF receptor-associated adaptor proteins, which are known to mediate NF-{kappa}B and AP-1 activation. Whilst mutation of sequences in the PXQXT motif undoubtedly impairs both TRAF-binding and NF-{kappa}B-activating functions (Devergne et al., 1996; Sandberg et al., 1997), flanking sequences are clearly also important (Devergne et al., 1996; Eliopoulos et al., 1997; Sandberg et al., 1997). Sequence changes on either side of the PXQXT motif were observed at position 189, where Gln was substituted by Pro in 68 % of NPC and 67 % of NPI samples, at position 192 Ser->Thr in 91 % NPC and 67 % NPI and at position 212 Gly->Ser in 96 % NPC and 50 % NPI.

Our results demonstrate that EBV isolates that are present in Chinese and Taiwanese NPC patients carry a sequence that varies from that of B95-8 in the A2-restricted T-cell epitope sequence in LMP-1 (aa 125–133). A recent report demonstrated that this variant sequence is not recognized by epitope-specific T cells; this correlates with reduced binding of the variant peptide sequence to HLA A2 molecules (Duraiswamy et al., 2003). This finding is in accord with the predicted HLA A2 binding score, shown in Table 2. The prevalence of an HLA A2-restricted ‘epitope-loss variant’ of LMP-1 in highly A2-prevalent southern Chinese and Taiwanese populations may therefore contribute to immune escape by malignant cells that express LMP-1.

Prevalence of a specific MHC haplotype in a given population has been implicated to provide CTL-driven immune selection, contributing to the emergence of genetic variants of EBV. This was first suggested by the finding that virus isolates from highly HLA A11-positive populations, including the Chinese population, were mutated specifically in two immunodominant A11-restricted CTL epitopes (De Campos-Lima et al., 1993, 1994). These results suggested that EBV variants lacking the immunodominant A11-restricted epitopes have enjoyed a selective advantage in these highly A11-positive populations. In contrast to CTL responses that are specific for A11-restricted epitopes in EBV, the T-cell response to A2-restricted epitopes in LMP-1 is relatively weak (Khanna et al., 1998; Duraiswamy et al., 2003). Therefore, it is unlikely that immune pressure would account for the emergence of this epitope-loss variant. Nevertheless, in NPC cells where EBV gene expression is restricted to just EBNA-1, LMP-1 and LMP-2, immune control must target viral antigens that are subdominant for T-cell responses. In this context, malignant cells that carry an LMP-1 epitope-loss variant may have an increased ability to avoid host T-cell immune surveillance. Loss of this epitope also has important implications for the design of T cell-based therapies for NPC (Chapman et al., 2001).

The type A strain of EBV is the most common strain found in Asian, as well as western, populations. Type B virus is found in approximately 20 % of healthy virus carriers from Taiwan and China (J.-C. Lin, J.-M. Cherng and H.-J. Lin, unpublished results). Therefore, in this study, it is interesting to note that virtually all NPC isolates studied (96 %) were of type A, in contrast to only 67 % of NPI isolates (P<0·05). Given that only type A strains carry the epitope-loss sequence in LMP-1, it will be important to determine whether the predominance of this strain in NPC is the result of immune pressure and/or increased transforming potential of this viral subtype (Rickinson et al., 1987).


   ACKNOWLEDGEMENTS
 
We thank Professor Alan Rickinson for his critical reading of and comments on this manuscript and Drs N.-T. Lin and T.-D. Lee for their technical assistance. This work was supported in part by an Institutional Grant of Tzu Chi University, TCMRC 8608, and grants from the National Science Council of the Republic of China, NSC 88-2314-B-320-008 and NSC 88-2318-B-320-001-M51.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baer, R., Bankier, A. T., Biggin, M. D. & 9 other authors (1984). DNA sequence and expression of the B95-8 Epstein–Barr virus genome. Nature 310, 207–211.[Medline]

Baichwal, V. R. & Sugden, B. (1988). Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein–Barr virus. Oncogene 2, 461–467.[Medline]

Baichwal, V. R. & Sugden, B. (1989). The multiple membrane-spanning segments of the BNLF-1 oncogene from Epstein–Barr virus are required for transformation. Oncogene 4, 67–74.[Medline]

Chapman, A. L. N., Rickinson, A. B., Thomas, W. A., Jarrett, R. F., Crocker, J. & Lee, S. P. (2001). Epstein–Barr virus-specific cytotoxic T lymphocyte responses in the blood and tumor site of Hodgkin's disease patients. Implications for a T-cell-based therapy. Cancer Res 61, 6219–6226.[Abstract/Free Full Text]

Chen, M. L., Tsai, C. N., Liang, C. L., Shu, C. H., Huang, C. R., Sulitzeanu, D., Liu, S. T. & Chang, Y. S. (1992). Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein–Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population. Oncogene 7, 2131–2140.[Medline]

Cheung, S.-T., Lo, K.-W., Leung, S. F., Chan, W.-Y., Choi, P. H. K., Johnson, P. J., Lee, J. C. K. & Huang, D. P. (1996). Prevalence of LMP1 deletion variant of Epstein–Barr virus in nasopharyngeal carcinoma and gastric tumors in Hong Kong. Int J Cancer 66, 711–712.[CrossRef][Medline]

De Campos-Lima, P.-O., Gavioli, R., Zhang, Q.-J., Wallace, L. E., Dolcetti, R., Rowe, M., Rickinson, A. B. & Masucci, M. G. (1993). HLA-A11 epitope loss isolates of Epstein–Barr virus from a highly A11+ population. Science 260, 98–100.[Medline]

De Campos-Lima, P.-O., Levitsky, V., Brooks, J., Lee, S. P., Hu, L. F., Rickinson, A. B. & Masucci, M. G. (1994). T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein–Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues. J Exp Med 179, 1297–1305.[Abstract]

de Thé, G. (1982). Epidemiology of Epstein–Barr virus and associated diseases in man. In The Herpesviruses, vol. 1, pp. 25–103. Edited by B. Roizman. New York: Plenum.

Devergne, O., Hatzivassiliou, E., Izumi, K. M., Kaye, K. M., Kleijnen, M. F., Kieff, E. & Mosialos, G. (1996). Association of TRAF1, TRAF2, and TRAF3 with an Epstein–Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-{kappa}B activation. Mol Cell Biol 16, 7098–7108.[Abstract]

Duraiswamy, J., Burrows, J. M., Bharadwaj, M., Burrows, S. R., Cooper, L., Pimtanothai, N. & Khanna, R. (2003). Ex vivo analysis of T-cell responses to Epstein–Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions. J Virol 77, 7401–7410.[Abstract/Free Full Text]

Edwards, R. H., Seillier-Moiseiwitsch, F. & Raab-Traub, N. (1999). Signature amino acid changes in latent membrane protein 1 distinguish Epstein–Barr virus strains. Virology 261, 79–95.[CrossRef][Medline]

Eliopoulos, A. G., Stack, M., Dawson, C. W., Kaye, K. M., Hodgkin, L., Sihota, S., Rowe, M. & Young, L. S. (1997). Epstein–Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-{kappa}B pathway involving TNF receptor-associated factors. Oncogene 14, 2899–2916.[CrossRef][Medline]

Eliopoulos, A. G., Blake, S. M. S., Floettmann, J. E., Rowe, M. & Young, L. S. (1999). Epstein–Barr virus-encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF2. J Virol 73, 1023–1035.[Abstract/Free Full Text]

Fielding, C. A., Sandvej, K., Mehl, A., Brennan, P., Jones, M. & Rowe, M. (2001). Epstein–Barr virus LMP-1 natural sequence variants differ in their potential to activate cellular signaling pathways. J Virol 75, 9129–9141.[Abstract/Free Full Text]

Floettmann, J. E. & Rowe, M. (1997). Epstein–Barr virus latent membrane protein-1 (LMP1) C-terminus activation region 2 (CTAR2) maps to the far C-terminus and requires oligomerisation for NF-{kappa}B activation. Oncogene 15, 1851–1858.[CrossRef][Medline]

Fries, K. L., Miller, W. E. & Raab-Traub, N. (1996). Epstein–Barr virus latent membrane protein 1 blocks p53-mediated apoptosis through the induction of the A20 gene. J Virol 70, 8653–8659.[Abstract]

Gires, O., Kohlhuber, F., Kilger, E. & 7 other authors (1999). Latent membrane protein 1 of Epstein–Barr virus interacts with JAK3 and activates STAT proteins. EMBO J 18, 3064–3073.[Abstract/Free Full Text]

Gorski, J. (1990). HLA analysis by PCR/SSOPH. In The HLA System: a New Approach, pp. 73–78. Edited by J. Lee. New York: Springer-Verlag.

Hahn, P., Novikova, E., Scherback, L. & 7 other authors (2001). The LMP1 gene isolated from Russian nasopharyngeal carcinoma has no 30-bp deletion. Int J Cancer 91, 815–821.[CrossRef][Medline]

Hammarskjöld, M. L. & Simurda, M. C. (1992). Epstein–Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-{kappa}B activity. J Virol 66, 6496–6501.[Abstract]

Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E. & Rickinson, A. (1991). Induction of bcl-2 expression by Epstein–Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65, 1107–1115.[Medline]

Henry, S., Sacaze, C., Berrajah, L., Karray, H., Drira, M., Hammami, A., Icart, J. & Mariame, B. (2001). In nasopharyngeal carcinoma-bearing patients, tumors and lymphocytes are infected by different Epstein–Barr virus strains. Int J Cancer 91, 698–704.[CrossRef][Medline]

Hu, L.-F., Zabarovsky, E. R., Chen, F., Cao, S.-L., Ernberg, I., Klein, G. & Winberg, G. (1991). Isolation and sequencing of the Epstein–Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma. J Gen Virol 72, 2399–2409.[Abstract]

Huen, D. S., Henderson, S. A., Croom-Carter, D. & Rowe, M. (1995). The Epstein–Barr virus latent protein-1 (LMP-1) mediates activation of NF-{kappa}B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Oncogene 10, 549–560.[Medline]

Itakura, O., Yamada, S., Narita, M. & Kikuta, H. (1996). High prevalence of a 30-base pair deletion and single-base mutations within the carboxy terminal end of the LMP-1 oncogene of Epstein–Barr virus in the Japanese population. Oncogene 13, 1549–1553.[Medline]

Izumi, K. M. & Kieff, E. D. (1997). The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-{kappa}B. Proc Natl Acad Sci U S A 94, 12592–12597.[Abstract/Free Full Text]

Izumi, K. M., McFarland, E. C., Ting, A. T., Riley, E. A., Seed, B. & Kieff, E. D. (1999). The Epstein–Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-{kappa}B activation. Mol Cell Biol 19, 5759–5767.[Abstract/Free Full Text]

Khanna, R., Burrows, S. R., Nicholls, J. & Poulsen, L. M. (1998). Identification of cytotoxic T cell epitopes within Epstein–Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2 supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes. Eur J Immunol 28, 451–458.[CrossRef][Medline]

Kieser, A., Kaiser, E. & Hammerschmidt, W. (1999). LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2. EMBO J 18, 2511–2521.[Abstract/Free Full Text]

Li, S. N., Chang, Y. S. & Liu, S. T. (1996). Effect of a 10-amino acid deletion on the oncogenic activity of latent membrane protein 1 of Epstein–Barr virus. Oncogene 12, 2129–2135.[Medline]

Lin, J.-C., Lin, S.-C., De, B. K., Chan, W.-P., Evatt, B. L. & Chan, W. C. (1993). Precision of genotyping of Epstein–Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C, and EBER): predominance of type A virus associated with Hodgkin's disease. Blood 81, 3372–3381.[Abstract]

Lin, J.-C., Lin, S.-C., Luppi, M., Torelli, G. & Mar, E.-C. (1995). Geographic sequence variation of latent membrane protein 1 gene of Epstein–Barr virus in Hodgkin's lymphomas. J Med Virol 45, <1`?show=[to]>183–191.[Medline]

Lung, M. L., Chang, R. S., Huang, M. L., Guo, H.-Y., Choy, D., Sham, J., Tsao, S. Y., Cheng, P. & Ng, M. H. (1990). Epstein–Barr virus genotypes associated with nasopharyngeal carcinoma in southern China. Virology 177, 44–53.[CrossRef][Medline]

Miller, W. E., Edwards, R. H., Walling, D. M. & Raab-Traub, N. (1994). Sequence variation in the Epstein–Barr virus latent membrane protein 1. J Gen Virol 75, 2729–2740.[Abstract]

Miller, W. E., Earp, H. S. & Raab-Traub, N. (1995). The Epstein–Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J Virol 69, 4390–4398.[Abstract]

Mitchell, T. & Sugden, B. (1995). Stimulation of NF-{kappa}B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein–Barr virus. J Virol 69, 2968–2976.[Abstract]

Mosialos, G., Birkenbach, M., Yalamanchili, R., VanArsdale, T., Ware, C. & Kieff, E. (1995). The Epstein–Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80, 389–399.[Medline]

Murono, S., Inoue, H., Tanabe, T., Joab, I., Yoshizaki, T., Furukawa, M. & Pagano, J. S. (2001). Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells. Proc Natl Acad Sci U S A 98, 6905–6910.[Abstract/Free Full Text]

Parker, K. C., Bednarek, M. A. & Coligan, J. E. (1994). Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 152, 163–175.[Abstract/Free Full Text]

Raab-Traub, N. (1992). Epstein–Barr virus and nasopharyngeal carcinoma. Semin Cancer Biol 3, 297–307.[Medline]

Rickinson, A. B., Young, L. S. & Rowe, M. (1987). Influence of the Epstein–Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells. J Virol 61, 1310–1317.[Medline]

Sandberg, M., Hammerschmidt, W. & Sugden, B. (1997). Characterization of LMP-1's association with TRAF1, TRAF2, and TRAF3. J Virol 71, 4649–4656.[Abstract]

Sandvej, K., Gratama, J. W., Munch, M., Zhou, X.-G., Bolhuis, R. L. H., Andresen, B. S., Gregersen, N. & Hamilton-Dutoit, S. (1997). Sequence analysis of the Epstein–Barr virus (EBV) latent membrane protein-1 gene and promoter region: identification of four variants among wild-type EBV isolates. Blood 90, 323–330.[Abstract/Free Full Text]

Sung, N. S., Edwards, R. H., Seillier-Moiseiwitsch, F., Perkins, A. G., Zeng, Y. & Raab-Traub, N. (1998). Epstein–Barr virus strain variation in nasopharyngeal carcinoma from the endemic and non-endemic regions of China. Int J Cancer 76, 207–215.[CrossRef][Medline]

Trivedi, P., Hu, L. F., Chen, F., Christensson, B., Masucci, M. G., Klein, G. & Winberg, G. (1994). Epstein–Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue. Eur J Cancer 30A, 84–88.[Medline]

Walling, D. M., Shebib, N., Weaver, S. C., Nichols, C. M., Flaitz, C. M. & Webster-Cyriaque, J. (1999). The molecular epidemiology and evolution of Epstein–Barr virus: sequence variation and genetic recombination in the latent membrane protein-1 gene. J Infect Dis 179, 763–774.[CrossRef][Medline]

Wang, D., Liebowitz, D. & Kieff, E. (1985). An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43, 831–840.[Medline]

Wang, F., Gregory, C., Sample, C., Rowe, M., Liebowitz, D., Murray, R., Rickinson, A. & Kieff, E. (1990). Epstein–Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J Virol 64, 2309–2318.[Medline]

Yoshizaki, T., Sato, H., Furukawa, M. & Pagano, J. S. (1998). The expression of matrix metalloproteinase 9 is enhanced by Epstein–Barr virus latent membrane protein 1. Proc Natl Acad Sci U S A 95, 3621–3626.[Abstract/Free Full Text]

Received 6 October 2003; accepted 2 February 2004.



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