Analysis of Epstein–Barr virus (EBV) nuclear antigen 1 subtypes in EBV-associated lymphomas from Brazil and the United Kingdom

Jane MacKenzie1, Diane Gray1, Roberto Pinto-Paes2, Luis F. M. Barrezueta2, Alison A. Armstrong1, Freda A. Alexander3, Duncan J. McGeoch4 and Ruth F. Jarrett1

LRF Virus Centre, Department of Veterinary Pathology, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK1
Department of Pathology, Santa Casa de São Paulo, São Paulo, CEP 01277, Brazil2
Department of Public Health Sciences, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK 3
MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, UK4

Author for correspondence: Jane MacKenzie.Fax +44 141 330 5733. e-mail j.mackenzie{at}vet.gla.ac.uk


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EBNA-1 is the only viral protein consistently expressed in all cells latently infected by Epstein–Barr virus (EBV). There is a high frequency of sequence variation within functionally important domains of EBNA-1, with five subtypes identified. Individuals may be infected with multiple EBV strains (classified according to EBNA-1 subtype), but Burkitt's lymphoma (BL) tumours carry a single subtype and exhibit some subtype preference. Subtype variation has also been related to geographical location. In the present study EBNA-1 polymorphisms were examined in a series of haematological malignancies from two distinct geographical regions, Brazil and the United Kingdom. Nucleotide sequence analysis of the carboxy-terminal region of EBNA-1 in 34 cases revealed six distinct sequences, some of which are novel. A new subtype, named V-Ala, was identified. EBNA-1 subtype in tumours differed markedly according to geographical location. In contrast to previous studies, we found evidence of EBNA-1 sequence variation within individual BL tumour samples.


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Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus which is associated with several distinct lymphomas including Burkitt's lymphoma (BL) (Epstein et al., 1964 ; Magrath, 1990 ), AIDS-related lymphomas (ARLs) (Hamilton-Dutoit et al., 1993 ) and Hodgkin's disease (HD) (Weiss et al., 1987 ; Armstrong et al., 1992 ). Expression of EBV in latency is restricted to the two latent membrane proteins (the LMPs) and to a group of six nuclear antigens (the EBNAs) (Kieff, 1996 ). The pattern of expression of these proteins varies in different tumours; however, every latently infected cell expresses EBNA-1 and in BL EBNA-1 is the only consistently detected protein (Rowe et al., 1992 ). EBNA-1 is required in trans for virus replication during latency (Yates, 1996 ) and transcriptional activation of the BamHI C latent promoter (Sugden & Warren, 1989 ).

Recent studies have shown a high frequency of sequence variation in the C terminus of EBNA-1 (Snudden et al., 1995 ; Wrightham et al., 1995 ; Bhatia et al., 1996 ; Gutiérrez et al., 1997a ; Chen et al., 1998 , 1999 ; Habeshaw et al., 1999 ). This region is essential for DNA binding and dimerization of EBNA-1 (Ambinder et al., 1991 ; Bochkarev et al., 1996 ). In a study by Bhatia et al. (1996) mutations were frequently detected between amino acids 467 and 583. These workers classified the mutants into two main groups termed prototype (similar to the prototype isolate from the B95-8 cell line) and variant, which differ by up to 15 amino acids within this region. These groups were further classified according to the amino acid at position 487. By use of this classification five subtypes have been identified thus far (Guti érrez et al., 1997 a). There are two prototype strains with either an alanine residue at position 487 (P-Ala) as in B95-8, or a threonine residue (P- Thr). The three variant strains have leucine (V-Leu), valine (V-Val) or proline (V-Pro) at this position. Multiple strains were found in samples of peripheral blood lymphocytes (PBLs) and oral secretions but only a single EBNA-1 subtype was detected in individual BL tumours (Bhatia et al., 1996 ; Gutiérrez et al., 1997a ). In African BL, P-Thr was the most common subtype whereas V-Leu, which was never found in PBLs, was the most frequently detected subtype in American BL.

To examine whether certain EBNA-1 subtypes are preferentially associated with lymphomas from distinct geographical locations or with different types of lymphoma, we determined the C-terminal sequence of EBNA-1 for 34 EBV-associated lymphomas from Brazil (12 BL, 3 HD and 5 ARLs) and the United Kingdom (2 BL and 12 HD). The EBV status of each was established by EBV EBER in situ hybridization with a commercial kit (Hybaid) or as previously described (Armstrong et al ., 1992 ). DNA was extracted by standard methods from fresh (UK) or paraffin-embedded (Brazil) material (Trainor et al ., 1982 ; Shimizu & Burns, 1995 ). PCR was done with the following primers:



These amplify a fragment of 211 bp, encoding amino acids 468–538 of EBNA-1 (EBV genome nucleotide positions 109351–109561), including 10 nucleotide positions that have been shown to be polymorphic in previous studies. PCR was performed in a 100 µl reaction containing 1x UlTma reaction buffer, 1 mM each primer, 40 mM dNTPs, 1·5 mM MgCl2 and 3 units of UlTma DNA polymerase (PE Biosystems) for 40 cycles. UlTma DNA polymerase was used as it has a proof-reading function. Hot-starts were performed using AmpliWax PCR Gems (PE Biosystems). Amplification products were subjected to electrophoresis in 8% polyacrylamide gels and fragments were extracted from gel slices by the `crush–soak' method for direct use in nucleotide sequencing reactions (Sambrook et al., 1989 ). In cases where this method gave insufficient material, PCR products were cloned into pSK Bluescript. DNA sequence was determined on both strands using fluorescent cycle sequencing reactions (PE Biosystems) with the amplification primers or, in the case of cloned fragments, primers representing standard vector sequences. Products were analysed on an ABI Prism 310 Genetic Analyser (PE Biosystems).

The analysis of 34 cases of lymphoma yielded six independent sequences. A comparison of these sequences and those from previous studies is shown in Fig. 1. The prototype sequence of B95-8 was detected in four cases and a related sequence, 3477, was detected in one case. These two sequences are both classified as P-Ala. The sequence found in the cell line AG876, and defined by Bhatia et al . as V-Leu, was detected in 13 cases. In a further 13 cases a P- Thr sequence was detected and is referred to as 1042. This sequence carries four nucleotide substitutions (leading to two amino acid substitutions) relative to the P-Thr sequence detected in the studies by Bhatia et al. The remaining two sequences have an alanine residue at position 487 but are sufficiently different from P-Ala to merit classification as a new subtype, V-Ala. Sequence 3340, found in two cases, has four nucleotide substitutions relative to B95-8, each leading to an amino acid substitution in the 120 bp sequence examined. Sequence 3478, detected in a single case, has six nucleotide changes, three of which are silent and three of which lead to amino acid substitutions. Both sequences have an asparagine at position 502. This amino acid is unique to variant subtypes.



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Fig. 1. Polymorphisms detected in a 120 bp sequence of EBNA-1 from position 109408–109527 are aligned. Only those codons in which a substitution has occurred, relative to the sequence of B95- 8, are shown. Substitutions are shown in bold. Sequences marked * were unique to this study and those marked # were detected in this study but have also been detected elsewhere. The four digit figures refer to the number assigned to one of the patients in whom that sequence was detected. The AM, PA and MT sequences were detected in the study by Wrightham et al. (1995) , c15 is a sequence described by Snudden et al. (1995) and P- Ala', P-Ala''', P-Thr' and V-Leu' are sequences detected in the study by Chen et al. (1998) . The remaining sequences were reported by Bhatia et al . (1996) and Gutiérrez et al. (1997a ).

 
The distribution of sequences is shown in Table 1. Of the Brazilian cases, 15/20 carried a variant EBNA-1 subtype whereas 13/14 UK cases contained a prototype subtype; this subtype difference between locales is highly statistically significant (P value=0·0001). These data are consistent with the recent findings of Habeshaw et al. (1999) , who also found a marked geographical variation in subtype distribution.


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Table 1. Distribution of EBNA-1 subtypes by geographical location and lymphoma type

 
Of the 12 cases of BL from Brazil, 11 carried a variant subtype (nine V-Leu and two V-Ala). The remaining case typed as a P-Thr. BL, particularly EBV-associated BL, is rare in the UK. Although 47 cases of BL and Burkitt's-like lymphoma were recruited into this study, on review only two were found to be EBV-associated BL; both were P-Ala. There is a clear preponderance of variant over prototype subtypes in BL from Brazil whereas the situation appears reversed in the UK. More cases of BL from the UK would have to be examined to confirm this trend. The predominance of prototype sequences in BL from the UK extends to non-BL. Of the 12 cases of HD examined, 11 carried a prototype sequence (nine P-Thr and two P-Ala); the remaining case was V- Leu. The pattern in Brazilian non-BL is less clear. Of the three cases of HD examined two were V-Leu and one was P-Thr, and of the five ARL cases, two were P-Thr, one was P-Ala, one V-Leu and one V-Ala.

Bhatia et al. detected a very low frequency of P-Ala in BL tumours from either Africa or America and have proposed that this subtype is incompatible with malignancy (Gutiérrez et al., 1997b ). However, we found 4/14 lymphoma cases from the UK, including the two cases of BL, contained the B95-8 P-Ala sequence. A related sequence was found in one case of Brazilian ARL (3477). Habeshaw et al. (1999) found the B95-8 sequence in 1/3 cases of European BL they examined and Chen et al. (1998) detected P-Ala sequences in 3/17 EBV-associated gastric carcinomas from the US. Thus this subtype of EBV can be associated with a malignant phenotype.

Initial reports described five EBNA-1 subtypes, each with a relatively invariant sequence (Bhatia et al., 1996 ; Gutiérrez et al., 1997a ). It is now clear that there are broader limits on variation in EBNA-1 sequence than previously suggested. We therefore analysed the degree of relatedness of the 18 independent nucleotide sequences, specifying amino acids 487–526 of EBNA-1 from this and previous studies, using the maximum likelihood phylogenetic program, DNAML, from the Phylip package (Felsenstein, 1981 ). As can be seen from Fig. 2, the eight variant sequences form one group in the tree (left end as drawn) and the ten prototype sequences another (right end). The categorization of these sequences into prototype and variant is thus valid but it is clear that classification should not be based simply on the amino acid present at residue 487. Of the 16 cases in this study with an EBNA-1 sequence located on the left of the tree, 15 were from Brazil; similarly, of the 18 cases located on the right of the tree, 13 were from the UK. We found no correlation between EBNA-1 subtype and EBV subtypes 1 and 2 (data not shown), consistent with previous studies (Wrightham et al., 1995 ; Bhatia et al., 1996 ; Habeshaw et al., 1999 ).



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Fig. 2. An unrooted, maximum likelihood tree was obtained by analysing the 18 independent 120 bp EBNA-1 sequences from this and previous studies with the DNAML program from the Phylip package (Felsenstein, 1981 ). The input order was randomized and the program was asked to search for the best tree including global rearrangements. The default settings for transition/transversion ratio, empirical base frequencies and one category of substitution rate were used. Variant sequences partition to the left of the dashed line, prototype sequences to the right. The broken arrow indicates the position of the AG876 sequence.

 
In 13 of the 34 cases examined nucleotide sequence could not be determined directly from fragments. It was therefore necessary to clone these PCR amplification products, and the sequence of a number of independent clones was analysed (range 3–14) in each case. In most cases the same sequence was found for each independent clone. In six cases a number of different sequences were detected (range 2–5). Where a mixture of sequences was detected, the most frequent sequence (at least half of the clones) is the one assigned to the case as reported above. In three cases of HD, one or two nucleotide substitutions were detected in a minority of the clones. A more complicated pattern was observed in the remaining three cases [two BL (both V-Leu) and one ARL (V-Ala) all from Brazil]. In the two cases of BL the majority of sequences were of the previously reported V-Leu subtype; the remaining clones from each case yielded unique and independent sequences. These sequences varied from the V-Leu subtype by 1–10 nucleotides with some sequences in each case being highly related to the B95-8 P-Ala sequence. The third case with a mixture of sequences was 3478, a case of ARL from Brazil, which carried a unique V- Ala sequence in the majority of clones (8/13); the remaining clones all had a G->A substitution in codon 492. In previous studies of BL only a single EBNA-1 sequence was detected in tumours; however, these results were derived from direct sequencing of PCR products and minor species may therefore have been missed. In a recent study of nasal lymphomas a high level of EBNA-1 heterogeneity was found within some samples (Gutiérrez et al., 1998 ). There are several possible explanations for the presence of multiple sequences. Sequence variability may result from misincorporation of nucleotides during PCR amplification. This explanation is unlikely in the present study as a proof-reading enzyme was used; furthermore, in one of our cases the cloned sequences varied by up to 10 nucleotides. Gutiérrez et al. have excluded such technical artefacts by performing independent, repeated PCRs and by assaying reaction products by single- stranded conformation polymorphism (K. Bhatia, personal communication). It is also possible that minor species represent EBV genomes present in infiltrating lymphocytes rather than in the tumour cells themselves. Gutiérrez et al. (1998) favoured the idea that multiple sequences were a result of ongoing mutations in the nasal lymphomas.

We have demonstrated that the C terminus of EBNA-1 is more heterogeneous than initial studies suggested. It is clear that the distribution of the different EBNA-1 species depends on the geographical location of the lymphomas under analysis.

The study was designed to examine EBNA-1 variation in lymphomas and PBL samples were not collected. We are aware that this limits the conclusions we can draw regarding the tumorigenic potential of the subtypes. However, this does not invalidate the observation that there are strong geographical associations with subtype in tumours.

Heterogeneity in herpesviruses genes is not unprecedented. Mutational hotspots in the LMP-1 gene have been intensively studied since it was observed that EBV from the CAO cell line, which carries a 30 bp deletion and additional mutations, has increased transforming efficiency compared to B95-8 virus (Li et al., 1996 ). The STP gene of herpesvirus saimiri and the K1 gene of human herpesvirus-8 also show marked sequence variation and are associated with transformation (Zong et al., 1997 ; Biesinger et al., 1992 ). Further studies are clearly required to determine the clinical significance and biological consequences of sequence variation in the EBNA-1 gene.


   Acknowledgments
 
This work was supported by the Leukaemia Research Fund as part of a specialist programme. Alison Armstrong is supported by a Kay Kendall Leukaemia Fund fellowship. We thank Kishor Bhatia, Gillian Habeshaw and Alan Rickinson for valuable comments and for making unpublished results available. We thank Rob Clayton and Jacqui Perry for their technical assistance.


   Footnotes
 
The GenBank accession numbers of the sequences reported in this paper are AF120226AF120239.


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Received 19 April 1999; accepted 13 July 1999.