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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Abstract |
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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:
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These amplify a fragment of 211 bp, encoding amino acids 468538 of EBNA-1 (EBV genome nucleotide positions 109351109561), 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 `crushsoak' 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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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 487526 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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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.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 April 1999;
accepted 13 July 1999.