Mutations in the Epstein-Barr virus latent membrane protein-1 (BNLF-1) gene in spontaneous lymphoblastoid cell lines: effect on in vitro transformation associated parameters and tumorigenicity in SCID and nude mice

Abstract

Aims-(1) To study the frequency of putative malignancy associated point mutations and a 30 base pair (bp) deletion in exon 3 of the C-terminus of the Epstein-Barr virus (EBV) encoded latent membrane protein (LMP)-1 (BNLF-1) gene in wild type EBV strains. (2) To assess the influence of these mutations on the tumorigenicity of lymphoblastoid cell lines (LCL).Methods-Eight spontaneous EBV (wild type) infected LCL were established from seven subjects. Deletions and single base mutations in the C-terminus of the BNLF-1 gene were demonstrated using bi-directional solid phase dideoxy sequencing following PCR amplification of viral DNA from the LCL. Tumorigenicity of the LCL was assessed in SCID and nude mice. Serum dependent growth and ability to form colonies in soft agarose were assessed for representative LCL.Results-All LCL showed sequence differences compared with the prototypic EBV strain B95-8. The 30 bp deletion could be detected in three of eight LCL and a 69 bp deletion (including the 30 bp deletion) was identified in an additional LCL. A range of single base mutations (including those described previously in association with EBV related neoplasias) was also seen in some of the LCL. In transformation studies, the genetic variations did not seem to influence the in vitro behaviour of the LCL. In the tumorigenicity studies, the presence of the 30 bp deletion had no influence on the behaviour of the LCL which were, as expected, tumorigenic in SCID mice but not in nude mice. In contrast, the LCL carrying the 69 bp deletion was tumorigenic in both SCID and nude mice.Conclusions-Genetic changes described previously in the C-terminus of the LMP-1 gene in various malignancy derived EBV strains are also present frequently in wild type viruses and do not simply define tumour specific EBV strains. Changes within this region may, however, still be important for the tumorigenicity of LMP-1 and thus play a role in EBV oncogenesis.