Sequence analysis of Epstein-Barr virus (EBV) early genes BARF1 and BHRF1 in NK/T cell lymphoma from Northern China

Abstract

Background: NK/T cell lymphoma is an aggressive lymphoma almost always associated with EBV. BamHI-A rightward open reading frame 1 (BARF1) and BamHI-H rightward open reading frame 1 (BHRF1) are two EBV early genes, which may be involved in the oncogenicity of EBV. It has been found that V29A strains, a BARF1 mutant subtype, showed higher prevalence in NPC, which may suggest the association between this variation and nasopharyngeal carcinoma (NPC). To characterize the sequence variation patterns of the Epstein-Barr virus (EBV) early genes and to elucidate their association with NK/T cell lymphoma, we analyzed the sequences of BARF1 and BHRF1 in EBV-positive NK/T cell lymphoma samples from Northern China.

Methods: In situ hybridization (ISH) performed for EBV-encoded small RNA1 (EBER1) with specific digoxigenin-labeled probes was used to select the EBV positive lymphoma samples. Nested-polymerase chain reaction (nested-PCR) and DNA sequence analysis technique were used to obtain the sequences of BARF1 and BHRF1. The polymorphisms of these two genes were classified according to the signature changes and compared with the known corresponding EBV gene variation data.

Results: Two major subtypes of BARF1 gene, designated as B95-8 and V29A subtype, were identified. B95-8 subtype was the dominant subtype. The V29A subtype had one consistent amino acid change at amino acid residue 29 (V → A). Compared with B95-8, AA change at 88 (L → V) of BHRF1 was found in the majority of the isolates, and AA79 (V → L) mutation in a few isolates. Functional domains of BARF1 and BHRF1 were highly conserved. The distributions of BARF1 and BHRF1 subtypes had no significant differences among different EBV-associated malignancies and healthy donors.

Conclusion: The sequences of BARF1 and BHRF1 are highly conserved which may contribute to maintain the biological function of these two genes. There is no evidence that particular EBV substrains of BARF1 or BHRF1 is region-restricted or disease-specific.

Fig. 1

BARF1 variations in 47 NK/T cell lymphoma specimens. Numbers across the top correspond to the amino acid positions under which the B95-8 prototype amino acid and nucleotide sequence are listed. The specimens showing identical sequences to each other are listed by a representative isolate in the left column, and the numbers in the parentheses after the name denote the number of isolates carried identical sequence with the representative isolate

Fig. 2

Schematic diagram of amino acid sequence variations in BARF1 protein. The amino acid sequence of BARF1 protein derived from B95-8 is listed. The previously identified BARF1/c-fms homology domain, transforming domain and CTL epitopes of BARF1 are underlined at the corresponding regions. Numbers indicate amino acid positions; asterisks indicate mutant amino acids. The mutated amino acid residues to B95-8 are indicated in boxes. Frequency of the mutations are also showed

Fig. 3

BHRF1 variations in 53 NK/T cell lymphoma specimens. Numbers across the top correspond to the amino acid positions under which the B95-8 prototype amino acid and nucleotide sequence are listed. Different patterns are noted to the left column, while the specimens showing identical sequences to each other are listed by a representative isolate in the second column. The followed numbers in the parentheses denote the amount of the identical sequences

Fig. 4

Schematic diagram of amino acid sequence variations in BHRF1 protein. The amino acid sequence of BHRF1 protein derived from B95-8 is listed. The previously identified BH1-3 domain are put into the rectangles. α2-α5 helices are underlined at the corresponding regions. Numbers indicate amino acid positions; asterisks indicate mutant amino acids. The mutated amino acid residues to B95-8 are indicated in boxes. Frequencies of the mutations were also showed