Sequence analysis of the Epstein-Barr virus (EBV) BRLF1 gene in nasopharyngeal and gastric carcinomas

Epstein-Barr virus (EBV) has a biphasic infection cycle consisting of a latent and a lytic replicative phase. The product of immediate-early gene BRLF1, Rta, is able to disrupt the latency phase in epithelial cells and certain B-cell lines. The protein Rta is a frequent target of the EBV-induced cytotoxic T cell response. In spite of our good understanding of this protein, little is known for the gene polymorphism of BRLF1.

BRLF1 gene was successfully amplified in 34 EBV-associated gastric carcinomas (EBVaGCs), 57 nasopharyngeal carcinomas (NPCs) and 28 throat washings (TWs) samples from healthy donors followed by PCR-direct sequencing. Fourteen loci were found to be affected by amino acid changes, 17 loci by silent nucleotide changes. According to the phylogenetic tree, 5 distinct subtypes of BRLF1 were identified, and 2 subtypes BR1-A and BR1-C were detected in 42.9% (51/119), 42.0% (50/119) of samples, respectively. The distribution of these 2 subtypes among 3 types of specimens was significantly different. The subtype BR1-A preferentially existed in healthy donors, while BR1-C was seen more in biopsies of NPC. A silent mutation A/G was detected in all the isolates. Among 3 functional domains, the dimerization domain of Rta showed a stably conserved sequence, while DNA binding and transactivation domains were detected to have multiple mutations. Three of 16 CTL epitopes, NAA, QKE and ERP, were affected by amino acid changes. Epitope ERP was relatively conserved; epitopes NAA and QKE harbored more mutations.

This first detailed investigation of sequence variations in BRLF1 gene has identified 5 distinct subtypes. Two subtypes BR1-A and BR1-C are the dominant genotypes of BRLF1. The subtype BR1-C is more frequent in NPCs, while BR1-A preferentially presents in healthy donors. BR1-C may be associated with the tumorigenesis of NPC.