Sequence analysis of Epstein-Barr virus EBNA-2 gene coding amino acid 148-487 in nasopharyngeal and gastric carcinomas

Abstract

Background: The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) plays a key role in the B-cell growth transformation by initiating and maintaining the proliferation of infected B-cell upon EBV infection in vitro. Most studies about EBNA-2 have focused on its functions yet little is known for its intertypic polymorphisms.

Results: Coding region for amino acid (aa) 148-487 of the EBNA-2 gene was sequenced in 25 EBV-associated gastric carcinomas (EBVaGCs), 56 nasopharyngeal carcinomas (NPCs) and 32 throat washings (TWs) from healthy donors in Northern China. Three variations (g48991t, c48998a, t49613a) were detected in all of the samples (113/113, 100%). EBNA-2 could be classified into four distinct subtypes: E2-A, E2-B, E2-C and E2-D based on the deletion status of three aa (294Q, 357K and 358G). Subtypes E2-A and E2-C were detected in 56/113 (49.6%), 38/113 (33.6%) samples, respectively. E2-A was observed more in EBVaGCs samples and subtype E2-D was only detected in the NPC samples. Variation analysis in EBNA-2 functional domains: the TAD residue (I438L) and the NLS residues (E476G, P484H and I486T) were only detected in NPC samples which located in the carboxyl terminus of EBNA-2 gene.

Conclusions: The subtypes E2-A and E2-C were the dominant genotypes of the EBNA-2 gene in Northern China. The subtype E2-D may be associated with the tumorigenesis of NPC. The NPC isolates were prone harbor to more mutations than the other two groups in the functional domains.

Figure 1

Aa 148-487 of the EBNA-2 gene sequence variations in EBVaGC, NPC biopsies and TWs of healthy donors in Northern China. The aa (148-487) and their corresponding nucleotide sequence variations of the EBNA-2 in the EBVaGC, NPC and TW from healthy donors in Northern China. Numbers on the top correspond to the aa and nucleotide positions of the gene under which the B95-8 prototype aa and nucleotide sequences are listed. Different subtypes are noted on the left of the table, while the specimens showing identical sequences to each other are represented by a sample in that group listed in the second column (such as GC85, T257...). The number in the parenthesis separated by "/" denotes the number of sample isolates with the identical sequence from EBVaGC, NPC and TW, respectively. '*' stands for the deletion of an aa. The previously reported mutations at positions 49102 and 49136 were not detected in isolates from our area. A 51bp deletion was at nucleotide 49102 and a triplet insertion of ctc was at nucleotide 49136 [29].

Figure 2

Amino acid sequence of the aa 148-487 in EBNA-2 gene and the mutated amino acids in isolates from EBVaGCs, NPCs and TWs in Northern China. Schematic illustration of the aa 148-487 of EBNA-2. The functional motifs (self-associational domain, NLS, RG domain and TAD) are underlined. Numbers indicate aa positions. The silent mutated aa positions are shown in green color and non-silent mutated sequences are in red color. The following numbers separated by "/" denote the number of the identical sequences from EBVaGC, NPC and TWs, respectively. The blue alphabets represent CR5, CR6 and RG regions of EBNA-2, respectively. The "Del" indicates the deletion of the aa.

Figure 3

The schematic model of aa 148-487 EBNA-2 involved in functional domains. Schematic aa 148-487 of EBNA-2 illustrating the relation of functional domains in this study. The aa number of self-association domain, CR5, CR6, NLS, RG, AD (TAD and NLS) are indicated. And the CR5, CR6, RG, AD domains are represented by ashy rectangle.