Epstein-Barr virus (EBV) nuclear antigen (EBNA)-4 mutation in EBV-associated malignancies in three different populations

Abstract

Different ethnic groups with a high human leukocyte antigen (HLA)-A11 prevalence have been shown to experience a high rate of Epstein-Barr virus (EBV) infection, EBV-associated malignancies, and Epstein-Barr nuclear antigen (EBNA)-4 mutations. The epitopes 399-408 and 416-424 of EBNA-4 are major antigenic epitopes that elicit an HLA-A11 cytotoxic T lymphocyte (CTL) response to EBV infection. Mutations selectively involving one or more nucleotide residues in these epitopes affect the antigenicity of EBNA-4, because the mutant EBV strains are not recognized by the HLA-A11-restricted CTLs. To investigate these mutations in common EBV-associated malignancies occurring in different populations, we studied the mutation rate of epitopes 399-408 and 416-424 of EBNA-4 in 25 cases of EBV-associated Hodgkin's disease (HD), nine cases of AIDS-related non-Hodgkin's lymphoma, and 37 cases of EBV-associated gastric carcinoma (GC) from the United States, Brazil, and Japan. We found one or more mutations in these two epitopes in 50% (6/12) of United States HD, 15% (2/13) of Brazilian HD, 50% (6/12) United States GC and 28% (7/25) Japanese GC, and 22% (2/9) of United States AIDS-lymphoma. Similar mutations were found in 30% (3/10) of United States reactive, 0% (0/6) of Brazilian reactive, and 25% (2/8) Japanese reactive tissues. The most frequent amino acid substitutions were virtually identical to those seen in previously reported isolates from EBV-associated nasopharyngeal carcinomas and Burkitt's lymphomas occurring in high prevalence HLA-A11 regions. However, only 2/28 (7%) mutations occurred in HLA-A11-positive patients. Our studies suggest that: 1) EBNA-4 mutations are a common phenomenon in EBV-associated HD, GC, and AIDS-lymphoma; 2) the mutation rate does not vary in these geographic areas and ethnic groups; 3) EBNA-4 mutations in EBV-associated United States and Brazilian HD, United States and Japanese GC, and United States AIDS lymphomas are not related to patients' HLA-A11 status.

Figure 1.

Schematic of nested PCR and Bi-PASA. The genomic HLA-A11 specific region was amplified by nested PCR. cg nucleotides at 559 and 560 were used as mutant alleles, which are exclusively seen in HLA-A1101, A1102 and A1103 and few other HLA-As (also see Figure 6). The positions of the two outer (P,Q) and the two inner (A,B) Bi-PASA primers are indicated. The solid arrows indicate the direction of Bi-PASA. The wavy arrows in the A and B primers represent 5′ tails. The bands containing the mutant alleles (AQ) were cut out for DNA sequencing.

Figure 2.

Sequence of epitopes 399-408 and 416-424 of A11-restricted EBNA-4 epitopes in HD. Corresponding nucleotides and amino acids of prototype B95.8 are at top. Changes of nucleotides and amino acids and their positions of HD cases relative to B95.8 sequence are indicated. Only epitopes 399-408 and 416-424 of EBNA-4 are shown. *This HD case was type B EBV-positive. All other cases were type A EBV positive. US: United States; HD: Hodgkin’s disease; GC: Gastric carcinoma; BRA: Brazilian; REA: Reactive lymphoid lesions.

Figure 3.

Sequence of epitopes 399-408 and 416-424 of A11-restricted EBNA-4 epitopes in GC. Corresponding nucleotides and amino acids of prototype B95.8 are at top. Changes of nucleotides and amino acids and their positions of GC cases relative to B95.8 sequence are indicated. Only epitopes 399-408 and 416-424 of EBNA-4 are shown. All cases are type A EBV-positive. The three US reactive cases are the same cases as those in Figure 2 ▶ . US: United States; HD: Hodgkin’s disease; GC: Gastric carcinoma; REA: Reactive lymphoid lesions.

Figure 4.

Sequence of epitopes 399-408 and 416-424 of A11-restricted EBNA-4 epitopes in US AIDS-related non-Hodgkin’s lymphoma. Corresponding nucleotides and amino acids of prototype B95.8 are at top. Changes of nucleotides and amino acids and their positions of GC cases relative to B95.8 sequence are indicated. Only epitopes 399-408 and 416-424 of EBNA-4 are shown. The first case is type A EBV-positive and the second case is type B EBV-positive. US: United States; AIDS: acquired immunodeficiency syndrome.

Figure 5.

F1 and F2 mutations seen in a US HD case. Part of sequencing gel shows substitutions of Ala in the prototype AA-399 (F1) (GCG) by Ser (TCA), and Val in AA-400 (F2) (GTG) by Leu (TTG). The mutant DNA sequence is shown in the first column, and prototype DNA sequence showed in the second column.