Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes

Abstract

Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed <2% nucleotide divergence from the B95.8 prototype while type 2 sequences remained even closer to the contemporary African prototype Ag876. However, type 1 isolates could clearly be divided into families based on linked patterns of sequence divergence from B95.8 across all four EBNA loci. Different patterns of IVT and AVF variation were associated with the different type 1 families, and there was additional epitope diversity within families. When the EBNA3 gene sequences of type 1 Chinese strains were subject to computer-based analysis, particular codons within the A11-epitope-coding region were among the few identified as being under positive or diversifying selection pressure. From these results, and the observation that mutant epitopes are consistently nonimmunogenic in vivo, we conclude that the immune selection hypothesis remains viable and worthy of further investigation.

FIG. 1.

Sequence changes in the entire EBNA3B gene of type 1 Chinese EBV strains relative to the Caucasian prototype 1 B95.8 EBNA3B gene. Strains are aligned vertically under the B95.8 prototype and are identified in the left-hand column of each block; strains within the same box in that column have identical sequences. All other columns represent individual EBNA3B codons (numbered at the top) where a nucleotide change relative to the B95.8 sequence was detected in one or more Chinese viruses. The nucleotide changes are shown in boldface type and resulting amino acid changes are also shown in boldface type; unchanged nucleotides and amino acids are not in boldface type. To illustrate the familialrelationships among the Chinese strains, blocks of the Li family sequence are shown in light shading, blocks of the Wu family sequence are shown in medium shading, and blocks of sporadic (Sp) sequence are shown in dark shading. One Chinese virus strain, C1, is entirely B95.8-like. Strains C4 to C10 constitute the Li family, strains C11 to NPC11 constitute the Wu family, strains C5, C17, and NPC15 are Wu/Li recombinants, and strains C6 to C13 represent various Wu/Sp recombinants. Codons lying within the AVF (399 to 408) and IVT (416 to 424) epitope regions are identified above the relevant columns. The 60-bp repeat locus with EBNA3B is also identified, and the number of repeats in each virus strain is shown.

FIG. 1.

Sequence changes in the entire EBNA3B gene of type 1 Chinese EBV strains relative to the Caucasian prototype 1 B95.8 EBNA3B gene. Strains are aligned vertically under the B95.8 prototype and are identified in the left-hand column of each block; strains within the same box in that column have identical sequences. All other columns represent individual EBNA3B codons (numbered at the top) where a nucleotide change relative to the B95.8 sequence was detected in one or more Chinese viruses. The nucleotide changes are shown in boldface type and resulting amino acid changes are also shown in boldface type; unchanged nucleotides and amino acids are not in boldface type. To illustrate the familialrelationships among the Chinese strains, blocks of the Li family sequence are shown in light shading, blocks of the Wu family sequence are shown in medium shading, and blocks of sporadic (Sp) sequence are shown in dark shading. One Chinese virus strain, C1, is entirely B95.8-like. Strains C4 to C10 constitute the Li family, strains C11 to NPC11 constitute the Wu family, strains C5, C17, and NPC15 are Wu/Li recombinants, and strains C6 to C13 represent various Wu/Sp recombinants. Codons lying within the AVF (399 to 408) and IVT (416 to 424) epitope regions are identified above the relevant columns. The 60-bp repeat locus with EBNA3B is also identified, and the number of repeats in each virus strain is shown.

FIG. 2.

Sequence changes in parts of the EBNA3A (codons 114 to 320) and EBNA3C (codons 121 to 293) genes of type 1 Chinese EBV strains relative to the Caucasian prototype 1 B95.8 sequence. The different virus strains are aligned vertically under the B95.8 prototype by using the same format as adopted in Fig. 1, with the nucleotide and amino acid changes identified in boldface type and the different blocks of family sequences identified by different degrees of shading as described in the legend to Fig. 1.

FIG. 3.

Sequence changes in a part of the EBNA2 gene (codons 109 to 259) of type 1 Chinese EBV strains relative to the Caucasian prototype 1 B95.8 sequence. The data are derived from a subset of the full panel of Chinese strains including five members of the Li family (NPC3 to C10), two Wu/Li recombinants (C5 and NPC15), four members of the Wu family (CT1 to NPC11), and three Wu/Sp recombinants (C15 to C13). The different virus strains are aligned vertically under the B95.8 prototype, and the nucleotide and amino acid changes and different blocks of family sequences are identified as described in the legend to Fig. 1.

FIG. 4.

Sequence changes in the EBNA2 (codons 109 to 259), EBNA3A (codons 114 to 320), EBNA3B (entire gene), and EBNA3C (codons 121 to 293) genes of type 2 Chinese EBV strains relative to the African prototype 2 Ag876 sequence. Virus strains are aligned vertically under the Ag876 prototype, and strains in the same box have identical sequences. Nucleotide and amino acid changes are identified as described in the legend to Fig. 1. Note that only the C19 and C20 strains are type 2 at all four gene loci. The other three strains are type 1-type 2 recombinants with type 2 sequences only at the EBNA3A, 3B, and 3C loci (C18) or only at the EBNA3B and 3C loci (NPC13 and NPC14).

FIG. 5.

Phylogenetic tree based on EBNA3 sequences. The tree shown was obtained by the neighbor-joining method (PHYLIP program Neighbor) by using maximum-likelihood distances between pairs of DNA sequences in the concatenated alignment of EBNA3A (part), EBNA3B (whole), and EBNA3C (part) sequences. The horizontal branches represent substitutions per nucleotide site, with the scale indicated at the foot of the tree. Branches for type 1 intratypic recombinants are drawn in grey (see text). The tree is rooted between types 1 and 2, with the branch joining types 1 and 2 (dashed line) compressed with respect to the rest of the figure. On the right, the AVF and IVT epitope sequences for each virus strain are indicated (using the same nomenclature as in Table 3), and viruses of the Li and Wu families, Wu/Li recombinants, Wu/Sp recombinants, and type 2 viruses are identified. wt, wild type; wt¹, Caucasian B95.8 prototype 1 epitope sequence; wt², African Ag876 prototype 2 epitope sequence.