Genetic Patterns Found in the Nuclear Localization Signals (NLSs) Associated with EBV-1 and EBV-2 Provide New Insights into Their Contribution to Different Cell-Type Specificities

Abstract

The Epstein-Barr virus (EBV) is a globally dispersed pathogen involved in several human cancers of B-cell and non-B-cell origin. EBV has been classified into EBV-1 and EBV-2, which have differences in their transformative ability. EBV-1 can transform B-cells into LCL more efficiently than EBV-2, and EBV-2 preferentially infects T-cell lymphocytes. The EBNA3A oncoprotein is a transcriptional regulator of virus and host cell genes, and is required in order to transform B-cells. EBNA3A has six peptide motifs called nuclear localization signals (NLSs) that ensure nucleocytoplasmic protein trafficking. The presence of multiple NLSs has been suggested to enhance EBNA3 function or different specificities in different cell types. However, studies about the NLS variability associated with EBV types are scarce. Based on a systematic sequence analysis considering more than a thousand EBNA3A sequences of EBV from different human clinical manifestations and geographic locations, we found differences in NLSs' nucleotide structures among EBV types. Compared with the EBNA3A EBV-1, EBNA3A EBV-2 has two of the six NLSs altered, and these mutations were possibly acquired by recombination. These genetic patterns in the NLSs associated with EBV-1 and EBV-2 provide new information about the traits of EBNA3A in EBV biology.

Keywords: EBV classification; EBV nuclear antigen EBNA 3A (EBNA3A); Epstein–Barr virus (EBV); nuclear localization signal (NLS); phylogeny; recombination.

Figure 1

ML phylogenetic tree reconstruction of 1006 EBV EBNA3A sequences (2835 nt) from the 5 continents. In pink are the sequences classified as EBV-1; in lilac are the sequences classified as EBV-2. The two sequences (NPCT021 and NPCT049) closely related to the EBV-2 clade are in burgundy, and are depicted by a burgundy arrow. The zoom area shows in detail the EBV-2 sequences with their associated metadata. Blue triangles represent Chilean sequences identified in this study. The numbers inside the triangle indicate the number of sequences. The scale bar indicates nucleotide substitutions per site.

Figure 2

Schematic representation of EBNA3A amino acid architecture. Coding (exons E1 and E2) and non-coding (intron I) regions of EBNA3A are in the center of the image. The intron region’s upper zoom area shows the five unique nucleotides and three-nucleotide deletions (arrows) shared only with the EBV-2 sequences. At the bottom of the image are the EBNA3A nuclear location signals (NLSs), with the respective canonical amino acids of each of the six motifs in dark blue. Non-canonical NLSs with crucial changes are highlighted in orange (NLS3 and NLS4). The amino acid changes that would not directly affect the non-canonical motifs are in light blue. The amino acid changes that would directly affect the non-canonical motifs are highlighted in red. The green triangle indicates the four EBV-2-typical deletions identified in this study, and the amino acid deletions are highlighted with a light blue background. The numbers inside the triangles (1, 8, 5, and 5′) indicate the number of amino acids deleted. There were two deletions of five amino acids: the first deletion is indicated with the number 5, and the second with 5′.

Figure 3

Phylogenetic tree and predicted recombination blocks in the EBNA3A gene. Phylogenetic tree reconstruction based on non-recombinant SNPs of 1006 EBV EBNA3A sequences on the Figure’s left side. The red blocks (I–V) indicate recombination areas common in multiple sequences that share descent at the bottom of the image. Below the red blocks are the positions corresponding to the EBNA3A nucleotide sequence. Sequences with predicted recombination blocks are in the zoomed area with their associated metadata. Only EBV-2 sequences had recombinant blocks predicted. Chilean sequences identified in this study are represented by blue triangles. The numbers inside the triangle indicate the number of sequences.