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. 2016 Jan 1;7(2):214-24.
doi: 10.7150/jca.13150. eCollection 2016.

Genome-wide Analysis of Epstein-Barr Virus (EBV) Integration and Strain in C666-1 and Raji Cells

Kai Xiao  1 , Zhengyuan Yu  2 , Xiayu Li  3 , Xiaoling Li  1 , Ke Tang  2 , Chaofeng Tu  2 , Peng Qi  2 , Qianjin Liao  4 , Pan Chen  4 , Zhaoyang Zeng  1 , Guiyuan Li  1 , Wei Xiong  1
Affiliations

Genome-wide Analysis of Epstein-Barr Virus (EBV) Integration and Strain in C666-1 and Raji Cells

Kai Xiao et al. J Cancer. .

Abstract

EBV is a key risk factor for many malignancy diseases such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). EBV integration has been reported, but its scale and impact to cancer development is remains unclear. C666-1 (NPC cell line) and Raji (BL cell line) are commonly studied EBV-positive cancer cells. A rare few EBV integration sites in Raji were found in previous research by traditional methods. To deeply survey EBV integration, we sequenced C666-1 and Raji whole genomes by the next generation sequencing (NGS) technology and a total of 909 breakpoints were detected in the two cell lines. Moreover, we observed that the number of integration sites was positive correlated with the total amount of chromosome structural variations (SVs) and copy number structural variations (CNVs), and most breakpoints located inside or nearby genome structural variations regions. It suggested that host genome instability provided an opportunity for EBV integration on one hand and the integration aggravated host genome instability on the other hand. Then, we respectively assembled the C666-1 and Raji EBV strains which would be useful resources for EBV-relative studies. Thus, we report the most comprehensive characterization of EBV integration in NPC cell and BL cell, and EBV shows the wide range and random integration to increase the tumorigenesis. The NGS provides an incomparable level of resolution on EBV integration and a convenient approach to obtain viral strain compared to any research technology before.

Keywords: Burkitt lymphoma (BL); C666-1; Epstein-Barr Virus (EBV); Nasopharyngeal carcinoma (NPC); Raji; Whole genome sequencing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
The workflow for analysis Epstein-Barr virus (EBV) integration and assembly of EBV genomes in C666-1 and Raji cells.
Figure 2
Figure 2
Summary of somatic genomic alterations in C666-1 and Raji cells. Various types of somatic alterations in C666-1(A) and Raji (B) genomes using circus plots. Tracks from outer to inner represent the following: (1) chromosome karyotype diagram; (2) SNP heatmap; (3) InDel heatmap; (4) virus integration sites (integrations within SV or CNV regions were represented in red, integrations near SV or CNV regions were represented in yellow, others were represented in green); (5) CNV diagram (red, outer of baseline: gain; inner of baseline: loss); (6) SV insertion (yellow); (7) SV deletion(blue); (8) SV invertion; (9) SV intra-chromosomal translocation (represent in blue) and inter-chromosomal translocation (represent in black).
Figure 2
Figure 2
Summary of somatic genomic alterations in C666-1 and Raji cells. Various types of somatic alterations in C666-1(A) and Raji (B) genomes using circus plots. Tracks from outer to inner represent the following: (1) chromosome karyotype diagram; (2) SNP heatmap; (3) InDel heatmap; (4) virus integration sites (integrations within SV or CNV regions were represented in red, integrations near SV or CNV regions were represented in yellow, others were represented in green); (5) CNV diagram (red, outer of baseline: gain; inner of baseline: loss); (6) SV insertion (yellow); (7) SV deletion(blue); (8) SV invertion; (9) SV intra-chromosomal translocation (represent in blue) and inter-chromosomal translocation (represent in black).
Figure 3
Figure 3
Correlation analysis of EBV integration and chromosome instability in C666-1 and Raji cells. A. The number of EBV integration sites versus the number of SVs and CNVs on C666-1 chromosomes. B. The number of EBV integration sites versus the number of SVs and CNVs on Raji chromosomes. C. Summary of distance between EBV integration sites and SVs and CNVs sites in C666-1 and Raji.
Figure 4
Figure 4
EBV integration sites and breakpoints in the EBV genomes of C666-1 and Raji cells. The circus plots representing the EBV genomes from C666-1 (A) and Raji (B) cells. Tracks from outer to inner represent the following: (1) foward ORFs (blue); (2) repeat regions (black); (3) reward ORFs (purple); (4) number of integration sites per Kilobases of EBV genomes (up to 10 sites, orange); (5) integration sites (green), some of integration sites were identified two or more times in C666-1 or Raji genome.
Figure 4
Figure 4
EBV integration sites and breakpoints in the EBV genomes of C666-1 and Raji cells. The circus plots representing the EBV genomes from C666-1 (A) and Raji (B) cells. Tracks from outer to inner represent the following: (1) foward ORFs (blue); (2) repeat regions (black); (3) reward ORFs (purple); (4) number of integration sites per Kilobases of EBV genomes (up to 10 sites, orange); (5) integration sites (green), some of integration sites were identified two or more times in C666-1 or Raji genome.
Figure 5
Figure 5
Sequencing depths and SNVs of EBV genome in C666-1 and Raji. Tracks from outer to inner represent the following: (1) foward ORFs(blue); (2) repeat regions(black); (3) reward ORFs (purple); (4) sequencing depths (up to 1800 reads, orange); single nucleotide variations (green) compared to B95-8 strain (5), reference sequence (NC_007605, 6), HKNPC1 strain (7), GD2 strain (8), AG876 strain (9) and GD1 strain (10). A. C666-1; B. Raji
Figure 5
Figure 5
Sequencing depths and SNVs of EBV genome in C666-1 and Raji. Tracks from outer to inner represent the following: (1) foward ORFs(blue); (2) repeat regions(black); (3) reward ORFs (purple); (4) sequencing depths (up to 1800 reads, orange); single nucleotide variations (green) compared to B95-8 strain (5), reference sequence (NC_007605, 6), HKNPC1 strain (7), GD2 strain (8), AG876 strain (9) and GD1 strain (10). A. C666-1; B. Raji
Figure 6
Figure 6
Phylogenetic analyses on whole EBV genomes and protein-encoding genes EBNA-1, LMP1, and BZLF1. A. Phylogenetic tree base on whole genome of 20 EBV strains, poorly aligned and highly divergent sequences were masked by Gblocks. Phylogenetic trees based on protein sequence alignment of EBNA1 (B), LMP1 (C), and BZLF1 (D) were generated. Phylogenetic analysis was performed using MEGA software (version 5), by Neighbor-joining (NJ) algorithm. Divergence scale, in numbers of substitution per site, is shown under each tree.
Figure 7
Figure 7
LMP1 protein sequences variation in different EBV strains. LMP1 protein sequences Alignment were generated using Megalign (DNAstar). Variant positions are marked in yellow. Functional domains of the gene are indicated above the consensus sequence.
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