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Comparative Study
. 2013 Jan;87(2):1172-82.
doi: 10.1128/JVI.02517-12. Epub 2012 Nov 14.

Whole-genome sequencing of the Akata and Mutu Epstein-Barr virus strains

Zhen Lin  1 , Xia Wang, Michael J Strong, Monica Concha, Melody Baddoo, Guorong Xu, Carl Baribault, Claire Fewell, William Hulme, Dale Hedges, Christopher M Taylor, Erik K Flemington
Affiliations
Comparative Study

Whole-genome sequencing of the Akata and Mutu Epstein-Barr virus strains

Zhen Lin et al. J Virol. 2013 Jan.

Abstract

Using a simple viral genome enrichment approach, we report the de novo assembly of the Akata and Mutu Epstein-Barr virus (EBV) genomes from a single lane of next-generation sequencing (NGS) reads. The Akata and Mutu viral genomes are type I EBV strains of approximately 171 kb in length. Evidence for genome heterogeneity was found for the Akata but not for the Mutu strain. A comparative analysis of Akata with another four completely sequenced EBV strains, B95-8/Raji, AG876, Mutu, and GD1, demonstrated that the Akata strain is most closely related to the GD1 strain and exhibits the greatest divergence from the type II strain, AG876. A global comparison of latent and lytic gene sequences showed that the four latency genes, EBNA2, EBNA3A, EBNA3B, and EBNA3C, are uniquely defining of type I and type II strain differences. Within type I strains, LMP1, the latency gene, is among the most divergent of all EBV genes, with three insertion or deletion loci in its CTAR2 and CTAR3 signaling domains. Analysis of the BHLF1 and LF3 genes showed that the reading frames identified in the B95-8/Raji genome are not conserved in Akata (or Mutu, for BHLF1), suggesting a primarily non-protein-coding function in EBV's life cycle. The Akata and Mutu viral-genome sequences should be a useful resource for homology-based functional prediction and for molecular studies, such as PCR, RNA-seq, recombineering, and transcriptome studies. As an illustration, we identified novel RNA-editing events in ebv-miR-BART6 antisense transcripts using the Akata and Mutu reference genomes.

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Figures

Fig 1
Fig 1
Workflow for Akata and Mutu EBV genome sequencing. (A) Percentages of aligned EBV and human reads in virus genome-enriched DNA fraction. Ratios of EBV genomes to cellular genomes and average fold coverage depth values are shown below pie charts. (B) Data analysis pipeline for de novo EBV genome assembly.
Fig 2
Fig 2
Genome assembly results for Akata EBV. Circos (30) was used to display data and features on the Akata EBV genome. (Track 1 [outermost]) Coding exons for latent genes (red), lytic genes (blue), and RPMS1/A73 (green). (Track 2) Repeat and regulatory elements (black). (Track 3) Signal maps displaying coverage depth at each genomic position (plotted in log scale [0 to 10,000 reads]). (Track 4) Contigs generated by de novo assembly (purple). *, contig containing an artificially expanded (iterated) IR1 repeat region for (better) visualization (the original contig contains approximately 1.5 copies of IR1 repeat units). (Track 5) Repeat regions reconstituted by iterating individual solved repeats (green). (Track 6) Sequences validated or solved by PCR cloning/Sanger sequencing (black). ds-DNA, double-stranded DNA.
Fig 3
Fig 3
Genome assembly results for Mutu EBV. Circos (30) was used to display data and features on the Mutu EBV genome. (Track 1 [outermost]) Coding exons for latent genes (red), lytic genes (blue), and RPMS1/A73 (green). (Track 2) Repeat and regulatory elements (black). (Track 3) Signal maps displaying coverage depth at each genomic position (plotted in log scale [0 to 10,000 reads]). (Track 4) Contigs generated by de novo assembly (purple). *, contig containing an artificially expanded (iterated) IR1 repeat region for (better) visualization (the original contig contains approximately 1.5 copies of IR1 repeat units). (Track 5) Repeat regions reconstituted by iterating individual solved repeats (green). (Track 6) Sequences validated or solved by PCR cloning/Sanger sequencing (black).
Fig 4
Fig 4
Akata EBV genomes are heterogenous. Screen shots of IGV-displayed aligned reads are shown at the two high-confidence variant locations in the Akata EBV genome. Reads are represented by horizontal gray bars, and variant bases are indicated within the read bars. The genomic position and heterogenous percentage of each variant are indicated above the read bars. The constructed Akata reference genome sequence is displayed at the bottom of the alignments.
Fig 5
Fig 5
Comparative analysis of type I and type II viral genomes with the Akata EBV genome. Variations (indels and SNVs) between each viral genome and the Akata EBV genome were identified using cross_match (15). The position of each variation is displayed below the Akata EBV gene annotations and is indicated by vertical red bars. Regions with high variation densities (at EBNA2, EBNA3A, EBNA3B, EBNA3C, and LMP1) are shaded for better visualization.
Fig 6
Fig 6
Phylogenetic analysis of EBV coding genes at the amino acid level. Divergence data were obtained using Megalign (DNAStar). Intratype I divergence values represent the divergence between the indicated strain genome and the B95-8/Raji genome (NC007605.1) (4, 5). Type I-type II divergence values represent the divergence between the indicated strain and the type II strain, AG876 (DQ279927.1) (19). (A) Divergence at latency gene loci; (B) intratype I divergence of lytic genes; (C) interstrain divergence of lytic genes.
Fig 7
Fig 7
Variation of LMP1 protein sequences. (A) Alignment of LMP1 protein sequences derived from 6 EBV genomes was generated using Megalign (DNAStar). Variant positions are highlighted in yellow. Functional domains are indicated above the consensus sequence. C-term, C terminus. (B) Phylogenetic tree of LMP1 sequences from the indicated 6 EBV genomes.
Fig 8
Fig 8
The BHLF1 and LF3 genes are noncoding in the Akata and Mutu EBV genomes. Predicted BHLF1 ORFs (A) and LF3 ORFs (B) in the Akata and Mutu EBV genomes are indicated above and below the predicted B95-8/Raji BHLF1 and LF3 ORFs. Start codons (blue) and stop codons (red) are displayed for all three frames in zoomed regions of the Akata and Mutu BHLF1 gene (A) and LF3 gene (B).
Fig 9
Fig 9
Detection of RNA-editing events in ebv-miR-BART-6 regions in Akata and Mutu EBV transcriptomes. A-to-I (G) RNA-editing evidence for sense and antisense transcripts spanning the ebv-miR-BART6-3p region is shown. T-to-C changes displayed as genome browser data represent antisense A-to-I (G) changes (validated by strand-specific sequence data [unpublished]).
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