Quantitative and qualitative RNA-Seq-based evaluation of Epstein-Barr virus transcription in type I latency Burkitt's lymphoma cells

Abstract

RNA-seq provides a rich source of transcriptome information with high qualitative and quantitative value. Here, we provide a pipeline for Epstein-Barr virus (EBV) transcriptome analysis using RNA-seq and we apply it to two type I latency cell lines, Mutu I and Akata. This analysis revealed substantial average expression levels of many lytic genes in predominantly latent cell populations. The lytic transcripts BHLF1 and LF3 were expressed at levels greater than those for 98% of all cellular polyadenylated transcripts. Exon junction mapping accurately identified the Qp-derived EBNA1 splicing pattern, lytic gene splicing, and a complex splicing pattern within the BamHI A region.

FIG. 1.

Visualization of RNA-seq coverage across the EBV genome. Coverage (Wiggle) files generated from SAMMate and the EBV annotation file were loaded onto the Integrated Genome Viewer (IGV [http://www.broadinstitute.org/igv/], developed at the Broad Institute). The y axis shows the number of reads mapping to each location of the genome. (A) Whole-genome view; (B) zoomed view of the intergenic region between the BMRF2 and BSLF2 genes; (C) lack of reads corresponding to the EBNA2 locus. The data range for coverage data was set to 20 (for Mutu I cells) or 30 (for Akata cells), meaning that maximal peaks represent genomic positions where there were at least 20 or 30 reads that crossed that position.

FIG. 2.

RPKM values for EBV genes in Mutu I (A) and Akata (B) cells. Mutu I cell results are the averages from two technical replicates (TR) from each of two separate RNA preparations. Error bars indicate the standard deviation for each gene. (C) The number and percentage of genes showing higher RPKM values than those for BHLF1 and LF3 in Mutu I and Akata cells out of a total of 22,803 annotated cellular and viral genes.

FIG. 3.

Illustration of specificity for RNA-seq in assessing EBV transcriptomes. The total number of reads that mapped to the EBV genome per 10 million mapped reads from the EBV-positive cell lines, Akata and Mutu I, and the EBV-negative cell lines, A549 and MCF7. No EBV-specific reads in either of the EBV-negative cell lines were identified. RNA1 and RNA2 refer to biological replicate RNA samples from Mutu I cells. TR1 and TR2 refer to technical sequencing replicates.

FIG. 4.

Visualization of junction evidence for EBNA1 (A), BZLF1 (B), and BLLF1/BLLF2 (C) genes. Junction (browser extensible data [BED]) files were generated by the junction mapper TopHat as outlined in file S1 in the supplemental data.