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. 2012 Feb;86(3):1458-67.
doi: 10.1128/JVI.06537-11. Epub 2011 Nov 16.

Identification of new viral genes and transcript isoforms during Epstein-Barr virus reactivation using RNA-Seq

Monica Concha  1 , Xia Wang, Subing Cao, Melody Baddoo, Claire Fewell, Zhen Lin, William Hulme, Dale Hedges, Jane McBride, Erik K Flemington
Affiliations

Identification of new viral genes and transcript isoforms during Epstein-Barr virus reactivation using RNA-Seq

Monica Concha et al. J Virol. 2012 Feb.

Abstract

Using an enhanced RNA-Seq pipeline to analyze Epstein-Barr virus (EBV) transcriptomes, we investigated viral and cellular gene expression in the Akata cell line following B-cell-receptor-mediated reactivation. Robust induction of EBV gene expression was observed, with most viral genes induced >200-fold and with EBV transcripts accounting for 7% of all mapped reads within the cell. After induction, hundreds of candidate splicing events were detected using the junction mapper TopHat, including a novel nonproductive splicing event at the gp350/gp220 locus and several alternative splicing events at the LMP2 locus. A more detailed analysis of lytic LMP2 transcripts showed an overall lack of the prototypical type III latency splicing events. Analysis of nuclear versus cytoplasmic RNA-Seq data showed that the lytic forms of LMP2, EBNA-2, EBNA-LP, and EBNA-3A, -3B, and -3C have higher nuclear-to-cytoplasmic accumulation ratios than most lytic genes, including classic late genes. These data raise the possibility that at least some lytic transcripts derived from these latency gene loci may have unique, noncoding nuclear functions during reactivation. Our analysis also identified two previously unknown genes, BCLT1 and BCRT2, that map to the BamHI C-region of the EBV genome. Pathway analysis of cellular gene expression changes following B-cell receptor activation identified an inflammatory response as the top predicted function and ILK and TREM1 as the top predicted canonical pathways.

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Figures

Fig 1
Fig 1
(A) Human exonic, intronic, and intragenic read distributions for uninduced and induced nuclear and cytoplasmic sequencing data. (B) Nuclear and cytoplasmic RNA-Seq coverage data at the human ZFR locus. The y axis is the number of reads spanning each genomic coordinate (x axis). Four known isoforms for ZFR are shown at the bottom of the figure. Thick lines represent coding sequences, medium thickness lines represent untranslated regions, and thin lines with leftward arrows represent introns.
Fig 2
Fig 2
(A) Fold induction of EBV genes in cytoplasmic Akata fractions. Red boxes indicate latency genes. (B) Western blot analysis of the immediate-early and early Zta, Rta, and BMRF1 proteins performed as a quality control prior to sequencing of RNAs. (C) Percentages of human and EBV reads in uninduced and induced cytoplasmic Akata fractions.
Fig 3
Fig 3
(A) Read coverage data (Novoalign) from uninduced and induced Akata cells and splicing evidence (from TopHat) from induced Akata cells are shown for the BZLF1/Zta locus. The y axis is the number of reads spanning each genomic coordinate (x axis). Thick lines represent coding sequences, medium thickness lines represent untranslated regions, and thin lines with leftward arrows represent introns. Evidence for the canonical BZLF1 splicing are represented by 818 and 339 reads spanning intron 1 and intron 2, respectively. Eighty-one reads spanning the exon 1-exon 3 splicing event correspond to the dominant-negative variant RAZ. (B) Coverage data and splicing evidence at the BLLF1 (gp350/gp220) locus show 101 reads spanning a novel splicing event. The peak within the annotated BLLF1 intron probably does not represent a stand-alone exon since no junction-spanning reads were identified at the peak edges. Instead, these reads are likely attributable to the unspliced version of BLLF1. (C) Relative expression of the BLLF1 splice variant and the alternative splice as determined by quantitative RT-PCR. RNAs from uninduced or induced Akata or Mutu I cells and from the type III latency cell lines Jijoye, X50-7, and JY are shown.
Fig 4
Fig 4
(A) Read coverage across the LMP1 locus illustrates the lytic LMP1 transcript structure in induced Akata cells and the classic type III latency LMP1 transcript structure in JY cells. The y axis is the number of reads spanning each genomic coordinate (x axis). Thick lines represent coding sequences, medium thickness lines represent untranslated regions, and thin lines with leftward arrows represent introns. Red values are the number of reads that span the indicated junctions. Coverage data were derived from Novoalign, and junction evidence was taken from TopHat. (B) Quantitative RT-PCR was used to determine the ratio of transcripts spanning exon 2 to exon 3 versus exon 1 to 2 in cytoplasmic fractions from uninduced or induced Akata cells and the type III latency cell lines JY, Jijoye, and X50-7.
Fig 5
Fig 5
(A) Unique coverage and splicing of LMP2 transcripts in induced Akata cells versus the type III cell line JY. The y axis is the number of reads spanning each genomic coordinate (x axis). Thick lines represent coding sequences, medium thickness lines represent untranslated regions, and thin lines with leftward arrows represent introns. Red values are the number of reads that span the indicated junctions. Coverage data were derived from Novoalign, and junction evidence was taken from TopHat. (B) Ratio of sequential splicing events to total coverage across LMP2 locus in induced Akata cells and JY cells. Total coverage calculation excluded regions of LMP2 which overlaps with BMRF1. (C) Real-time RT-PCR validation of alternative splicing in induced Akata and induced Mutu I cells.
Fig 6
Fig 6
Higher nuclear retention of latency genes (sans EBNA1 and LMP1) than lytic genes following B-cell receptor activation in Akata cells.
Fig 7
Fig 7
(A) Coverage across new transcript region in induced Akata cells. Red arrows represent primers used for 5′ RACE. Green arrows represent PCR primers used for quantitative RT-PCR. (B) Real-time RT-PCR analysis (values are relative to the no-RT control in uninduced and induced conditions) of BCLT1 expression (B) and BCRT2 (C) and 5′ RACE identification of start sites. 5′ RACE products were cloned and sequenced. The start site was determined to be identical using either primer.
Fig 8
Fig 8
(A) Quantitative RT-PCR validation of selected cellular gene changes following BCR activation. (B) Top cellular functions predicted to be influenced by expressed cellular genes with changes of >4-fold. (C) Top canonical pathways predicted to be influenced by expressed cellular genes with changes of >4-fold.
Fig 9
Fig 9
Illustration of EBV genome coverage gap where the genome has been split for subsequent EBV RNA-Seq analysis.
Fig 10
Fig 10
(A) Higher nuclear retention of BCRT2 and BCLT1 than the average of lytic genes following B-cell receptor activation in Akata cells. (B) Real-time RT-PCR validation of cytoplasmic to nuclear BCLT1 and BCRT2 transcript detection relative to actin RNA in induced Akata and Mutu I cells.
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