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. 2015 Jan;89(1):713-29.
doi: 10.1128/JVI.02570-14. Epub 2014 Oct 29.

High-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project

Subing Cao  1 , Michael J Strong  1 , Xia Wang  1 , Walter N Moss  2 , Monica Concha  1 , Zhen Lin  1 , Tina O'Grady  1 , Melody Baddoo  1 , Claire Fewell  1 , Rolf Renne  3 , Erik K Flemington  4
Affiliations

High-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project

Subing Cao et al. J Virol. 2015 Jan.

Abstract

Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.

Importance: Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

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Figures

FIG 1
FIG 1
HTLV-1, EBV, KSHV, and murine type C retrovirus (Mu-C-retro) detection in 50 lymphoma cell lines. (A) A heat map shows the number of detected viral reads per million unique mapped reads (VPMM) in the 50 cell lines. Color intensity represents relative VPMM across all cell lines. (B) Histogram of VPMM for each virus in the respective virus-positive cell lines.
FIG 2
FIG 2
EBV read coverage in EBV-positive lymphoma cell lines. The vertical axis represents the number of reads aligning to each nucleotide position. The linear EBV annotation was split between BBLF2/3 and the BGLF3.5 lytic genes instead of the terminal repeats to facilitate the analysis of coverage and splicing for the LMP2 gene. Blue bars represent lytic genes, red bars represent latent genes, green bars represent noncoding genes, aquamarine bars represent microRNAs, and black bars represent nongene features.
FIG 3
FIG 3
Analysis of transcription in the EBV BamHI W intronic and oriLyt regions. (A) Hierarchical clustering analysis of EBV gene expression shows expression in the BamHI W intronic region and oriLyt regions more closely resembles latency gene expression than lytic gene expression. Overlapping genes were excluded from analysis due to uncertainty of read mapping. The Raji cell line was excluded from the analysis due to deletion of a number of lytic genes. The top horizontal axis shows distance between cell lines based on EBV transcriptome patterns. (B) Read coverage in the BamHI W repeat region. The vertical axis represents the number of reads aligned to each nucleotide position. Only the beginning of the repeat region is shown to better illustrate coverage. Ribodepleted strand-specific JY RNA-seq data (JY-se for transcription in sense direction and JY-as for antisense direction) suggests the BamHI W intronic region is transcribed from the sense direction. (C) Read coverage in the right oriLyt (oriLyt-Rt) and left oriLyt (oriLyt-Lt) regions. Transcription of oriLyt regions is in the sense direction in JY cells.
FIG 4
FIG 4
Analysis of transcription in the EBV oriP-BCRF1/vIL-10 gene region. (A) Transcription level at the oriP-BCRF1 region is higher in Hs 611.T than in other EBV-positive cell lines. The vertical axis represents the number of reads aligned to each nucleotide position. BBRF3 is used as a reference gene to illustrate the discordance of oriP-BCRF1 expression with other lytic gene expression. (B) Transcription in oriP-BCRF1 region in Hs 611.T cells is validated by qRT-PCR analysis. (C) Strand-specific qRT-PCR analysis shows that the oriP-BCRF1 region is predominantly transcribed in the sense direction. The primers used for analysis are indicated in panel A.
FIG 5
FIG 5
Evidence of new splicing of BARTs in the BamHI A region of the EBV genome. (A) Canonical splicing of BARTs is shown for each cell line. (B) Novel splicing events identified in each cell line. Each bar represents a spliced-out intron with color intensity (black to red) reflecting read abundance for each splicing event (total read number was shown below each bar). Only introns with more than five reads are shown.
FIG 6
FIG 6
Disruption of BACH2 expression by EBV integration in Raji cells occurs through a splice donor sequestration mechanism. The vertical axis represents the number of reads aligned to each nucleotide position. The top panel shows read coverage and splicing data for the wild-type BACH2 gene in Namalwa and Raji cells (only canonical splicing is shown). The bottom panel shows the alignment of RNA-seq data to EBV-chr6 chimeric genome and the splicing events across the EBV-chr6 junction sites. Gel pictures show the presence of chimeric transcripts in Raji cells but not in the negative control Akata cells by RT-PCR analysis.
FIG 7
FIG 7
KSHV transcriptome analysis in BCP-1 cells. The vertical axis represents the number of reads aligned to each nucleotide position. Coverage across entire genome is represented in the bottom panel and expanded coverage views for the left, middle, and right expressed gene clusters are shown in the upper panels.
FIG 8
FIG 8
Evidence of sample cross-contamination. Coverage and single nucleotide variation spectrum exhibits different patterns that cluster according to their file names. (A) The left panel shows the numerical ordering of file names of cell line data with total number of reads mapping to the murine type C retrovirus. Alignments are shown in the right panel, with the vertical axis representing the number of reads aligned to each nucleotide position. (B) The left panel shows the numerical ordering of file names of cell line data with total number of reads mapping to the Moloney MuLV. Alignments are shown in the right panel, with the vertical axis representing the number of reads aligned to each nucleotide position.
FIG 9
FIG 9
Resolution of the murine retrovirus genome in DEL cells. DEL RNA-seq data were aligned to several closely related retrovirus genomes and the de novo-assembled DEL retrovirus genome. The vertical axis represents the number of reads aligned to each nucleotide position. Phylogenic tree (generated by Lasergene 10 MegAlign) shows the distance of sequence divergence between the DEL retrovirus and other analyzed retroviruses.
FIG 10
FIG 10
MuLV integration analysis in DEL cells. The top panel shows the coverage for chimeric read pairs aligning to the TNFRSF6B gene (their mates are mapped to the MuLV genome). The vertical axis represents the number of reads aligned to each nucleotide position. Mapped reads are shown below coverage frames, with pink representing rightward-oriented reads and blue representing leftward-oriented reads. The MuLV integration site is located in the second intron of the TNFRSF6B gene. The bottom panel shows the coverage of total reads aligned to the TNFRSF6B gene in the DEL cell line.
FIG 11
FIG 11
Resolution of HTLV-1 genomes in MJ and HuT 102 cells. The top panel shows the alignment of MJ RNA-seq reads to the HTLV-1 reference genome and the de novo-assembled HTLV-1 genome. The middle panel shows the alignment of HuT 102 RNA-seq reads to the HTLV-1 reference genome and the de novo-assembled HTLV-1 genome. A phylogenetic tree in the bottom panel shows the distance of sequence divergence between different HTLV-1 strains.
FIG 12
FIG 12
HTLV-1 integration analysis in HuT 102 and MJ cells. In HuT 102 and MJ cells, 14 and 66 integrations were detected, respectively, with 3 and 8, respectively, occurring upstream from exons containing AUG initiation codons. The coverage of chimeric reads and total reads are shown on upper and lower tracks for the integration into the IL-15 (HuT 102 cells) and the STAT5B (MJ cells) genes. The vertical axis represents the number of reads aligned to each nucleotide position. Mapped reads are shown below coverage frames, with pink representing rightward-oriented reads and blue representing leftward-oriented reads.
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