High-Throughput RNA Sequencing-Based Virome Analysis of 50 Lymphoma Cell Lines from the Cancer Cell Line Encyclopedia Project

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 crosscontamination 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. IMPORTANCEViruses 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 sh...

Host Gene Expression Is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BamHI A Rightward Transcript Region

“…6A). These data are in good agreement with the previously published splicing analyses performed using EBV gastric carcinoma tumors and EBV-infected lymphocytes, suggesting that the splicing pattern of the BARTs is relatively constant during EBV latency (13,28). These data suggest that the cDNA clone that was generated from the C15 tumor is also expressed in AGS-EBV cells, although it is not the most abundant isoform.…”

Latent Expression of the Epstein-Barr Virus (EBV)-Encoded Major Histocompatibility Complex Class I TAP Inhibitor, BNLF2a , in EBV-Positive Gastric Carcinomas

“…To investigate this issue further, we first performed a global virome analysis on the expanded cohort of patient GC RNA-seq (n ϭ 285) and whole-exome sequence (WXS) (n ϭ 352) data sets from TCGA (8), using a directed virome analysis approach that we have reported previously (9,10). For this analysis, all RNA-seq and WXS data sets were aligned to an index containing the human genome (Genome Reference Consortium GRCh37) plus 740 mammalian viral genomes, using the transcript aligner STAR (Spliced Transcripts Alignment to a Reference) (11) run with default options plus the clip5pNbases 6 and outFilterMultimapNmax 1000 command options (removes the first 6 bases of each read and filters out any reads that map to more than 1,000 regions of the genome, respectively).…”

The role of HIV integration in viral persistence: no more whistling past the proviral graveyard

“…The EBVtransformed cell line Raji is a well-described B cell line with substantial growth potential. DNA analyses revealed that Raji cells have an integrated copy of EBV in intron 1 of BACH2, and transcriptome analyses detected a loss of BACH2 expression in this cell line (112,113). Reintroduction of BACH2 expression in these cells results in a loss of clonogenic activity (114), suggesting downregulation of BACH2 facilitated persistence, clonal expansion, or both.…”