Avian Leukosis Virus Activation of an Antisense RNA Upstream of TERT in B-Cell Lymphomas

Abstract

Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering the expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep-sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) gene promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3,552 nucleotides (nt) upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation to TERT. Transcriptome-sequencing (RNA-seq) analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is upregulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives the expression of a chimeric transcript containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving overexpression of the TERT antisense RNA suggest it may have a role in tumorigenesis. IMPORTANCEThe data suggest that ALV integrations in the TERT promoter region drive the overexpression of a novel antisense RNA and contribute to the development of lymphomas. Avian leukosis virus (ALV) is a simple retrovirus that does not carry a viral oncogene but induces tumors by insertional mutagenesis (1, 2). ALV typically induces B-cell lymphomas, but can also induce erythroblastomas, hemangiomas, and myeloid tumors (1-3). Proviral integration can upregulate the expression of nearby genes via strong enhancers and promoter elements in the viral long terminal repeat (LTR) sequences. An advantage of using ALV as an insertional-mutagenesis tool is its relatively random integration pattern, with only a slight preference for actively transcribed sites (4-6). A reduced integration bias allows us to map proviral integrations in many genomic locations and to observe the selection of integration sites with oncogenic potential. Previous studies have shown common integration sites in ALV-induced lymphomas in the MYC, MYB, BIC (miR 155 precursor), and telomerase reverse transcriptase (TERT) genes (6-10). Highthroughput sequencing revealed multiple integration sites in a series of rapid-onset B-cell lymphomas (6). The TERT promoter region was identified as the most clonally expanded of these integrations, suggesting this is an early event in tumorigenesis (6). Twenty-six unique integration sites were identified in the region in multiple independent tumors (6).Telomerase is a ribonucleoprotein complex that adds repeat sequences to chromosome ends. It contains a catalytic protein component, TERT, as well as a noncoding telomerase RNA template component (TERC). Elevated telomerase activity has been detected in more than 90% of all hum...

ALV Integration-Associated Hypomethylation at the TERT Promoter Locus

“…These integrations are the most clonally expanded in a subset of tumors, suggesting that proviral integration into this region is an important early event in lymphoma development [ 3 , 9 ]. Tumors with TERT promoter integrations are associated with increased TERT expression [ 7 , 8 ]. The mechanism of this activation is presently under investigation.…”

“…Proviral integration can upregulate the expression of proximal genes through enhancer and promoter elements in the viral long terminal repeat (LTR) sequences [ 1 , 2 ]. Previous studies have shown common integration sites in ALV-induced lymphomas near MYC , MYB , BIC and, more recently, the telomerase reverse transcriptase (TERT) genes [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. Previously, we found that integrations in the TERT promoter region were one of the most clonally expanded—or most abundant unique—integrations in tumors tested from ALV infected chickens [ 3 , 9 ].…”

“…TAPAS transcripts corresponded to a region downstream of the common ALV integration site in the TERT promoter region. Expression of TAPAS was shown to be partly driven by the ALV proviral promoter, which was supported by the detection of spliced proviral transcripts fused with TAPAS [ 8 ]. Furthermore, the same subset of tumors was associated with increased TERT expression [ 7 , 8 ].…”

“…Expression of TAPAS was shown to be partly driven by the ALV proviral promoter, which was supported by the detection of spliced proviral transcripts fused with TAPAS [ 8 ]. Furthermore, the same subset of tumors was associated with increased TERT expression [ 7 , 8 ]. Thus, we studied the epigenetic state of the ALV provirus and the TERT promoter in chicken lymphomas with clonally expanded TERT promoter integrations.…”

Selection for avian leukosis virus integration sites determines the clonal progression of B-cell lymphomas

“…Therefore, loss of super-infection resistance could be involved in tumorigenesis. Alternatively, deletions in the env region of proviruses or mutations affecting env expression, identified in some tumors in previous work in our lab [ 18 ], might allow cells to overcome super-infection resistance.…”