Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5’LTR in tumors, however both proviruses are not transcriptionally silent. In BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. Using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all infected samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600bp (AS1-S) and a less abundant transcript of ~2200bp (AS1-L). Alternative splicing creates a second transcript of ~400bp (AS2) utilizing the first exon of AS1. Examination of protein coding potential showed AS2 to be non-coding, and the coding potential of AS1-S/L to be ambiguous, with a small potential open reading frame (ORF) of 264bp. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5' RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. The identification of these transcripts shows the BLV provirus to be far from silent in tumors and their consistent expression points to a vital role in the life cycle of the virus and its tumorigenic potential.