Identification and characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts reveals their constitutive expression in leukemic and pre-leukemic clones

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 and produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about {bsim}5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The 5LTRs of both the BLV and HTLV-1 proviruses are transcriptionally silent in tumors, however they are not entirely quiescent, with the HLTV-1 antisense transcript HBZ and the BLV microRNAs constitutively expressed in tumors. Here, using RNA-seq, we demonstrate that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of {bsim}600bp (aS1-S) and a less abundant longer transcript of {bsim}2200bp (AS1-L). Alternative splicing also creates a second transcript of {bsim}400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5 RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV.