Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs.
BACKGROUND
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 the proviruses are not transcriptionally silent. In the case of 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.


RESULTS
Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600 bp (AS1-S) and a less abundant transcript of ~2200 bp (AS1-L). Alternative splicing creates a second transcript of ~400 bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264 bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. 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. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species.


CONCLUSIONS
The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV.
['Durkin|Keith|K|', 'Rosewick|Nicolas|N|', 'Artesi|Maria|M|', 'Hahaut|Vincent|V|', 'Griebel|Philip|P|', 'Arsic|Natasa|N|', 'Burny|Arsène|A|', 'Georges|Michel|M|', 'Van den Broeke|Anne|A|0000-0002-5422-0549']
D000818:Animals D050976:Basic-Leucine Zipper Transcription Factors D002417:Cattle D016583:Enzootic Bovine Leukosis D059014:High-Throughput Nucleotide Sequencing D006801:Humans D001909:Leukemia Virus, Bovine D015448:Leukemia, B-Cell D016393:Lymphoma, B-Cell D035683:MicroRNAs D016372:RNA, Antisense D012367:RNA, Viral D012191:Retroviridae Proteins D012756:Sheep D020079:Terminal Repeat Sequences D014158:Transcription, Genetic
Bovine Leukemia Virus Deltaretrovirus RNA-seq High throughput sequencing Leukemogenesis Noncoding RNA Oxford Nanopore minION