title: Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Unit of Animal Genomics, GIGA-R, Université de Liège (ULg), 4000, Liège, Belgium.
authors.forename: Keith
authors.initials: K
authors.lastname: Durkin
authors.affiliation: Unit of Animal Genomics, GIGA-R, Université de Liège (ULg), 4000, Liège, Belgium.|Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), 1000, Brussels, Belgium.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rosewick
authors.affiliation: Unit of Animal Genomics, GIGA-R, Université de Liège (ULg), 4000, Liège, Belgium.
authors.forename: Maria
authors.initials: M
authors.lastname: Artesi
authors.affiliation: Unit of Animal Genomics, GIGA-R, Université de Liège (ULg), 4000, Liège, Belgium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Hahaut
authors.affiliation: Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, S7N 2Z4, Canada.|School of Public Health, University of Saskatchewan, Saskatoon, S7N 2Z4, Canada.
authors.forename: Philip
authors.initials: P
authors.lastname: Griebel
authors.affiliation: Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, S7N 2Z4, Canada.
authors.forename: Natasa
authors.initials: N
authors.lastname: Arsic
authors.affiliation: Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), 1000, Brussels, Belgium.
authors.forename: Arsène
authors.initials: A
authors.lastname: Burny
authors.affiliation: Unit of Animal Genomics, GIGA-R, Université de Liège (ULg), 4000, Liège, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Georges
authors.affiliation: Unit of Animal Genomics, GIGA-R, Université de Liège (ULg), 4000, Liège, Belgium. anne.vandenbroeke@bordet.be.|Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), 1000, Brussels, Belgium. anne.vandenbroeke@bordet.be.
authors.forename: Anne
authors.identifier: 0000-0002-5422-0549
authors.initials: A
authors.lastname: Van den Broeke
mesh_terms: D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D002417:Cattle; D016583:Enzootic Bovine Leukosis / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D001909:Leukemia Virus, Bovine / Q000235:genetics*; D015448:Leukemia, B-Cell / Q000821:virology*; D016393:Lymphoma, B-Cell / Q000821:virology*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012191:Retroviridae Proteins / Q000235:genetics; D012756:Sheep; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; C479751:HBZ protein, human T-cell leukemia virus type I; D035683:MicroRNAs; D016372:RNA, Antisense; D012367:RNA, Viral; D012191:Retroviridae Proteins
keywords: Bovine Leukemia Virus; Deltaretrovirus; High throughput sequencing; Leukemogenesis; Noncoding RNA; Oxford Nanopore minION; RNA-seq