Natural polymorphisms in the bovine leukemia virus microRNA cluster modulate miRNA expression and host regulatory pathways

Abstract

Bovine leukemia virus (BLV) encodes a cluster of viral microRNAs (miRNAs) that remain abundantly expressed dur- ing latency, when viral protein production is restricted. Naturally occurring single-nucleotide polymorphisms (SNPs) within this locus are common, yet their functional consequences for miRNA output and host gene regulation remain poorly defined. Peripheral blood leukocytes from 53 naturally BLV-infected cattle were analyzed, and the 554-nt BLV miRNA locus was amplified and sequenced. Field-derived variants containing mutations within RNA polymerase III promoter motifs, seed regions, and termination signals were selected for functional evaluation. Reference and vari- ant loci were co-expressed with a BLVΔ-miRNA infectious clone in HEK293T cells under controlled conditions. Mature miRNA levels were quantified by stem-loop reverse-transcription quantitative polymerase chain reaction (RT–qPCR), and global host transcriptional responses were assessed using oligonucleotide microarrays. Predicted miRNA targets were identified using bioinformatic analyses. Sequence analysis identified 84 polymorphic sites, with a substantial proportion mapping to Pol III regulatory elements and seed regions. Variant loci displayed altered accumulation of selected mature miRNAs and shifts in predicted seed-dependent target repertoires. Transcriptome profiling revealed variant-associated modulation of innate immune-signaling components, interferon-responsive genes, antigen-presentation pathways, tumor-suppressor networks, and extracellular matrix-related processes. Enrichment analysis demonstrated a statistically significant overlap between predicted miRNA targets and downregulated tran- scripts. Natural polymorphisms within the BLV miRNA cluster modulate miRNA expression and are associated with dis- tinct host regulatory signatures in a controlled experimental system. These findings suggest that sequence variation in the viral miRNA locus may contribute to differential host–virus interactions and influence mechanisms supporting viral persistence.