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

Bovine leukemia virus (BLV) encodes a cluster of viral microRNAs (miRNAs) that remain abundantly expressed during 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 variant 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 transcripts. Natural polymorphisms within the BLV miRNA cluster modulate miRNA expression and are associated with distinct 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.