Identification of novel RNA polymerase III promoters in bovine leukemia virus miRNA cluster by cross-taxa analysis of small non-coding RNAs

Abstract

Background Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that induces enzootic bovine leukosis. A defining feature of BLV is its viral miRNA cluster, which is transcribed atypically by RNA polymerase III via internal type 2 promoters rather than through the canonical Pol II pathway. These miRNAs accumulate to high levels within infected lymphocytes and can alter expression of a variety of host genes involved in lymphocyte proliferation and impose leukemogenic processes. Results Here, we present a comprehensive in silico characterization of new A-box and B-box promoter motifs within the BLV miRNA-coding region. As the first step, a taxonomically diverse dataset of small non-coding RNAs (tRNAs, SINEs, and other ncRNAs) was assembled to derive position-weight matrices and corresponding IUPAC consensus sequences for type 2 internal Pol III promoter motifs. Using these models, all available BLV miRNA cluster sequences were scanned to identify and map A-box-like and B-box-like elements and to reconstruct the underlying promoter architecture. Our analyses reveal a noncanonical BLV promoter organization: overlapping degenerate A-box variants—most frequently three distinct elements—reside within the pre-miRNA hairpin region, whereas B-box elements were positioned downstream of the Pol III termination signal, effectively excluded from the mature transcript. Conclusions Despite motif degeneration, critical nucleotide positions remained strongly conserved, indicating evolutionary pressure to preserve Pol III recruitment while accommodating viral genome constraints. These findings fill a crucial gap in understanding of BLV Pol III promoter architecture and provide a foundation for future studies on how unconventional promoter configurations regulate viral miRNA expression and virus–host interactions