A small polypeptide encoded by antisense long non-coding RNAs mediates bovine leukemia virus replication

Bovine leukemia virus (BLV) induces a lymphoproliferative disease in ruminant species. The provirus constitutively transcribes antisense RNAs (AS1-S/L and AS2) from a promoter located in the 3′ long terminal repeat (LTR).

To elucidate the role of antisense transcription in BLV pathogenesis, a reverse genetics strategy was employed using a proviral mutant (A1) carrying a two-nucleotide substitution that selectively disrupts antisense transcription. In both infected sheep and cell culture models, the A1 mutation markedly reduced antisense RNA expression and altered disease progression. Integrated transcriptomic, confocal microscopy, and immunohistochemical analyses identified HMCN1, encoding the extracellular matrix protein Hemicentin-1, as a gene specifically upregulated in B lymphocytes infected with the A1 mutant. RNA interference experiments further demonstrated that HMCN1 regulates cellular adhesion, migration, and survival. Notably, whereas the wild-type provirus efficiently induced leukemia/lymphoma, the A1 mutant failed to trigger malignant transformation, highlighting a key contribution of antisense transcription to BLV-driven oncogenesis.

Although the antisense RNAs have been predicted to be non-coding, AS1 contains a 264 bp open reading frame (AS-ORF). To investigate whether this AS-ORF encodes a polypeptide, the presence of anti-AS1 antibodies was analyzed in sera from BLV-infected sheep and bovines. Data show that antibodies recognizing the AS1 polypeptide are detected in the sera, supporting its expression in vivo. Gaussia luciferase complementation experiments showed that the AS1-ORF protein interacted with the Tax protein and enhanced basal transcription from the viral promoter. Luciferase reporter assays showed that AS1 activated the AP-1 signaling pathway. Mutation of the initiation codon of the AS-ORF reduced viral infectivity of a BLV provirus in cell culture and reduced proviral loads in experimentally infected sheep.

Together, these findings demonstrate that BLV antisense transcription is a major determinant of viral persistence. They further reveal that the antisense transcript AS1 encodes a previously unrecognized functional protein that contributes to viral replication and pathogenesis.