Biogenesis of virus‐derived piRNAs in Bombyx mori BmN4 cells: Evidence for incidental processing of viral RNA

Insects rely on RNA interference (RNAi) pathways for antiviral defense, primarily through small interfering RNAs (siRNAs). However, PIWI-interacting RNAs (piRNAs), traditionally associated with transposon silencing, have been observed to target viruses (vpiRNAs) in some cell lines. One example regards the Bombyx mori N4 (BmN4) cells, which are persistently infected with viruses such as Bombyx mori latent virus (BmLV). The biogenesis and functional role of these vpiRNAs remain unclear, prompting questions about whether they represent a dedicated antiviral mechanism. This study employs bioinformatic analyses of publicly available small RNA sequencing (sRNA-seq) archives to elucidate vpiRNA biogenesis modalities. We examined sRNA length distributions, genomic mapping, nucleotide biases, and 5' read distances in total sRNA pools, as well as in PIWI protein immunoprecipitates, including those subject to experimental manipulations of key factors [e.g., Zucchini (ZUC), Trimmer (TRIM), Nibbler]. Results reveal that BmLV-derived vpiRNAs are predominantly sense-oriented, 27-28 nt in length, 2'-O-methylated, and bound to both SIWI and AGO3, with TRIM being essential for 3' maturation. They lack clear piRNA pathway fingerprints and ping-pong signatures, indicating primary biogenesis dominance over secondary/tertiary pathways. ZUC processes viral RNAs into intermediates (~70-90 nt) without nucleotide biases, similarly to endogenous mRNAs, suggesting that processing might result from high RNA abundance and mitochondrial colocalization. Our results indicate that vpiRNAs in BmN4 cells possibly emerge as a byproduct of the piRNA biogenesis machinery incidentally processing abundant viral transcripts. This study highlights the BmN4 cell line as a model for studying sRNA biogenesis and reconsiders the hypothesis that vpiRNAs may represent a dedicated antiviral response in these cells.