Functional characterization of a novel protein-coding circular RNA, circRNA_1193, from the mAAP gene in silkworm and its role in antiviral defense against BmCPV

ABSTRACT A novel circular RNA, circRNA_1193, which originates from the membrane alanyl aminopeptidase-like gene in silkworms, was explored for its potential function and regulatory mechanism. We validated the presence of circRNA_1193 in Bombyx mori cytoplasmic polyhedrosis virus (BmCPV)-infected silkworm ovary cell line (BmN) cells through a combination of reverse transcription polymerase chain reaction, Northern blotting, and in situ hybridization. CircRNA_1193 exhibited tissue-specific expression, being highly enriched in the midgut and Malpighian tubules, and displayed a specific response to BmCPV infection, but not to Bombyx mori nucleopolyhedrovirus or lipopolysaccharide. Functional analyses revealed that the overexpression of circRNA_1193 suppressed BmCPV replication, whereas its knockdown increased viral replication. Bioinformatic analyses revealed potential internal ribosome entry sites, m6A methylation sites, and open reading frames (ORFs) within circRNA_1193, suggesting its potential coding capacity. We confirmed the translation of the ORF by constructing a DsRed reporter vector and demonstrating DsRed expression in transfected cells. Furthermore, a mutation of the start codon within circRNA_1193 abolished its antiviral activity, highlighting the crucial role of the translated protein, which is 35 kDa and is designated as VSP35. Furthermore, our data suggest that the formation of circRNA_1193 relies on reverse complementary flanking sequences. These findings unveil a novel protein-coding circular RNA in silkworms that plays a critical role in antiviral defense.IMPORTANCEThis study identified a novel circular RNA, circRNA_1193, in the silkworm Bombyx mori, and revealed its critical role in antiviral defense against Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). We demonstrated that circRNA_1193 exhibits tissue-specific expression, is upregulated in response to BmCPV infection, and possesses antiviral activity. Importantly, we show that circRNA_1193 encodes the viral protein VSP35, which is essential for its antiviral function. These findings provide new insights into the complex regulatory mechanisms of circular RNAs in antiviral immunity and underscore the potential of circular RNAs as therapeutic targets in viral diseases. The identification of a protein-coding circular RNA with antiviral activity in B. mori has broader implications for understanding the evolution and diversity of host defense mechanisms against viruses.