BmEAK7 inhibits BmNPV infection through enhanced cellular phagocytosis

The silkworm EAK7 protein (Bombyx mori enhancer-of-akt-1-7, BmEAK7) is a member of the EAK (enhancers of akt-1) protein family. Mammalian EAK7 (mEAK7) acts as a positive activator of mTOR and functions as an evolutionarily conserved lysosomal membrane protein. In this study, BmEAK7 was cloned and identified from the silkworm midgut. Sequencing results showed that its open reading frame (ORF) is 1356 bp in full length, encoding a protein of 451 amino acids (Aa) BmEAK7 localizes to the cell membrane and cytoplasm, and is expressed in all silkworm tissues. Relatively high expression levels were detected in the midgut, testis, Malpighian tubules, and fat body. After infection with Bombyx mori nucleopolyhedrovirus (BmNPV), BmEAK7 expression was significantly upregulated in the midgut and fat body. Further experiments revealed that overexpression of BmEAK7 significantly suppressed BmNPV infection and replication. In contrast, knocking down BmEAK7 expression promoted the virus's infection and replication. To investigate the underlying immune pathways, we detected and analyzed the expression levels of key genes in BmEAK7-overexpressing cells infected with BmNPV. Results indicated that the expression levels of key mTOR pathway genes (BmAkt, BmTOR1, BmFoxO, BmeIF4E, and BmS6) were significantly upregulated. Meanwhile, the protein expression of mTOR, Akt, and their phosphorylated forms (p-mTOR at Ser²⁴⁴⁸ and p-Akt) was markedly enhanced. In contrast, Bm4EBP1 expression was notably downregulated. Conversely, knockdown of BmEAK7 produced experimental results that were the complete opposite of those obtained with BmEAK7 overexpression. This is the first report confirming that BmEAK7 possesses antiviral activity in silkworms. Mechanistically, BmEAK7 modulates the mTOR signaling pathway to enhance BmFoxO expression, thereby inhibiting BmNPV replication and proliferation. Additionally, BmEAK7 promotes cell proliferation and migration, and regulates endocytic pathways to improve phagocytic capacity. These effects collectively enhance cellular immunity. This study establishes a foundation for understanding BmEAK7's function and its mechanism in resisting BmNPV infection. It also provides a potential target for developing transgenic silkworm strains with antiviral properties.