Identification of Bombyx mori nucleopolyhedrovirus bm58a as an auxiliary gene and its requirement for cell lysis and larval liquefaction

Bombyx mori nucleopolyhedrovirus orf58a ( bm58a) and its homologues are highly conserved in genomes of all sequenced group I alphabaculoviruses and its function is still unknown. Transcriptional analysis revealed that bm58a is a very late gene initiated from a late transcriptional start motif TAAG. To examine its role in the virus, a bm58a knockout virus (v(Bmbm-58a-KO-PH-GFP)) was generated through homologous recombination in Escherichia coli. Analysis of fluorescence microscopy, titration assays and electron microscopy examination showed that the deletion of bm58a did not affect viral replication and occlusion bodies formation in vitro, indicating that bm58a is not required for viral propagation. However, v(Bmbm-58a-KO-PH-GFP) did not result in cell lysis when wild-type virus infected cells began to lyse, and the v(Bmbm-58a-KO-PH-GFP) infected cells remained intact until 2 weeks post-infection. Quantification of polyhedra release from cells confirmed this observation. Accordingly, though deletion of bm58a did not reduce Bombyx mori nucleopolyhedrovirus infectivity in vivo in bioassays, it did significantly disrupt the larval liquefaction, reducing the level of polyhedra release from infected host. Immunofluorescence analysis demonstrated that Bm58a was predominantly localized on the cellular membrane at the late stage of infection, which may contribute to its function of facilitating cell lysis and larval liquefaction. Our results suggest that although bm58a is not essential for viral propagation as an auxiliary gene, it is a key factor of virus-induced cell lysis and larval liquefaction in vitro and in vivo.