Summary auto-generated
This study characterized the Bombyx mori nucleopolyhedrovirus (BmNPV) ORF56 gene, which encodes a structural protein of occlusion-derived virus (ODV). Researchers detected Bm56 transcripts at 12 hours post-infection and protein at 16 hours post-infection in infected cells. Western blot analysis confirmed that Bm56 is a nucleocapsid component of ODVs, and confocal microscopy showed it localizes to the outer nuclear membrane and intranuclear regions. To investigate Bm56 function, researchers generated a Bm56-knockout bacmid through homologous recombination in E. coli. The deletion did not affect budded virus (BV) production in cultured BmN cells, indicating the gene is dispensable for in vitro BV replication. However, electron microscopy revealed that Bm56 deletion disrupted occlusion-body morphogenesis. In larval bioassays, the knockout virus showed similar infectivity to wild-type virus, but required 16-18 hours longer to achieve lethal effects. These findings demonstrate that while Bm56 is not essential for BV production in cultured cells, it facilitates efficient viral replication and spread in living insects.
Key findings
- Bm56 is a late-transcribed structural protein component of ODV nucleocapsids, not budded virus particles
- Deletion of Bm56 does not affect budded virus production in cultured cells but severely impairs occlusion-body morphogenesis
- Bm56-deleted virus maintains infectivity (LD50) in silkworm larvae but requires 16-18 hours longer to kill them compared to wild-type virus
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Abstract
Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase–Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16–18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.