Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.