ORF40 (bm40) of the Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a homologue of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) AC51 and is a highly conserved gene in sequenced alphabaculoviruses. To investigate the role of bm40 in the baculovirus infection cycle, a bm40 knockout BmNPV bacmid was constructed via homologous recombination in Escherichia coli. Western blotting analysis revealed that bm40 is a late gene during virus infection. Compared with wild-type and repair viruses, the knockout virus exhibited a single-cell infection phenotype. Titration assays confirmed that no infectious budded viruses (BVs) were produced due to the bm40 deletion, while there was no effect on viral DNA replication. Electron microscopy revealed that Bm40 is required for nucleocapsid egress from the nucleus to the cytoplasm, nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and subsequent embedding of ODVs into polyhedra. Confocal microscopy showed that Bm40 was predominantly localized in the nuclei from 48 h post-infection and subsequently condensed on the nuclear membrane and polyhedra at the late phase of infection. Taken together, these results demonstrate that Bm40 plays an essential role in BV production and ODV envelopment in the BmNPV infection cycle.
Chilo iridescent virus (CIV), officially named invertebrate iridescent virus 6 (IIV6), is a nucleocytoplasmic virus with a ~212-kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the CIV virion consists of 54 virally encoded proteins. In this study, we identified the interactions between the structural proteins using the yeast two-hybrid system. We cloned 47 structural genes into both bait and prey vectors, and then analysed the interactions in Saccharomyces cerevisiae strain AH109. A total of 159 protein–protein interactions were detected between the CIV structural proteins. Only ORF 179R showed a self-association. Four structural proteins that have homologues in iridoviruses (118L, 142R, 274L and 295L) showed indirect interactions with each other. Seven proteins (138R, 142R, 361L, 378R, 395R, 415R and 453R) interacted with the major capsid protein 274L. The putative membrane protein 118L, a homologue of the frog virus 3/Ranagrylio virus 53R protein, showed direct interactions with nine other proteins (117L, 229L, 307L, 355R, 366R, 374R, 378R, 415R and 422L). The interaction between 118L and 415R was confirmed by a GST pull-down assay. These data indicate that 415R is a potential matrix protein connecting the envelope protein 118L with the major capsid protein 274L.