Characterization of baculovirus repeated open reading frames (bro) in Bombyx mori nucleopolyhedrovirus

The baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) contains five related open reading frames (ORFs). Recent sequence analyses of several other baculovirus genomes reveal that these ORFs belong to a unique multigene family called the baculovirus repeated ORFs (bro) family. Here we have characterized these five genes from BmNPV at the transcriptional and translational levels. Reverse transcription-PCR and primer extension analyses indicated that transcription of all bro genes occurs by 2 to 4 h postinfection (p.i.) and reaches maximal levels between at 8 and 12 h p.i. Transcription of all genes is initiated between 50 and 70 nucleotides upstream of the start codon, at a characteristic C(T)AGT motif. Expression of a cat reporter gene under the control of each bro promoter provides evidence that a viral factor(s) is required for the transcription of all bro genes. Immunoblot analysis indicated that a population of BRO proteins is produced vigorously between at 8 and 14 h p.i. Immunohistochemical analysis by confocal microscopy showed that BRO proteins are localized in both the nucleus and the cytoplasm at 8 h p.i. Four BmNPV mutants, in which the bro-a, bro-b, bro-c, and bro-e genes were individually inactivated, were successfully isolated. However, exhaustive efforts failed to isolate a bro-d-deficient mutant. Similarly, it was not possible to isolate a double-deletion bro-a bro-c mutant. The bro-d gene may play an irreplaceable functional role(s) during viral infection, while bro-a and bro-c may functionally complement each other.