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

Abstract

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.

FIG. 1

bro gene family in baculoviruses and alignment of BROs. (A) Presence of the bro family in various baculoviruses. The maps show the genome organization of bro genes in various baculoviruses. The arrowheads denote the direction of transcription. Baculoviruses aligned was as follows. Ac, AcNPV; Bm, BmNPV; Op, OpNPV; Ld, LdNPV; Xc, XcGV. (B) Alignment of amino acid sequences of BmNPV BROs and AcNPV BRO (accession no. L22858). The alignment was conducted with the Clustal X program and modified by using Boxshade. The alignment of three or more identical amino acids is indicated by white letters within black boxes, and three or more similar residues are shown in shaded boxes. Dashes indicate gaps.

FIG. 2

RT-PCR analysis of bro genes. Total RNA was extracted from mock-infected BmN cells (Mi) or cells at 2, 4, 8, 12, and 24 h p.i. RT-PCR was performed with five sets of the specific primers for each bro either with (+) or without (−) reverse transcriptase.

FIG. 3

Transcriptional analysis of bro genes. (A) Comparison of 5′-flanking regions of bro genes. The capital letters indicate three or more identical nucleotides, and the lowercase letters indicate less than three identical nucleotides or nonidentical sequences. The primer extension products are denoted by boldface and dotted sequences, indicating the transcriptional initiation sites. (B) Primer extension mapping of bro transcripts. Total RNA was isolated from BmNPV-infected BmN cells after mock infection or at 4, 8, 12, 24, 48 h p.i. (lanes 1 to 6). The sequencing ladders were generated by using the plasmids shown in Table 1. The small arrows beside the sequence indicate the initiation sites of transcription.

FIG. 4

CAT activity of bro promoter constructs in BmN cells. Reporter plasmids which contain the promoter region of each bro were cotransfected with (+) or without (−) BmNPV DNA into BmN cells. Lane 1 shows a reference standard of the acetylated substrate provided from the manufacturer; lane 2 indicates the activity from the CAT reporter plasmid without a promoter showing no CAT activity in the presence of BmNPV; and lane 3 shows no CAT activity from BmNPV DNA-transfected cells. Cells were collected at 72 h p.i. and used for the CAT assay.

FIG. 5

Western blot analysis of BROs in BmNPV-infected BmN cells. Extracts were prepared from mock- or BmNPV-infected cells collected at the indicated times postinfection. Cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with anti-BRO antibodies. Arrows show the immunoreactive bands. Size markers (kilodaltons) are indicated are shown on the right.

FIG. 6

Distribution of BRO proteins in BmNPV-infected BmN cells. (A) Intracellular localization of BRO proteins. The top panels show BRO immunofluorescent images, and the bottom panels show differential interface contrast images of the same fields as the top ones. Higher magnifications of specific cells are shown on the right-hand panel in each block. BmN cells were mock infected (a) or infected with BmNPV for 8 h (b) and subjected to immunohistochemistry. (B) Subcellular distribution of BRO proteins. The nuclear fraction (N) and cellular fraction (C) of BmNPV-infected BmN cells (14 h p.i.) were separated and subjected to Western blotting.