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Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

Bombyx mori Bidensovirus (BmBDV), a bipartite virus possesses two single-stranded linear DNAs (VD1 and VD2) and shows high pathogenic ability to Bombyx mori. Previous research found that the genes of nonstructural protein ns1 and ns2 were in the same transcript. To investigate the mechanism of transcriptional regulation of ns1 and ns2 genes, the 5'-flanking sequence (289 nt) of ns1 gene, encompasses the regions of the common terminal sequence (CTS) and the predicted P5 promoter from the 5'-terminus of the viral genome to the transcription initiation site of the ns1 gene was cloned and fused to the upstream of the luciferase reporter gene. The luciferase reporter assay showed that the 53 nt CTS of VD1 and VD2 can downregulate the activity of P5 by 13.3 %. The comparison in different cell lines showed that P5 possessed high promoter activity in BmN and Hi5 cell lines. Interestingly, P5 also had high activity in Hela cells, a kind of cancer cell of human. Subsequent truncated promoter analysis showed that the 31 nt (-236 to -206 nt) sequence is very important to P5 for the activity down to 36.5 % after deletion of it. While the activity also remained 26.5 % after the deletion of the TATA box, suggesting that the promoter is TATA independent. Moreover, in order to further understand the activity intensity of P5, a comparison with other three promoters, B. mori actin3 (Bm-actin3), B. mori nuclear polyhedrosis virus (BmNPV) immediate early 1 gene promoter (BmNPV-ie-1), and a synthetic promoter (3xP3) was carried out, the result indicated that the activity of P5 was weaker than that of anyone of them.