Functiomal Analysis Of BmNPV ORF71and ORF54

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus which specifically infects the domestic silkworm. The BmNPV genome was128,413nucleotides long and contained136open reading frames (ORFs) encoding predicted proteins of over60amino acids. In this study, we constructed Bm71-disrupted and Bm54-deleted virus and analysed the viral replication in cultured cells, the ability to kill the host larvae, and the morphogenesis in the ultrathin sections of the mutant-virus-transfected cell. The main results were as follows:1. Bm71located at66714-67517nt, contains804bp, encode a267amino-acid peptide with predicted molecular weight of30kDa. There are three early transcriptional motifs CAGT at434,401,288nt, three early transcriptional motifs GATA at298,193,154nt and one late transcriptional motif TAAG at295nt upstream of the Bm71start codon. At490nt downstream of the stop codon TAA, A typical polyadenylation signal (AATAAA) was located. No signal peptide sequence and transmembrane region were found, but, N-glycosylation sites at116and190amino-acid and Kinase specific phosphorylation sites at177amino-acid were predicted. Bm71polypeptide was shown to contain a number of sequence motifs that included a RING finger-like domain, a leucine zipper/basic region and an acidic domain.2. The cat cassette was inserted into the PstI site (426nt from the start codon) of the Bm71ORF coding region through homologous recombinantion. The ph and egfp gene was transpositional recombined to the ph locus of bacmid. The deleted, repair and wildtype bacmid was successfully constructed.3.Bm71-disrupted bacmid could successfully transfect Bm5cells and could produce infectious BV but the BV titer of the Bm71-D virus was reduced by approximately5-fold. The bioassay data indicated that the Bm71-D virus has a similar LD50and a longer LT50for killing the B. mori larvae compared with the WT and Bm71-R viruses. The disruption of Bm71did not obviously affect occlusion body morphogenesis.4. The predicted ORF of Bm54is2,418bp which encodes a protein of805amino-acid residues with molecular weight of93kDa and a theoretical pI of5.30. At25and217nt upstream of the start codon of Bm54ORF, two baculovirus consensus late transcriptional start motifs (TAAG) were found, suggesting that Bm54might be a late gene. In addition, a typical polyadenylation signal (AATAAA) was located at nt299downstream of the stop codon of the ORF. Motif analysis revealed that a desmoplakin N-terminus domain was found. No signal peptide sequence, nuclear localization signal or transmembrane domain was found.5. Transcriptional analysis by RT-PCR revealed that a PCR product with predicted size of505bp was detectable at3h p.i., and was still stable at72h p.i.. This result indicated that Bm54was transcribed at early and late times. The Bm54protein expression results revealed that a band with an apparent molecular mass of93kDa presented a strong antiserum reaction. This band was detectable as early as6h p.i., increased to high levels at120h p.i.6. The subcellular localization analysis of Bm54showed that extensive fluorescence localized in the cytoplasm at72h p.i. and weak staining was detected in the nucleus. Bm54gene encodes a structural protein associated with the ODVs.7. A Bm54-null bacmid was constructed through λ Red homologous recombinant in Escherichia coli. At the Bm54locus, the cat cassette was used to replace a519bp region of the Bm54gene,835bp of the5’end and1,065bp of the3’end were retained so that the deletion would not affect transcription of the adjacent genes(dnapol and lef-3).8. The Bm54-deleted virus produced non-infectious budded virus (BV) and produced normal nucleocapsids but defect polyhedra.9. Bm71and Bm54was not the baculovirus core set genes, the gene-deleted virus can produce nucleocapsids in cultured cells. However, the Bm71-deficient virus gained a lower infectivity in both cultured cells and Bombyx mori larvae, the cells transfected with viral DNA lacking Bm54produced non-infectious BVs and the affected polyhedra morphogenesis.