Molecular biological studies on multipulication of BmNPV and viral gene

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We have determined the nucleotide sequence and located the in vivo transcript termini of Bombyx mori nucleopolyhedroviruses (BmNPV) 25K gene. In BmNPV, few polyhedra(FP) mutants by mutation of the 25K gene have not been identified. In this study, we have confirmed that the disruption of BmNPV 25K gene induces its FP mutant phenotype. We have cloned and sequenced the 25K gene of three BmNPV isolates ; BmSNPV-H which produces wild type polyhedra, BmSNPV-T which produces cubic polyhedra occluding relatively small number of viruses, and BmMNP V-La which produces a number of unusual polyhedra in midgut cells. The full-length cDNA of the 25K gene of BmNPV-T and -La has a 645 bp protein-coding region with 126 bp of 5' untranslated region and 135bp of 3' untranslated region before the start of the poly(A) tail. The amino acid sequence deduced from nucleotide sequence of 25K gene of BmNPV-T was the same as that of BmNPV-La, indicating that the variations in polyhedron formation observed between … More BmNPV-Tand -La are not associated with mutation of the 25K gene. The substitution of one amino acid and the deletion of another amino acid were recognized in the deduced amino acid sequence of BmNPV-H, compared with sequences of BmNPV-T and -La. Our primer extension experiment indicated that transcription initiation site ofthe BmNPV 25K gene is at a position adjacent to the early promoter CAGT element located prior to the gene coding region, unlike other NPV systems where transcription initiation sites of 25K gene are within the baculovirus late promoter ATAAG element.We. determined the amounts of viral DNA, polyhedrin mRNA and polyhedrin in the midgut epithelial cells infected with BmNPV, in order to study the poor production of polyhedra in these cells. When the appearance and accumulation of polyhedrin mRNA in the midgut epithelial cells were investigated by northern blot analysis, polyhedrin mRNA were detected as early as 6 h post-infection (p. L At 12 h p. L, amount of polyhedrin mRNA increased in the midgut epithelial cells. Quantitative PCR of virus DNA in the midgut epithelial and fat body cells at 72 h p. i. indicated that amount of the virus DNA in midgut epithelial cells was close to that in fat body cells, suggesting that virus DNA synthesis occurred in midgut epithelial cells. Quantitative RT-PCR for polyhedrin mRNAs in the midgut epithelial and fat body cells at 72 h p. i. indicated that amounts of the mRNA in both cells did not differ signficantly each other, suggesting that the transcription of polyhedrin gene occurred markedly in midgut epithelial cells. Western blot analysis showed that polyhedrin was produced in midgutepithelial cells, but its amountwas much lower than thatin fatbody cells. It is concluded that low level of at translation of polyhedrin gene may be related to poor polyhedron formation in midgut epithelial cells. Less