Characterization Of Bm41 And Bm51

Until now, the genomes of 48 baculoviruses have been sequenced. Bombyx morinucleopolyhedrovirus(BmNPV) is one of the most studied baculoviruses; ManyBmNPV genes including Bm67,Bm56,Bm9, have been characterized; however,some genes remain unclear. In this study, two BmNPV genes, Bm41 and Bm51,conseved in Lepidoptera baculoviruses are characterized. The gene transcription, geneexpression, sub-cellular location, and gene disruption were adopted to characterizethese two genes. The object of this study is to enhance our understanding ofbaculovirus molecular biology. The main results are as following:1. Functional analysis of BmNPV orf41(Bm41)Bombyx mori nucleopolyhedrovirus(BmNPV) ORF41(Bm41), homologous toAc52, is a gene present in most lepidopteran nucleopolyhedroviruses. The Bm41 islocated at 39204-39788nt in the genome of BmNPV T3 strain. Its ORF are 585bp inlength and is predicted to encode a 194 amino acid peptide with a deduced molecularweight of 23.3kDa.Bm41 transcripts and encoded protein in BmNPV-infected cells can be detectedfrom 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 isnot a viral structural protein and is detected in both the nuclei and cytoplasm ofinfected cells. A Bm41-disrupted virus(vBm^De) and a repaired virus(vBm^Re) weregenerated to investigate the function of Bm41. The results showed that Bm41 wasessential for viral replication, and the disruption of Bm41 resulted in a much lowerviral titer. Transmission electron microscopy revealed that disruption of Bm41affected normal nucleocapsid envelopment and polyhedra formation in the nucleus.The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression.The disrupted virus reduced BmNPV infectivity in an LD50 bioassay and took 18-23h longer to kill larvae than wild-type virus in an LT50 bioassay.2. Functional analysis of BmNPV orf51(Bm51)BmNPV ORF 51(Bm51) is a gene present in many lepidopteran NPVs. TheBm51 is located at 45,935-46,402nt in the genome of BmNPV T3 strain. Its ORF are468 bp in length and is predicted to encode a 155 amino acid peptide with a deducedmolecular weight of 18.5kDa. Transcripts of Bm51 were detected from 4.5 through 72hour post infection(h p.i.) by RT-PCR. The corresponding protein was detected from6 to 72 h p.i. in BmNPV-infected BmN cells by western blot analysis using apolyclonal antibody against Bm51. Western blot assay of occlusion-derived virus andbudded virus(BV) preparations revealed that Bm51 encodes a 23-kDa structuralprotein that is associated with BV and is located in the envelope fraction of buddedvirions. The protein was temporarily called BV-E23. In addition immunofluorescencemicroscopy demonstrated that the protein was present within the cytoplasm and nucleiin virus-infected cells. In conclusion, the available data suggest that Bm51 is afunctional ORF of BmNPV and encodes a protein expressed in the early stage of theinfection cycle that is associated with the BV envelope.