Characterization Of Three Baculovirus Core Genes From Bombyx Mori Nucleopolyhedrovirus

The family Baculoviridae is a highly selective pathogen in arthropods, mainly in insects of the order Lepidoptera. Recently, the baculovirus have been successfully developed as expression vector system, recombinant bio-insecticide, gene transfer vector and surface display vector, providing huge benefits to nature science development. So far, the genomes of 28 baculoviruses, including 21 nucleopolyhedroviruses and 7 granuloviruses, have been sequenced. By comprehensive comparisons of all the sequenced baculovirus genomes, 30 genes are revealed as baculovirus core genes, some of which have been proved to be involved in virus replication, transcription and structural assemblage, and several other genes whose function ara unknown. In this study, three baculovirus core genes, Bombyx mori nucleopolyhedrovirus (BmNPV) ORF79 , ORF118 and ORF56 were characterized. The gene transcription, gene expression, sub-cellular location, virus structural location and gene knock-out were adopted to characterize these three genes. The object of this study is to enhance our understanding of baculovirus molecular biology. The results were as following: 1. The analysis of Bm 79The Bm79 is located at 76132-76680nt in the genome of BmNPV T3 strain. It contains 549 nts and is predicted to encode a 182 amino acid peptide with a deduced molecular weight of 20.9 kDa. A baculovirus consensus late transcriptional start motif TTAAG is found at 145 nt upstream of the start codon ATG. Two typical polyadenylation signals (AATAAA) are found downstream of the stop codon TAA. Computer analysis predicted a confident signal peptide at the N-termini (1-22 residues).Temporal regulation of the Bm79 transcript was examined by Northern blot analysis using total RNA isolated from BmNPV-infected BmN cells. The result revealed one transcript was detectable as early as 12 h p.i., reached peak at 72 h p.i., and still remained highly steady till 96 h p.i..The antiserum against Bm79 was prepared by expressing Bm79 in Escherichia.coli. The expression of Bm79 protein in infected-cells was analyzed by Western blot using anti-Bm79 serum. The result demonstrated that a positive band was detectable asearly as 24 h p.i., increased to high levels at 48 h p.i. and lasted till 72 h p.i.. From above data, we conclude that Bm79 is a baculovirus late expression gene. This conclusion was confirmed when coupled with Ara-C assay. However, the molecular weight of the detected protein was larger than the predicted putative Bm79 gene product (20.9 kDa). The tunicamycin treatment showed the increased molecular weight was not due to the iV-glycosylation.Furthermore, Western blot was performed to determine if Bm79 was a structural protein. The immuno-reactive band was only present in ODVs instead of BVs and Bm79 was further located in ODV-E. These results indicate that Bm79 is a specific-ODV protein, and this was further confirmed by IEM using ultrathin sections of polyhedra reacted with anti-Bm79 serum.The sub-cellular localization of the Bm79 proteins was investigated by immmunofluorescence. The fluorescence was closely concentrated within the nucleoplasm and no fluorescence was detected in the cytoplasm. Thus, the Bm79 protein displayed an intranuclear localization, where ODV obtains its envelope protein. Therefore, this observation indirectly supports the evidence that Bm79 is specific for ODV envelope protein.To further investigate the function of Bm 79, the ï¿¡m79-deleted (76324-76415nt) virus was constructed by the phage X, Red system and Bac-to-Bac system. The null-virus, BmBacA79-PH, was unable to infect host cell line. However, the rescue-virus with Bm79 gene recovered, BmBacA79-PH-79Re, can't rescue this disability. Thus in this study, we conclude that Bm79 deletion destroyed the other nearby genes' function, and an effective strategy should be developed.2. The analysis of Bmll8The Bmll8 is located at 113396-114826nt in the genome of BmNPV T3 strain. It is 1431nts in length, and is predicted to encode a 476 amino acid peptide with Mr=S5.5 kDa. A baculovirus consensus late transcriptional start motif TTAAG is found at 18nt upstream of the start codon ATG. Computer analysis predicted a confident cell attachment site at the C-termini.The BmlJ8 transcriptional analysis was examined by Northern blot analysis using total RNA isolated from BmNPV-infected BmN cells. The result revealed two transcripts with molecular weight of 2.0nt and 3.0nt, respectively. The two transcripts were detectable as early as 12 h p.i., and remained highly steady till 96 h p.i.. The Bmll8 gene was expressed in E.coli, and the antiserum against Bmll8 was prepared. Western blot analysis revealed two Bmll8 products with molecular weight of 56kDaand 60kDa respectively, which were detected as early as 16h p.i.. Thus, Bmll8 is a baculovirus late expression gene. Additionally, Bmll8 fused with GFP was used to visualize its sub-cellular location, and Bmll8 was located associated with nuclear membrane, shaped like a ring zone.Furthermore, Bmll8 gene was knocked out by replacing with the reporter genes of chloramphenicol acetyltransferase {cat) and green fluorescence protein (gfp). The transfection of the null-virus DNA, BmBacA118-PH, was presented to single-cell infection with fluorescence and polyhedron appearance. Thus, the Bmll8 recovered virus, BmBacAll8-PH-118Re, was constructed. The amount of infected cells with fluorescence and polyhedra continuously increased after the cells transfected with BmBacAll8-PH-118Re DNA, which demonstrated that the virus rescued the disability successfully. This phenomenon was confirmed by the second generation infection. Thus it is concluded that the Bmll8 gene is an essential gene for BV production. 3. The analysis of Bm56The Bm56 is located at 54058-54462nt in the genome of BmNPV T3 strain. It is 405nt in length, and is predicted to encode a 134 amino acid peptide with Mr=15.8 kDa. A baculo virus consensus late transcriptional start motif ATAAG is found at 68nt upstream of the start codon ATG. Computer analysis predicted a confident transmembrane domain (TM) at the C-termini.The RT-PCR analysis of Bm56 demonstrated that Bm56 transcribes as early as 12h p.i., peaked at 16h p.i., and keep highly steady at late phase. The Bm56 gene was expressed in E.coli, and the antiserum against Bm56 was prepared. Two Bm56 encoding proteins in the infected cells were detected with molecular weight of 16kDa and 42kDa, respectively. The two protein were appeared as early as 16h p.i.. Thus, Bm56 is a baculovirus late expression gene. The sub-cellular localization of the Bm56 proteins was investigated by immmunofluorescence. The Bm56 protein was detected to associated with the nuclear membrane, assuming determined by its TM domain.In addtion, a gene encoding RNA recognition protein {Ha39) from Helicoverpa armigera single nucleopolyhedrovirusvirus was characterized in this thesis. The Ha39 is located at 34732-35316nt in the genome of HearNPV Cl strain. It contains 585 nucleotides and is predicted to encode a 194 amino acid peptide with a molecular weight of 22.5 kDa. A baculovirus early consensus transcriptional start motif (CAGT) and a late transcriptional start motif (TAAG) are observed at 132 nt and 23 nt upstream of the start codon, respectively. Additionally, a typical polyadenylationsignal (AATAAA) is found at 15 nt downstream of the translation stop codon TGA. Computer analysis of Ha39 amino acid sequence predicts a confident eukaryotic RNA recognition motif (RRM) at aa 76-152. In the genome of HearNPV, Ha39 is the only gene predicted to encode such a protein.The RT-PCR and Northern blot analysis of Ha39 demonstrated that Ha39 transcribes as early as 3h p.i., peaked at 24h p.i., and keep steady at late phase. Western blot analysis revealed a Ha39 gene product with molecular weight of 23kDa was detected as early as 6h p.i.. Thus, Ha39 is a baculovirus early expression gene. Additionally, the sub-cellular localization of the Ha39 protein was investigated by immmunofluorescence. The fluorescence was present within the cytoplasm and no fluorescence was detected in the nucleoplasm. Furthermore, Western blot was performed to determine if Ha39 was a structural protein. The immuno-reactive band was only present in BVs instead of ODVs. This result indicates that Ha39 is a specific-BV protein.