Analysis Of ORF65and ORF91from Bombyx Mori Nucleopolyhedrvirus, A Biological Insecticide

Silkworm is an important economic insect, which produces a lot of cocoon for providing the material of a large quantity of silk fabric every year. To date, more and more auxiliary products and edible foods from silkworm have been comprehensively developed and exploited. As a representative member of Alphabaculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen of silkworm and causes serious economic losses to silk production. However, the genetically modified baculoviruses have been widely used as biopesticides with their advantage of secuity, long-term, environmental protection.Now, more and more baculovirus genes have been reported. However, the functions of some genes have not been described until now. The analysis and summarization of the functions of viral genes may not only helps us to understand the mechanism of baculovirus infection or reproduction, but also facilitates us in improving novel baculovirus expression vector systems and biocontrol pesticides by genetically modification.In this study, BmNPV orf65and orf91(Bm65and Bm91) were selected to study their roles in viral life cycle at multi-molecular levels, including their transcriptional level, expression level, and localization level in infected cells and virions. The deleted viruses of Bm65and Bm91were respectively achieved by homologous recombination in E.coli and transfection in BmN cells, which were important for further disclosing their roles in viral life cycle. The results in this study were summarized as follows:1. The results of Bm65:(1) Bioinformatics analysis showed that Bm65is a highly conservative gene, encoding an unknown104-amino acid protein with predicted molecular weight about12.2kD. Analysis of Bm65sequence revealed that a typical early transcription motif TATA located in the upstream of its initiation codon, implying that Bm65may be an early gene. Alignment result showed that the homologous sequences of Bm65present in the vast majority of Lepidoptera nuclear polyhedrosis viruses genome. InterProScan software analysis showed Bm65belongs to GIY-YIG-like endonuclease superfamily and may be associated with viral DNA replication.(2) Bm65fusion protein was expressed in E.coli and the antiserum to Bm65was prepared. Additionally, the Bm65was sucessfully expressed in sf-9cells, which was helpful to purify Bm65for further functional analysis in vitro.(3) RT-PCR analysis showed that the transcription profile of Bm65could be detected during6to72h postinfection (p.i.).5’-RACE analysis showed that the transcription initiation site of Bm65located at-14nucleotides upstream of ATG and only one transcript was found. Confocal microscopy analysis revealed that Bm65located both in the cytoplasm and nucleus in virus-infected cells.(4) Bm65was replaced by Cm using Red-recombination and screened by resistance to achieve Bn65-knockout bacmid. Furthermore, pBmBm65KO-GP, pBmWTGP and pBmBm65RepGPwere constructed by transposition. Fluorescence microscopy showed that Bm65deletion blocked the generation of infectious budding viruses in the host cells. qPCR analysis revealed that Bm65is non-essential for the viral DNA replication.2. The results of Bm91:(1) Bm91is a conserved gene in the lepidopteran-specific nucleopolyhedrovirus NPV, which encodes a105-amino-acid protein with a predicted molecular weight of11.8kD. Bioinformatic analysis showed that Bm91belongs to a llkD gene family and may be involved in the formation of virus particles. Sequence analysis revealed that a typical late transcription motif TTAAG located at-13nts upstream of the initiation codon, implying that Bm91may be a late gene.(2) Bm91was expressed and antiserum to Bm91was prepared. Transcriptional analysis showed the transcription of Bm91was detected from12to96h postinfection (p.i.). The transcription initiation site of Bm91was-12nucleotides upstream of ATG. The expression of Bm91in the infected BmN cells was detected from24h to96h postinfection (p.i.). Subcellular localization showed that Bm91located both in the cytoplasm and nucleus in virus-infected cells. Western blot analysis revealed that Bm91was a specific component of the ODV envelope. Although bioinformatics analysis showed there maybe two potential N-glycosylation modification sites in Bm91, N-glycosylation inhibition test revealed that there wa? no N-glycosylated modification inBm91.(3) Bm91-knockout bacmid was constructed to study its role in viral life cycle. Further analysis indicated that although Bm91deletion did not affect the generation of infectious viruses, it did delay the killing of the infected larvae. These above results indicated that Bm91gene encoded a specific component of ODV and was associated with virulence.These results may not only contribute to the knowledge of molecular biology of BmNPV and insect cells, which help us to further understand the mechanism of viral infection and viral propagation, but also facilitate the possible practical application of the baculo viruses.