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ABSTRACT: Bombyx mori nucleopolyhedrovirus (BmNPV) BmP95 is a highly conserved gene that is found in all of the sequenced baculovirus genomes and is also found in nudiviruses. To investigate the role of BmP95 in virus infection in vitro, a BmP95 deletion virus (vBmP95-De) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis indicated that the BmP95 deletion bacmid led to a defect in production of infectious budded virus (BV). However, deletion of BmP95 did not affect viral DNA replication. Electron microscopy showed that masses of aberrant tubular structures were present in the cells transfected with the BmP95 deletion bacmid, indicating that deletion of BmP95 affected the assembly of nucleocapsid. This defect could be rescued by insertion of full length of BmP95 into the polyhedrin locus of the BmP95-knockout bacmid, but not the N-terminal domain of BmP95. In combination, full length of BmP95 is essential for the BV production and is required for the nucleocapsid assembly.
Journal of General Virology 03/2013; · 3.36 Impact Factor
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ABSTRACT: Odv-e25 is a core gene of baculoviruses and encodes a 25.5 k Da protein located on both budded virus (BV) and occlusion-derived virus (ODV). Our previous study demonstrated that ODV-E25 was required for the formation of intranuclear microvesicles and ODV, and an odv-e25 deletion mutant could be rescued by re-expression of odv-e25 under its native promoter. To investigate the functions of ODV-E25 expression level on ODV formation, the promoter of ie-1 (pIE1), the odv-e25 native promoter, and the polyhedrin promoter (pPH) were used to direct odv-e25 expression. Our results showed that the production of ODV-E25 under its native promoter was higher than that under pIE1 but lower than that under pPH. Viral DNA replication and budded viruses (BVs) production showed that expression of odv-e25 under pIE1 and pPH could not completely repair the defects caused by the deletion of ODV-E25, while expression under its native promoter did. Electron microscopy showed that intranuclear microvesicles were found in all the constructs transfected cells except the odv-e25-null virus. However, mature ODVs only were detected in cells transfected with virus in which odv-e25 was expressed under its native or polyhedrin promoter. These results indicated that the formation occlusion-derived virus was affected by the expression level of ODV-E25.
Virus Research 01/2013; · 2.94 Impact Factor
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ABSTRACT: The peritrophic membrane lines the gut of most insects at one or more stages of their life cycles. It facilitates the digestive processes in the guts and protects from invasion by pathogens or food particles. In the current study, a novel PM protein, designated as BmMtch, was identified from the silkworm, Bombyx mori. The open reading frame of BmMtch is 888 bp in length, encoding 295 amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. They are localized on the 11th chromosome as single copy with one exon only. Quantitative real time PCR analysis (qRT-PCR) revealed that BmMtch was mainly expressed in larval fat bodies, Malpighian tubules, testis and ovaries, and could be detected through all stages of the life cycle of silkworm. Immuno-fluorescence analysis indicated that BmMtch was localized within the goblet cell of larval midgut. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch represents a novel member of insect PM proteins, without chitin-binding domains.
Molecular Biology Reports 10/2012; · 2.93 Impact Factor
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ABSTRACT: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e25 is a core gene found in all lepidopteran baculoviruses, but its function is unknown. In this study, we generated an odv-e25-knockout AcMNPV and investigated the roles of ODV-E25 in the baculovirus life cycle. The odv-e25 knockout was subsequently rescued by reinserting the odv-e25 gene into the same virus genome. Fluorescence microscopy showed that transfection with the odv-e25-null bacmid vAcBac(KO) was insufficient for propagation in cell culture, whereas the 'repair' virus vAcBac(RE) was able to function in a manner similar to that of the control vAcBac. We found that odv-e25 was not essential for the release of budded viruses (BVs) into culture medium, although the absence of odv-e25 resulted in a 100-fold lower viral titer at 24 h post-transfection (p.t.). Analysis of viral DNA replication in the absence of odv-e25 showed that viral DNA replication was unaffected in the first 24 h p.t. Furthermore, electron microscopy revealed that polyhedra were found in the nucleus, while mature occlusion-derived viruses (ODVs) were not found in the nucleus or polyhedra in odv-e25 null transfected cells, which indicated that ODV-E25 was required for the formation of ODV.
Archives of Virology 01/2012; 157(4):617-25. · 2.11 Impact Factor
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ABSTRACT: The insect peritrophic membrane (PM), separating midgut epithelium and intestinal contents, is protective lining for the epithelium and plays the important role in absorption of nutrients, and also is the first barrier to the pathogens ingested through oral feeding. In order to understand the biological function of silkworm larval PM, shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) approach was applied to investigate its protein composition. Total 47 proteins were identified, of which 51.1% of the proteins had the isoelectric point (pI) within the range of 5-7, and 53.2% had molecular weights within the range 15-45 kDa. Most of them were found to be closely related to larval nutrients metabolism and innate immunity. Furthermore, these identified proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process and cell localization. Most of the proteins had catalytic activity, binding activity and transport function. The knowledge obtained from this study will favour us to well understand the role of larval PM in larval physiological activities, and also help us to find the potential target and design better biopesticides to control pest, particularly the Lepidoptera insect.
Molecular Biology Reports 07/2011; 39(4):3427-34. · 2.93 Impact Factor
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ABSTRACT: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e66 is a core gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E66. The N-terminal 23 amino acid of the envelope protein ODV-E66 are sufficient to direct native and fusion proteins to induced membrane microvesicles and the viral envelope during infection with AcMNPV. In this study, an odv-e66-knockout bacmid can not express N-terminal hydrophobic domains was constructed via homologous recombination in Escherichia coli. The odv-e66 deletion had no effect on budded virus (BV) production and viral DNA replication in infected Sf9 cells. Larval bioassays demonstrated that injection of odv-e66 deletion BV into the hemocoel could kill P. xylostella larvae as efficiently as repaired and control viruses; however, odv-e66 deletion mutant resulted in a 50% lethal dose that was 10(3) higher than that of the repaired and control viruses when inoculated per os. These results indicated that ODV-E66 envelope protein most likely played an important role in the oral infectivity of AcMNPV, but is not essential for virus replication.
Virus Research 03/2011; 158(1-2):72-8. · 2.94 Impact Factor
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ABSTRACT: The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication.
Virus Research 01/2011; 155(1):69-75. · 2.94 Impact Factor
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ABSTRACT: The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.
Applied Microbiology and Biotechnology 02/2010; 87(1):289-95. · 3.42 Impact Factor
