Xingwei Xiang

Autographa californica multiple nucleopolyhedrovirus odv-e66 is an essential gene required for oral infectivity.

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.

The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

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.

Bombyx mori nucleopolyhedrovirus BmP95 plays an essential role in budded virus production and nucleocapsid assembly.

Bombyx mori nucleopolyhedrovirus (BmNPV) BmP95 is a highly conserved gene that is found in all of the baculovirus genomes sequenced to date 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 by 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 cells transfected with the BmP95 deletion bacmid, indicating that deletion of BmP95 affected assembly of the nucleocapsid. This defect could be rescued by insertion of full-length BmP95 into the polyhedrin locus of the BmP95-knockout bacmid but not the N-terminal domain of BmP95. Together, these results showed that full-length BmP95 is essential for BV production and is required for nucleocapsid assembly.

Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus: A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection.

Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with B. mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus; however, there are currently no reports about B. mori. Based on previous studies that have revealed that behavior is controlled by the central nervous system, the transcriptome profiles of the brains of BmNPV-infected and non-infected silkworm larvae were analyzed with the RNA-Seq technique to reveal the changes in the BmNPV-infected brain on the transcriptional level and to provide new clues regarding the molecular mechanisms that underlies BmNPV-induced ELA. Compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499, 117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B. mori. Our analyses suggested that these genes were related to the locomotor activity of B. mori via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection. These results provide new insight into the molecular mechanisms of BmNPV-induced ELA.

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

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.

Molecular cloning and functional characterization of the dual oxidase (BmDuox) gene from the silkworm Bombyx mori.

Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5′-terminal untranslated region (5′-UTR), an 802 bp 3′-terminal region (3′-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.

Immobilization of foreign protein in BmNPV polyhedra by fusion expression with partial polyhedrin fragments

Bombyx mori nucleopolyhedrovirus (BmNPV) produces large, proteinaceous crystal matrix named polyhedra, which occlude progeny virions which are produced during infection and protect virions from hostile environmental conditions. In this study, five overlapping N-terminal fragments of the BmNPV polyhedrin ORF were cloned and ligated with the foreign gene egfp, and five recombinant baculoviruses were constructed by BmNPV(Polh(+)) Bac-to-Bac baculovirus expression system was used to co-express the polyhedrin and fused protein. The results showed that the fusion proteins were highly expressed, and the foreign proteins fused with the 100aa fragment of polyhedrin could be embedded into polyhedra at a higher ratio. This study provides a new method for efficient preservation of useful proteins for the development of new biopesticide with toxin protein and delivery vector system of vaccines.

Immobilization of foreign protein into polyhedra of Bombyx mori nucleopolyhedrovirus (BmNPV)

In the late phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection, a large amount of polyhedra appear in the infected cell nucleolus, these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment. To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV, two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus (polh+) Bac-to-Bac system, designated as vBmBac(polh+)-enhanced green fluorescent protein (EGFP) and vBmBac(polh+)-LacZ, which can express the polyhedrin and foreign protein simultaneously. Light microscopy analysis showed that all viruses produced polyhedra of normal appearance. Green fluorescence can be apparently detected on the surface of the vBmBac(polh+)-EGFP polyhedra, but not the BmNPV polyhedra. Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra. As expected, the vBmBac(polh+)-LacZ polyhedra contained an amount of LacZ and had a higher β-galactosidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra. This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.

The formation of occlusion-derived virus is affected by the expression level of ODV-E25.

Odv-e25 is a core gene of baculoviruses and encodes a 25.5 kDa 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.

Proteomics identification and annotation of proteins of a cell line of Bombyx mori, BmN cells.

A cell line is an important experimental platform for biological sciences as it can basically reflect the biology of its original organism. In this study, we firstly characterized the proteome of cultured BmN cells, derived from Bombyx mori. Total 1478 proteins were identified with two or more peptides by using 1D (one-dimensional) SDS/PAGE and LTQ-Orbitrap. According to the gene ontology annotation, these proteins presented diverse pI values and molecular masses, involved in various molecular functions, including catalytic activity, binding, molecular transducer activity, motor activity, transcription regulator activity, enzyme regulator activity and antioxidant activity. Some proteins related to virus infection were also identified. These results provided us with useful information to understand the molecular mechanism of B. mori as well as antiviral immunity.