Shigetoshi Miyajima

Molecular Characterization of Bombyx mori Cytoplasmic Polyhedrosis Virus Genome Segment 4

ABSTRACT The complete nucleotide sequence of the genome segment 4 (S4) ofBombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2,Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.

Nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 8.

The segments 8 (S8) of the 10 double-stranded RNA genomes from Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strains I and H were converted into cDNAs, amplified by PCR, and cloned. The nucleotide sequence analysis of the two full-length S8 cDNAs showed that the segments consist of 1328 nucleotides encoding putative proteins (p44) of 390 amino acids with molecular masses of about 44 kDa, which have glutamic acid-rich and proline-rich domains in their central regions. They had quite high identity with each other: about 98% in nucleotide and amino acid sequences. The recombinant p44 expressed in BmN4 cells using the baculovirus vector was detected by immunoblot analysis. p44 was also confirmed with the same antiserum to be present in BmCPV-infected midgut cells, but not in polyhedra, virus virions and uninfected midgut cells, indicating that p44 is expressed as a nonstructural protein of BmCPV.

Characterization of aminopeptidase N from the brush border membrane of the larvae midgut of silkworm, Bombyx mori as a zinc enzyme

Three GPI-anchored proteins, aminopeptidase N, alkaline phosphatase and alkaline phosphodiesterase I were released from the midgut brush border membrane of Bombyx mori by phosphatidylinositol-specific phopholipase C and the aminopeptidase N was purified to a homogeneous state. N-terminus and 6 internal sequences, one of which possessed part of zinc-binding motif, showed homology with those from other species. The zinc content in purified aminopeptidase N was estimated as approximately 0.72 mol/mol of the protein and 1,10-phenanthroline completely inhibited the enzyme activity, suggesting zinc requirement for the activity. The aminopeptidase N activity was inhibited not only by probestin and actinonin, but also strongly depressed by amastatin, while leuhistin and bestatin were less inhibitory. These suggest that the active site of aminopeptidase N might be structurally different from those of mammals. Calcium and magnesium ions stimulated the aminopeptidase N activity, but copper ion was rather inhibitory. Zinc ion showed bi-modal effect on the activity, i.e., stimulatory at low concentration, but inhibitory at higher than 100 microM. This inhibition was completely restored by EDTA. These results suggest that the aminopeptidase N possesses two zinc ion-binding sites with high and low affinity as essential and inhibitory one, as well as some regulatory metal-binding sites.

Induction of anti-malarial transmission blocking immunity with a recombinant ookinete surface antigen of Plasmodium berghei produced in silkworm larvae using the baculovirus expression vector system

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA, B. mori larvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 could be purified from the hemolymph of each infected larva using affinity chromatography. Immunization of Balb/c mice with BmrPbs21 induced high anti-BmrPbs21 and anti--ookinete antibodies but low anti-EcrPbs21 antibody. In contrast, EcrPbs21 induced high anti--EcrPbs21 antibody but low anti-BmrPbs21 and anti-ookinete antibodies. This suggests that most B-cell epitopes on nPbs21 are conformational and that many of the linear epitopes in EcrPbs21 are not normally exposed in nPbs21. Oocyst formation in Anopheles stephensi mosquitoes, which fed on mice immunized with purified BmrPbs21 and infected with P. berghei, was blocked by 85.5-97.1%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.

Evaluation of Spim Insect Cells Using a Novel Baculovirus Expression Vector System Employing the Hyphantria Cunea NPV

Using a lepidopteran insect cell line, FRI-SpIm-1229 (Splm), and the Hyphantria cunea nucleopolyhedrovirus (HycuNPV), we have developed a novel baculovirus expression vector system. When SpIm cell suspension culture was adapted to a serum-free medium, Sf-900 II, and scaled up to 50 ml by the simple shaker culture, no significant reduction in the cell growth as well as HycuNPV replication was observed. We have constructed a HycuNPV polyhedrin promoter-basedtransfer vector plasmid, pHcMU1, and obtained a recombinantHycuNPVexpressing an insect peptide hormone (PTTH). The recombinantPTTH was N-glycosylated and secreted in the culture supernatant, suggesting that SpIm cells could recognize and process correctly both of the N-glycosylation and signalsequences. The expression level of PTTH in HycuNPV/SpIm cell system using serum-free Sf-900 II medium was comparable to that in the BmNPV/BmN4 cell system using TC-100 medium with 10% fetal bovine serum.

Production of a Transmission Blocking Vaccine Candidate of Rodent Malaria Plasmodium Berghei Using a Baculovirus Expression Vector System

An ookinete surface antigen Pbs21 of rodent malaria, Plasmodium berghei, was successfully produced in both cultured BmN4 cells as well as in the silkworm larvae using a baculovirus expression vector system of Bombyx mori. The recombinant Pbs21 was purified from larval hemolymph of the silkworm with an affinity column and used for immunization of mice. Anti-native Pbs21 was induced in immunized mice and transmission of P. berghei to mosquito Anopheles stephensi was strongly blocked in these mice. Immunoblotting analyses suggested that the recombinant Pbs21 produced in the silkworm larvae has a structure close to native Pbs21 and is a good candidate for a transmission blocking vaccine of the malaria parasite.