Motoko Ikeda

Molecular characterization of ovary trehalase of the silkworm, Bombyx mori and its transcriptional activation by diapause hormone.

We have isolated a cDNA encoding ovary trehalase of the silkworm, Bombyx mori. Sequence analyses revealed that the isolated cDNA contains 3143 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis showed a 3.0 kb transcript in developing ovaries carrying membrane-bound trehalase. A single copy of trehalase gene was present in the haploid genome of the silkworm. The effect of diapause hormone on the accumulation of trehalase mRNA was examined on developing ovaries in in vivo and in vitro conditions. The synthetic diapause hormone brought about a 6-fold increase in trehalase mRNA content in ovaries 4 h after injection. The similar increase was found in ovaries which were incubated in vitro with diapause hormone. Coincubation of ovaries with diapause hormone and actinomycin D could not increase the mRNA level in ovaries, and maintained a basal level which was found in ovaries incubated without diapause hormone. These results indicate that diapause hormone stimulates transcription of the trehalase gene in developing ovaries of the silkworm.

Induction of embryonic diapause and stimulation of ovary trehalase activity in the silkworm, Bombyx mori, by synthetic diapause hormone

Abstract Diapause hormones, BomDH-I[19-Cys] and BomDH-I[19-Trp], of the silkworm, Bombyx mori were chemically synthesized and used to confirm the physiological function proposed in our previous experiments performed on the partially purified hormone preparation. Injection of the synthetic hormone into day-4 pharate adults caused the moths to lay diapause eggs and the dose for a half maximal activity was estimated to be 10 pmol/pupa. The induced diapause eggs ceased embryogenesis at stage 8 as observed for natural diapause embryos. Embryogenesis was resumed by chilling at 5°C for 60 days or by HCl treatment, which was effective to break down natural diapause induced by the implantation of an active subesophageal ganglion. Synthetic diapause hormone stimulated trehalase activity in developing ovaries in both in vivo and in vitro conditions according to the same time course. The stimulated activity was comparable to that found in ovaries which developed in situ with the endogenous hormone. This diapause hormone action was suppressed by the addition of cycloheximide and actinomycin D to the incubation medium. Addition of EGTA to the medium reduced the hormonal stimulation of trehalase activity, whereas Ca 2+ addition rescued the hormone action on trehalase activity in ovaries. These results confirmed that diapause hormone is responsible for both the induction of embryonic diapause in laid eggs as well as the stimulation of trehalase activity in developing ovaries of silkworms.

Purification and characterization of proteases responsible for vitellin degradation of the silkworm, Bombyx mori

Hydrolytic activity of benzoyl-dl-arginine-p-nitroanilide (BApNA) by crude egg extracts exhibited two peaks during the latter period of embryogenesis of the silkworm, Bombyx mori. The first peak appeared at the time of marked degradation of vitellin, and consisted of two proteases. The two proteases were purified from day 8 eggs to homogeneity by DEAE-cellulose, Sephadex G-75 and native-gel electrophoresis. One enzyme had a molecular mass of 30,000 Da and the other 24,000 Da. The NH2-terminal amino acid sequences were different from one another and only the 30 k-protease showed the sequence common to the trypsin family. Both enzymes showed similar properties in Km values and optimum pH, but different temperature dependence. Inhibition spectrum and substrate specificity suggest that these enzymes belong to the trypsin-like serine protease family. The purified proteases cleaved two yolk proteins, vitellin and egg-specific protein, into small peptides through limited hydrolysis, but could not attack the 30 kDa proteins which are the second major yolk proteins of silkworms. Degradation of vitellin was characterized by the preferential hydrolysis of a 178 kDa large subunit. From the biochemical properties and developmental changes, these two proteases appear to be responsible for the degradation of vitellin in eggs undergoing embryogenesis.

Baculovirus genes modulating intracellular innate antiviral immunity of lepidopteran insect cells.

Innate immunity is essential for insects to survive infectious pathogens. In baculovirus-infected lepidopteran cells, apoptosis and global protein synthesis shutdown are major mechanisms of intracellular innate immunity that inhibit viral replication. In contrast, baculoviruses have evolved diverse genes and mechanisms to counter the antiviral immunity activated in infected cells. In this review, we summarize the current knowledge of the cellular antiviral pathways and the baculovirus genes that modulate antiviral immunity. The studies highlighted illustrate a high degree of diversity in both the cellular responses against viral infections and viral responses against intracellular antiviral immunity, providing an important basis of further studies in this field.

cDNA cloning, sequencing and temporal expression of the protease responsible for vitellin degradation in the silkworm, Bombyx mori.

1. We have cloned the cDNA encoding the vitellin (Vtn)-degrading protease (30 k Vtn protease and 24 k Vtn protease) of the silkworm, Bombyx mori, and determined the primary structure by sequencing the cDNA along with mRNA. 2. The deduced amino acid sequence comprised 264 amino acid residues and had high homology to the trypsin-like proteases of vertebrates and invertebrates. 3. Northern blot analysis using the cDNA as a probe revealed that the transcription of the Vtn protease gene occurred at the restricted stage of embryogenesis when the protease activity appeared. 4. The in vitro translation experiment demonstrated that a 32 kDa polypeptide was the primary translation product and the translation activity changed according to transcriptional activity. 5. By Western blotting using the antiserum against each Vtn protease, two enzymes were shown to share the common antigenicity, and the titer of both enzyme proteins changed closely related with activity of proteases. 6. These results led us to conclude that the biosynthesis of Vtn protease is regulated at the level of transcription.

Baculovirus IAP1 induces caspase-dependent apoptosis in insect cells

Baculoviruses encode inhibitors of apoptosis (IAPs), which are classified into five groups, IAP1-5, based on their sequence homology. Most of the baculovirus IAPs with anti-apoptotic functions belong to the IAP3 group, with certain exceptions. The functional roles of IAPs from other groups during virus infection have not been well established. We have previously shown that Hyphantria cunea multiple nucleopolyhedrovirus (HycuMNPV) encodes three iap genes, hycu-iap1, hycu-iap2 and hycu-iap3, and that only Hycu-IAP3 has anti-apoptotic activity against actinomycin D-induced apoptosis of Spodoptera frugiperda Sf9 cells. In the present study, we demonstrate that transient expression of Hycu-IAP1 is capable of inducing apoptosis and/or stimulating caspase-3-like protease activity in various lepidopteran and dipteran cell lines. Transient-expression assay analysis also demonstrates that not only Hycu-IAP1 but also IAP1s from Autographa californica MNPV, Bombyx mori NPV and Orgyia pseudotsugata MNPV (OpMNPV) are capable of inducing apoptosis, and that apoptosis induced by Hycu-IAP1 is precluded by the functional anti-apoptotic baculovirus protein Hycu-IAP3. In HycuMNPV-infected Spilosoma imparilis (SpIm) cells and OpMNPV-infected Ld652Y cells, caspase-3-like protease activity is markedly stimulated during the late stages of infection, and the caspase-3-like protease activity stimulated in HycuMNPV-infected SpIm cells is repressed by RNA interference-mediated silencing of hycu-iap1. In addition, initiator caspase Bm-Dronc, the B. mori homologue of Dronc, is cleaved upon transfection of BM-N cells with a plasmid expressing Hycu-IAP1. These results provide the first evidence that baculovirus IAP1s act to induce caspase-dependent apoptosis, possibly by replacing the cellular IAP1 that prevents Dronc activation.

Nucleotide sequence and transcriptional analysis of the DNA polymerase geneof Bombyx mori nuclear polyhedrosis virus

Abstract A gene encoding a putative DNA polymerase ( pol ) of Bombyx mori nuclear polyhedrosis virus (BmSNPV) was cloned and sequenced. The gene included an open reading frame (ORF) encoding a polypeptide of 988 amino acids with a predicted molecular mass of 114.65 kDa. The deduced amino acid sequence of the BmSNPV pol ORF showed an overall identity of 96 and 45% to those of the Autographa californica NPV (AcMNPV) pol ORF and the Lymantria dispar NPV poi ORF, respectively, and contained sequences conserved in a variety of eukaryotic and viral replicative DNA polymerases. The BmSNPV pol lacked a canonical TATAA element but contained a G + C-rich sequence in the transcriptional initiation region. Analyses by Northern blot hybridization, RNase protection assay, primer extension, and 3′ and 5′ RACE (rapid amplification of cDNA ends) showed that at least seven different transcripts of approximately 3.1 kb that shared a common 3′ end were expressed from the BmSNPV pol . The expression of these transcripts from BmSNPV pol was regulated differentially during virus infection. Transcription of five of the seven species initiated in the close vicinity of and within the motif 5′-GCGTGCT-3′. One transcript placed its initiation site within the motif 5′-AGAGCGT-3′ and the remaining one within the motif 5′-GGCGGTGG-3′. The motifs 5′-GCGTGCT-3′ and 5′-AGAGCGT-3′ have been identified in pol and other genes of AcMNPV as conserved sequences containing transcriptional initiation sites, whereas the motif 5′-GGCGGTGG-3′, which is arranged as a direct repeat in BmSNPV pol but not in AcMNPV pol , has not been defined as the sequence responsible for transcriptional initiation sites. The BmSNPV pol transcripts were detectable at 2 hr postinfection (p.i.), peaked at 10 hr p.i., and declined to a low level by 18 hr p.i. The expression of BmSNPV pol was not inhibited but delayed dramatically by the protein synthesis inhibitor cycloheximide upon treatment of infected cells, whereas aphidicolin, an inhibitor of DNA polymerase, inhibited BmSNPV pol transcription. These results suggest a complicated and unique mechanism for the regulation of BmSNPV pol expression.

Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori

We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.

Host Range Factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) Is an Essential Viral Factor Required for Productive Infection of NPVs in IPLB-Ld652Y Cells Derived from L. dispar

ABSTRACT Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.

Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line.

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuDeltaep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuDeltaep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV.