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PubMed1995

The wild-type Autographa californica nuclear polyhedrosis virus induces apoptosis of Spodoptera littoralis cells.

Spodoptera littoralis cells infected with the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) yielded significantly lower budded virus titers than Spodoptera frugiperda-infected cells and produced very low levels of polyhedrin. Relative to AcMNPV-infected S. frugiperda SF9 cells viral DNA replication was severely reduced in Spodoptera littoralis SL2 cells. Microscopic examination of SL2-infected cells revealed progressive cell blebbing starting at 6-8 hr postinfection and culminating in total cell destruction at 24 hr postinfection. The data suggested that AcMNPV-infected SL2 cells undergo apoptosis. The occurrence of an active apoptotic process in the infected cells was confirmed by: (1) observation of fragmentation of the cell nuclei stained with the specific fluorescent dye DAPI (4',6'-diamidino-2-phenylindole) and (2) the presence of low-molecular-weight DNA oligomers. Neither SL2 cells infected with S. littoralis nuclear polyhedrosis virus (SINPV) nor SF9 cells infected with AcMNPV, respectively, showed nuclear fragmentation or oligonucleosomal ladder formation.

Animals↗
PubMed1996

Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC.

Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI proteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.

Amino Acid Sequence↗
PubMed2005

Spodoptera littoralis caspase-1, a Lepidopteran effector caspase inducible by apoptotic signaling.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can successfully infect Spodoptera frugiperda SF9 cells, but in contrast, in Spodoptera littoralis SL2 cells it induces apoptosis aborting the infection. To understand better the mechanism of induction and execution of apoptosis in SL2 cells, we identified and characterized the first Spodoptera littoralis caspase, Sl-caspase-1. Sl-caspase-1 is an effector caspase that cleaves DEVD but not IETD and LEHD substrates, and the caspase-3 inhibitor DQMD-CHO inhibited this activity. It was involved in two apoptotic pathways induced by UV irradiation and virus infection. Moreover processing of Sl-caspase-1 was a determinant factor for baculovirus induction of apoptosis in SL2 cells. Since very little is known on the regulation of expression of Lepidopteran caspases, we studied Sl-caspase-1 expression after exposure to apoptosis stimuli. We found that triggering apoptosis in SL2 cells increased the steady-state level of Sl-caspase-1 without changing the level of sl-caspase-1 mRNA, suggesting that Sl-caspase-1 was post-transcriptionally up regulated. This regulation might occur as an early event in transduction of the apoptotic signal.

Amino Acid Sequence↗
PubMed2006

Delta11 desaturases of Trichoplusia ni and Spodoptera littoralis exhibit dual catalytic behaviour.

The Delta(11) desaturases found in moths such as Spodoptera littoralis play a critical role in the biosynthesis of their sex pheromones. The ability to functionally express these enzymes in yeast has allowed one to study the transformation of long-chain fatty acyl substrates to their 11-ene products in greater mechanistic detail. In this article, we report on the detection and quantitation of a minor 11-hydroxylated byproduct (0.1% of total fatty acids), which is formed by the Delta(11) desaturases found in Trichoplusia ni and Spodoptera littoralis. The position of the hydroxyl group was determined by characteristic mass spectral fragmentation of the trimethylsilyl derivatives and is in accord with predictions based on previous mechanistic investigations of the Spodoptera Delta(11) desaturase. The level of 11-hydroxylation was insensitive to the mode of desaturase expression (constitutive vs. induced) and the presence or absence of a b5-fusion domain. Our findings suggest that in future, a search for hydroxylated products should be included in functional analyses of insect desaturase genes.

Animals↗
PubMed2005

Nucleotide sequence and transcriptional analysis of the pif gene of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV).

Defective viruses, not transmissible alone, increase the transmissibility of complete genotypes in natural populations of Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV). The defective phenotype is associated with a 15 kb deletion, which includes the pif (per os infectivity factor) gene. The sequence of a 2.4 kb fragment that includes pif was determined. Multiple transcripts encompassing pif were detected by Northern blot analysis. RT-PCR and nuclease protection analysis demonstrated the presence of run-through transcripts starting upstream of pif. A 2.0 kb messenger started from a CTAAG promoter motif located 11 nt upstream of the pif start codon, and ended 450 nt downstream from the pif stop codon. This pif mRNA included a small downstream ORF (homologous to Se37). A transcript of 0.8 kb was detected that may correspond to a specific transcript from this small ORF. This transcript would start at a late consensus motif internal to pif coding sequences, ending at the same polyadenylation signal as the pif transcript. These transcription features resemble those of pif transcription in Spodoptera littoralis NPV, although the genomic location of pif is not equivalent in the two viruses. SfMNPV pif can encode a protein of 529 amino acids, closely related to Spodoptera exigua MNPV PIF.

Amino Acid Sequence↗
PubMed1997

Characterization of the polyhedrin gene of Spodoptera litura nuclear polyhedrosis virus.

The polyhedrin gene (polh) of a characteristically distinct Spodoptera litura nuclear polyhedrosis virus isolate (SIMNPV) is identified in the HindIII-F fragment of the viral DNA. The nucleotide sequence of the 1057 base pair (bp) region of this fragment contains an open reading frame (ORF) without any intervening sequence for coding a polypeptide of 246 amino acids. Analysis of the nucleotide sequence and deduced amino acid sequence indicate that this has more than 70% sequence identity to known polyhedrins. The coding region is preceded by an AT rich region containing the conserved late promoter motif TAAG. The upstream promoter and coding regions of this polh gene are more similar to polh of the NPVs of Spodoptera frugiperda (Sf), Spodoptera exigua (Se) and Panolis flamea (Pf).

Amino Acid Sequence↗
PubMed2006

SPODOBASE: an EST database for the lepidopteran crop pest Spodoptera.

BACKGROUND: The Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations. DESCRIPTION: The ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary. CONCLUSION: The SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL: http://bioweb.ensam.inra.fr/spodobase.

Animals↗
PubMed1998

Identification and characterization of spodoptera frugiperda furin: a thermostable subtilisin-like endopeptidase.

Spodoptera frugiperda (Sf9) cells are widely employed for high-level expression of heterologous recombinant genes from baculovirus vectors. Using a plasmid library encoding cDNA of Sf9 cells we have identified here the Spodoptera frugiperda analog of the proprotein convertase furin which plays an important role in posttranslational protein processing. Spodoptera frugiperda furin (Sfurin) is closest related to Drosophila melanogasterfurin with which it shares an extended cysteine-rich domain, whereas mammalian furin shows high homology only in the catalytic domain. Mammalian furin and Sfurin were further compared by expression from baculovirus vectors. Substrate specificity and inhibitor profiles are identical for Sfurin and mammalian furin, whereas calcium-dependence, pH-optimum, and thermostability differ. Cleavage of recombinant influenza virus hemagglutinin was significantly enhanced in Sf9 cells after overexpression of Sfurin.

Amino Acid Sequence↗
PubMed2005

[Construction of a baculovirus transfer vector and expression of baculovirus-mediated gfp gene in larvae of Spodoptera litura].

To construct a novel baculovirus expression system of Spodoptera litura multicapsid nucleopolyhedrovirus, the 5' end and 3' end-flanking fragments of ph gene were amplified from the genome DNA of SpltMNPV, Japan-C3 strain using two pairs of primers synthesized according to SpltMNPV China-G2 strain genome DNA sequence published in GenBank. To obtain the transfer vector pSplt-gfp, the fragment of gfp gene was inserted into this vector between two fragments tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully. It will be helpful to establish Spodoptera litura multicapsid nucleopolyhedrovirus expression system and more effective pesticide for Spodoptera litura.

Animals↗
PubMed1993

Phosphorylation of elongation factor-2 from the lepidopteran insect, Spodoptera frugiperda.

In mammalian cells, protein synthesis can be regulated at the level of elongation by the phosphorylation of elongation factor 2 (eEF-2) by a highly specific Ca2+/calmodulin-dependent kinase. In this report, we show that eEF-2 from a cell line derived from the insect, Spodoptera frugiperda, is a substrate for mammalian eEF-2 kinase and that phosphorylation is Ca(2+)-dependent. Furthermore, two-dimensional peptide mapping shows that the kinase phosphorylates the same sites in Spodoptera eEF-2 as those phosphorylated in the rabbit protein. However, we were unable to detect an eEF-2 kinase in Spodoptera cells.

Amino Acid Sequence↗
PubMed2026

Construction of an infectious clone of Spodoptera frugiperda densovirus and its biological characteristics.

Densoviruses are highly pathogenic to their insect hosts and have great potential for biocontrol. Spodoptera frugiperda densovirus (SfDV) was isolated from diseased larvae of Spodoptera frugiperda, while its biological functions remain unclear. Herein, we successfully constructed an infectious clone of SfDV. The S. frugiperda larvae transfected with the infectious clone exhibited anorexia, stunted growth, and reduced activity. Histopathological analysis further showed that the epidermis, fat body and trachea were infected instead of muscle and midgut tissues. Transmission electron microscopy (TEM) revealed that numerous virions of about 22 nm were distributed within both the nucleoplasm and cytoplasm of epidermal cells. Moreover, many virions were also found contained within vesicles in the cytoplasm. The replication kinetics of the rescued SfDV (rSfDV) was similar to that of the parental SfDV. The median lethal dose (LD50) and median lethal time (LT50) values of rSfDV were 6.63 × 107 viral genome copies (vgc), 5.23 d, respectively, which were also comparable to those of the parental SfDV. Taken together, the infectious clone of SfDV provides an important tool for further exploring the genome function, pathogenesis, and interactions with its hosts.

Animals↗
PubMed1996

Gaucher disease: functional expression of the normal glucocerebrosidase and Gaucher T1366G and G1604A alleles in Baculovirus-transfected Spodoptera frugiperda cells.

Gaucher disease is an inherited sphingolipidosis resulting from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1 as non-neuronopathic, type 2 as acute neuronopathic, and type 3 as subacute neuronopathic. We recently identified a rare mutation (G-->A at glucocerebrosidase cDNA nucleotide position 1604) [Choy et al., 1994a, Am J Med Genet 51:156-160] and a novel mutation (T-->G at glucocerebrosidase cDNA nucleotide position 1366) in two type 1 Gaucher patients by sequence analysis of the entire glucocerebrosidase coding region [Choy et al., 1994a, 1994b, Hum Mol Genet 3:821-823]. To demonstrate that these are deleterious and not neutral mutations, we cloned the full-length glucocerebrosidase cDNA of patients and of a normal control in the plasmid vector pAcUW1, recombined the human gene into the Baculovirus genome downstream of its polyhedron p10 promoter, and expressed the inserted gene in cultured cells of Spodoptera frugiperda transfected by recombinant Baculovirus. The levels of residual glucocerebrosidase activity determined in transfected cells with the Gaucher G1604A and T1366G alleles are 6.9% and 2.9% of that expressed by the normal allele (normal = 352.0 nmol/hr/mg protein or 100%). By comparison, the enzyme-specific activity expressed in transfected cells by 2 known Gaucher alleles, A1226G and T1448C, that are prevalent in type 1 and type 2 Gaucher disease are 23.4% and 3.3% of normal. No endogeneous glucocerebrosidase activity was detected in cultured cells transfected by either the wild-type Baculovirus or Baculovirus with the pAcUW1 plasmid vector without the glucocerebrosidase cDNA insert. These findings show that the Baculovirus expression system in cultured Spodoptera frugiperda cells is a suitable system for the functional expression and characterization of the normal and mutant glucocerebrosidase alleles. Moreover, the use of this expression system demonstrates that the G1604A and T1366G mutations are both deleterious mutations resulting in profoundly deficient glucocerebrosidase activity and subsequent Gaucher disease.

Adult↗
PubMed1995

Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae.

A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E. coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication. In S. exigua larvae, permissive Spodoptera spp. cultured cells, and nonpermissive D. melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i. Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae. Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry. Analysis of infected S. exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection. Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production. Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.

Animals↗
PubMed1999

Characterization of Spodoptera littoralis type B nucleopolyhedrovirus infection in selected insect cell lines.

We have examined Spodoptera littoralis type B nucleopolyhedrovirus (SpliNPV) infections in CLS79, Sf9, and Se1 cells derived from lepidopteran insects of the genus Spodoptera (Family: Noctuidae), Ld652Y cells from Lymantria dispar (Family: Lymantriidae), and Md210 cells from Malacosoma disstria (Family: Lasiocampidae). CLS79, Sf9, and Se1 cells were permissive for SpliNPV infection as these cell lines supported complete viral DNA replication, virus-specific transcription, and production of viable progeny. Neither Ld652Y nor Md210 cells supported production of viable SpliNPV progeny. Ld652Y cells supported limited viral DNA replication and displayed reduced and delayed transcription of viral-specific RNAs. Md210 did not support viral DNA replication and displayed dramatically reduced transcription of viral-specific RNAs. We used transient expression assays as an indirect measure of the translation of SpliNPV early gene products in Sf9, Ld652Y, and Md210 cells. While transactivation of viral promoter-mediated luciferase expression occurred in SpliNPV-infected Ld652Y cells, little to no transactivation activity was detected in SpliNPV-infected Md210 cells. Our data indicated that the block to productive SpliNPV infection in Ld652Y and Md210 cells may be at the level of viral RNA transcription and further suggested that host factors play an important role in productive SpliNPV infection.

Animals↗
PubMed2004

Development of a hybrid delta-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura.

A hybrid delta-endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known delta-endotoxins. The hybrid delta-endotoxin was created by replacing amino acid residues 530-587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III. The truncated delta-endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilised Cry1EC made from E. coli was 4- fold more toxic to the larvae of S. litura than Cry1Ca, the best known delta-endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. coli caused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilised from inclusion bodies caused mortality of S. litura with LC50 513 ng/ml semi synthetic diet. A synthetic gene coding for the hybrid delta-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes. Transgenic, single copy plants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1-0.7% of soluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in all stages of larval development. Hence, unlike in E. coli, the hybrid delta-endotoxin folded into a functionally active conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC50 58 ng/ml diet) compared to expression in E. coli.

Amino Acid Sequence↗
PubMed2005

Physical and partial genetic map of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genome.

A Nicaraguan isolate of Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) is undergoing field trials for control of this pest in the Americas. This isolate is composed of multiple genotypes, some of which are deletion mutants. Identification of the genetic changes in deleted genotypes cannot be accomplished without the construction of a detailed physical map. In the present study, combinations of restriction endonuclease analysis and Southern blot analysis was performed. This map was refined by sequencing the termini of cloned restriction fragments. The SfMNPV genome was estimated to be 129.3 kb, 8 kb larger than the previously characterized Sf-2 variant from the United States, due to a deletion between 14.8 and 21.0 m.u. in the physical map described in this study. A total of 27.92 kb were sequenced, which represented 21.5% of the whole genome and included 38 ORFs. Comparison with other sequenced baculoviruses revealed that SfMNPV displayed the highest sequence identity (66%) and gene arrangement (78%) with Spodoptera exigua MNPV, sharing 36 putative ORFs. In addition, the genome organization was similar to that of SeMNPV, with minor differences. Phylogenetic analysis confirmed the close relatedness between SeMNPV and SfMNPV, suggesting they evolved from a common ancestor.

Animals↗
PubMed2005

Identification of the apoptosis inhibitor gene p49 of Spodoptera litura multicapsid nucleopolyhedrovirus.

Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). Computer-assisted analysis indicated that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) ORF55 (designated as the p49 gene) display 79 and 31% amino acid identity with Spodoptera littoralis (Spli)MNPV P49 and Autographa californica (Ac)MNPV P35, respectively, Splt MNPV putative P49 contains a peptide cleavage site TVTDG recognized by death caspases. In marker rescue assay, Splt-p49 was able to suppress apoptosis induced by infection of a mutant AcMNPV deficient in p35 and rescue the mutant virus replication from apoptosis in Sf-9 cells.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of odv-e25 of Spodoptera litura multicapsid nucleopolyhedrovirus.

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) odv-e25 is 684 bp long, potentially encoding 227 amino acids with a predicted molecular weight of 24.9 kDa. Homology analysis indicated that SpltMNPV ODV-E25 has 35-65% amino acid identity with that of other known baculoviruses. RT-PCR results revealed that the odv-e25 is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (TTAAG). Western blot analysis of odv-e25 expression with an antiserum made against 6 x His tagged ODV-E25 expressed in Escherichia coli indicated that it was present as a doublet of approximately 27 kDa from 24 h through 96 h in SpltMNPV-infected Spli-221 cells. Similar results were seen on Western blots of Spodoptera exigua (Se)MNPV-infected Se301 cells. Immunofluorescence analysis showed that ODV-E25 was predominantly present in the cytoplasm of SpltMNPV-infected cells and localized to the envelopes of occlusion-derived virus.

Amino Acid Sequence↗