Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori.

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.

On the classification and nomenclature of baculoviruses: a proposal for revision.

Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

Developmental expression patterns of four chemosensory protein genes from the Eastern spruce budworm, Chroistoneura fumiferana.

Chemosensory proteins (CSPs) are associated with insect sensory organs, including the sensillum lymph in some cases. However, they are also commonly expressed in nonsensory tissues that lack gustatory and olfactory neurones. We characterized the sex and development specific expression patterns of four CSP genes from the Eastern spruce budworm (ESB) using Northern blots. CfumAY426540.2 was detected at high levels in adult moths. Conversely, CfumAY426538 was expressed in all stages except adult moths, and was most abundant during late stages of the 6th instar. CfumAY701858 was expressed in all stages, while CfumAY426539 was detected less frequently, at specific developmental stages such as the 5th to 6th instar moult. During a natural moult, and a premature moult induced by the ecdysteroid agonist tebufenozide, CfumAY701858 and CfumAY426539 were up-regulated, while CfumAY426538 appeared to be down-regulated. Our results suggest that some members of the CSP gene family from the ESB may be involved in development, including moulting.

Expression analysis of a modified factor X in stably transformed insect cell lines.

A modified Factor X protein was combined with a cellulose-binding domain tag and expressed in insect cell lines. The protein, CBDFX, was expressed and secreted into the medium. Stable, transformed Hi5 and Sf9 insect cell lines were generated and tested for production of secreted CBDFX. The highest Sf9 and Hi5 CBDFX-producing cell lines were scaled up to 2-liter fermentors to evaluate production of this recombinant protein. Secreted protein production levels reached 4 mg/liter for the stable, transformed Hi5 cell line and 18 mg/liter for the stable, transformed Sf9 cell line. The protein was properly processed as determined by amino terminal sequencing and bound well to the cellulose substrate Avicel. In addition the activated recombinant CBDFX(a) was capable of recognizing and efficiently processing a Factor X cleavage site.

Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif.

Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.

The baculovirus transcriptional transactivator ie0 produces multiple products by internal initiation of translation.

Ie0 is the only gene of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) that is known to be spliced. In this study, cDNAs of ie0 were isolated, cloned, and sequenced. It was observed that IE0 contains 35 amino acids (aa) added to the N-terminus of IE1. In addition, it was found that the leader sequence of ie0 contains a 4-aa minicistron. To functionally characterize IE0, ie0 cDNAs were expressed under control of either the ie1 or the ie0 promoter. Unexpectedly, examination of ie0 translation products revealed that the predominant product from ie0 mRNAs was not IE0, but IE1. Mutation analysis showed that IE1 translation was preferentially initiated from either of two AUGs found in the first 15 nucleotides (nt) of the ie1 ORF that are internal to the ie0 ORF. It is unknown whether the internal translation initiation occurs via a leaky scanning mechanism or by an internal ribosomal entry site. Transactivation analysis with constructs that had point mutations in the ie1 AUGs and were translated only as IE0 revealed that OpMNPV IE0 is a 14- to 15-fold stronger transactivator than IE1. IE0 was also shown to be autoregulatory and to transactivate early genes in an enhancer-independent or -dependent manner. These results suggest that differential expression of baculovirus early genes can be obtained by coexpression of IE0 and IE1 in infected cells, which may permit subtle regulation of specific sets of viral genes.

Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells.

Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.

Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems.

We have produced an active form of Schistocerca gregaria ion transport peptide (ITP) in an insect cell expression system. Transformed Drosophila Kc1 cells secreted a form of ITP into the cell culture medium that was proteolytically cleaved correctly at the amino (N)-terminus. Concentrated culture supernatant from transformed Kc1 and Hi5 cells had high biological activity when tested on isolated locust ilea. Conversely, ITP expressed by baculovirus-infected Sf9 cells was larger in size and had decreased specific activity compared to ITP produced by Kc1 cells due to incorrect cleavage of the peptide at the N-terminus in the baculovirus system. This demonstrates how processing of the secreted foreign protein (ITP) expressed under the late polyhedrin promoter is compromised in a baculovirus-infected cell. Transient transformation of Kc1 cells results in supernatants containing two forms of ITP; one form (A) co-elutes with synthetic ITP and the other form (B) has reduced electrophoretic mobility. In contrast, in stably transformed Kc1 cell supernatant, ITP is expressed in a single form, which has the same electrophoretic mobility and specific biological activity as form A produced by transiently transformed Kc1 cells. Arch.

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines.

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.

Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-L) in a baculovirus/insect cell system.

We expressed an N-terminally extended Schistocerca gregaria ion transport peptide (ScgITP) and its homologue (ion transport peptide-like; ITP-L) in insect Sf9 cells using baculovirus expression vectors. Antibodies raised against peptide fragments of ITP and ITP-L were used to detect and characterize the baculovirus expressed peptides (bacITP, bacITP-L). Biological activity of the expressed peptides was assayed using the highly specific bioassay for native ITP, namely the increase in ileal short-circuit current which is a measure of active Cl- transport. BacITP and bacITP-L expression was optimal in Sf9 cells infected at a multiplicity of infection of 1, grown in Grace's medium, and harvested 2-3 days after infection. Western blots showed that bacITP was 2 kDa larger than native or synthetic ITP. This difference was not due to glycosylation and could in part be attributed to post-translational cleavage of the ITP propeptide at a site 11 amino acids upstream of the cleavage site used by S. gregaria to produce native ITP. BacITP stimulated ileal short-circuit current but is significantly less active (270-fold) than synthetic ITP (synITP) possibly as a result of the N-terminal extension. Production of bacITP-L permitted us to show that it is not stimulatory in the bioassay but reduces the synITP response in vitro and thus may have some potential for enhancing the effectiveness of biological control agents such as baculoviruses.

4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes.

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa x Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.

A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines.

A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines. Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (ie2) promoter. Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines. A chimeric synthetic bacterial-OpMNPV ie promoter-Zeocin resistance gene cassette has been included to facilitate cloning in E. coli as well as the generation of stably transformed insect cell lines. The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines.

Characterization of the acidic domain of the IE1 regulatory protein from Orgyia pseudotsugata multicapsid nucleopolyhedrovirus.

This study presents a detailed analysis of the acidic N-terminal region of the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region of Autographa californica multicapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar and Spodoptera frugiperda cells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28-43 and amino acids 113-124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.

Baculovirus immediate-early promoter-mediated expression of the Zeocin resistance gene for use as a dominant selectable marker in dipteran and lepidopteran insect cell lines.

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 microg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 microg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3-4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by beta-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.

Characterization of a unique OpMNPV-specific early gene not required for viral infection in tissue culture.

opep-2 is an Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) early gene in the ie1-ie2 gene region for which there is no homolog in either the archetype virus, Autographa californica MNPV, or Bombyx mori NPV. opep-2 is transcribed immediately upon infection as three mRNAs which initiate from a early gene motif (TATA-N27-CAGT). The expression of multiple transcripts at very early times postinfection has only been previously described for the baculovirus early gene ie1, which produces spliced mRNAs. However, distinct from ie1, the multiple mRNAs of opep-2 are due to multiple termination sites and not splicing. Western blot analysis of steady-state levels of OPEP-2 showed that in OpMNPV-infected Ld652Y cells maximum levels are obtained at 8-12 hr postinfection (p.i.) prior to DNA replication. By 48 hr p.i. OPEP-2 is shut off and is undetectable. To aid in elucidating the function of this OpMNPV-specific gene an opep-2 deletion mutant was generated and was compared to wild-type virus to determine if its absence affects viral growth in Ld652Y tissue culture cells.

Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins.

Alternative transcriptional initiation as a novel mechanism for regulating expression of a baculovirus trans activator.

In this report, we show that the Orgyia pseudotsugata nuclear polyhedrosis virus p34 gene, which is homologous to the Autographa californica nuclear polyhedrosis virus PE-38 gene, is a trans activator. The predicted p34 protein contains a number of motifs that are similar to those found in other eukaryotic transcriptional trans activators, including a putative zinc finger DNA-binding domain, a glutamine-rich domain, and a leucine zipper. Northern (RNA) blot analysis showed that the p34 gene is expressed as a 1.1-kb mRNA from 1 to 48 h postinfection and as a 0.7-kb mRNA from 18 to 120 h postinfection. Mapping of these transcripts showed that they were 3' coterminal but initiated at different 5' start sites. The 1.1-kb transcript initiates at a baculovirus early gene motif (CACAGT) and encodes the entire p34 open reading frame (ORF). The 0.7-kb transcript initiates at a baculovirus late gene start site (GTAAG) internal to the p34 ORF. Western blot (immunoblot) analysis using p34 antisera showed that the 0.7-kb transcript is translated as an amino-terminally truncated 20-kDa form of the full length 34-kDa protein. Functional analysis indicated that the 34-kDa protein transcriptionally trans activates the IE-2 promoter whereas the 20-kDa protein does not. Therefore, p34 produces two functionally different proteins from the same ORF, using the novel mechanism of alternative transcriptional initiation.

Analysis of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus trans-activators IE-1 and IE-2 using monoclonal antibodies.

We have produced monoclonal antibodies against two Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) transcriptional trans-activators, IE-1 and IE-2. Temporal analysis of IE-1 and IE-2 proteins have shown that IE-1 continues to increase in steady-state levels from 0 to 120 h post-infection, whereas IE-2 declines by 36 h post-infection. At least five different electrophoretic forms of IE-1 are present in OpMNPV-infected LD652Y cells of which three appear to be from the non-spliced IE-1 gene. Cotransfection experiments also showed that IE-1 causes a reduction in the levels of IE-2 protein but concurrently IE-2 increases IE-1 expression. Western blot analysis indicated that a form of IE-1 copurified with budded virions but did not copurify with the polyhedron-derived virus.

Characterization of an early gene coding for a highly basic 8.9K protein from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus.

A new gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified that encodes a highly basic 8.9K protein. The gene called p8.9 is expressed as a 0.5 kb transcript by 1 h post-infection and initiates at an early gene motif. The promoter of the 0.5 kb transcript has two upstream elements, repeats I and II, which are similar to motifs previously characterized in the OpMNPV IE-2 gene and the Autographa californica nuclear polyhedrosis virus IE-N and PE38 genes. A second p8.9 transcript expressed from 8 to 72 h post-infection was shown to initiate 634 bp upstream from the early gene motif in a region that has no similarity to any previously described baculovirus promoter or initiation site. Transient assays utilizing a reporter gene have shown that the p8.9 early promoter is active in a Lymantria dispar (LD652Y) and Spodoptera frugipeda (Sf9) cell lines in the absence of other viral factors. In addition, it was also demonstrated that the p8.9 promoter is trans-activated by the OpMNPV IE-2 and p34 genes, but not by IE-1.

Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus.

A second immediate early (IE) regulatory gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified. The IE-2 gene which is homologous to the IE-N gene of Autographa californica MNPV was mapped to the HindIII A fragment of OpMNPV between 0.41 to 1.37 map units. The IE-2 gene codes for a predicted protein of 45,640 Da and analysis of the amino acid sequence shows that the protein has a highly basic amino terminal domain and a cysteine-rich domain that is similar to a zinc finger motif that is also found in the baculovirus proteins GC30 and PE-38. The IE-2 gene is expressed as a 1.3-kb transcript that was detectable by 0.5 hr postinfection (hr p.i.), reached maximum steady state levels by 6 hr p.i., and declined slightly by 48 hr p.i. Cis-acting 5' regulatory sequences were analyzed by deletion analysis of the IE-2 promoter linked to the reporter gene chloramphenicol acetyl transferase. Maximum expression was obtained when the IE-2 promoter contained sequence 275 bp upstream from the transcriptional start site. trans-Activation analysis revealed that IE-2 trans-activated the IE-1 promoter and in addition appeared to be autoregulatory.