Persistence and coexistence of engineered baculoviruses.

Baculoviruses, and in particular, the nucleopolyhedroviruses infect a wide range of arthropod hosts and have the potential to be used as biopesticides. However, one of the major drawbacks with these pathogens as biocontrol agents is that they have a slow response time. Alterations to the speed of kill and pathogen life history characteristics can influence the competitive outcome and persistence between wildtype and modified strains. Here, we explore, theoretically, how life-history modifications of pathogens can affect the epidemiology and ecology of strain coexistence. In particular, we show how under simple mass action disease transmission, life-history difference between strains are insufficient to allow coexistence. Additional heterogeneities in transmission are shown to be necessary to facilitate coexistence of wildtype and modified pathogen strains. We also illustrate how the patterns of infectivity of wildtype and modified strains can also affect long-term coexistence, and argue that appropriate assessment of genetic modifications must be presented in terms of relevant ecological theory.

Functional interaction between Cydia pomonella granulovirus IAP proteins.

Cydia pomonella granulovirus (CpGV) encodes for three inhibitor of apoptosis (IAP) proteins. One of them, CpIAP3, was previously identified based on its capacity to substitute for the p35 gene in blocking Autographa californica nucleopolyhedrovirus (AcMNPV)-induced apoptosis in SF21 cells. In this paper, the function of the other two CpGV IAP proteins, Cp94 and CpIAP5, was studied. Neither Cp94 nor CpIAP5, independently or together, can block actinomycin D-induced apoptosis in transient expression experiments. However, CpIAP5 has a significant stimulatory effect on the ability of CpIAP3 to block apoptosis. The mechanism of this stimulation remains to be elucidated. This is the first evidence of functional interactions between IAPs encoded by a single baculovirus.

Baculovirus expression vectors that incorporate the foreign protein into viral occlusion bodies.

Current baculovirus expression systems typically produce soluble proteins that accumulate within the infected insect cell or are secreted into the growth medium. A system has now been developed for the incorporation of foreign proteins, along with the matrix protein, polyhedrin, into baculovirus occlusion bodies. Initial studies showed that a recombinant virus expressing a translational fusion between polyhedrin and GFP did not form occlusion bodies. However, a baculovirus coexpressing native polyhedrin and the polyhedrin-GFP fusion protein formed occlusion bodies that fluoresced under UV light, demonstrating that they included the polyhedrin-GFP fusion protein. This was confirmed by immunoblot analysis. Thus, incorporation of a foreign protein into occlusion bodies depends on an interaction between native polyhedrin and the polyhedrin fusion protein. Electron microscopy demonstrated that the occlusion bodies containing GFP also incorporated virions as expected. These ColorPol occlusion bodies were as infectious to insect larvae as occlusion bodies produced by wild-type virus. This new system expands the capabilities for foreign gene expression by baculoviruses, which has implications for biopesticide design, novel vaccine delivery systems, and fusion protein purification applications.

Expression of the Cydia pomonella granulovirus iap3 gene.

The IAP3 protein of Cydia pomonella granulovirus (CpGV) was the first identified member of the baculovirus IAP family of proteins, which have been shown to block apoptosis in diverse systems. However, little is known of the expression and subcellular localisation of CpGV IAP3 during a viral infection. This study examined IAP3 in cells infected by CpGV and in cells infected by an Autographa californica nucleopolyhedrovirus (AcMNPV) recombinant that carried the CpGV iap3 gene. The levels of iap3 specific transcripts were monitored and production of the protein was assessed using an IAP3-specific antiserum. The data showed that iap3 is expressed during both early and late phases of infection, with a switch occurring from distal early transcription start sites to proximal late start sites. Protein levels are highest after DNA replication. IAP3 is localised exclusively in the cytoplasm. Subcellular fractionation experiments demonstrated that the protein is present in both soluble and membrane-bound cytosolic fractions. The membrane-bound fraction includes IAP3 that is associated with the mitochondria. However, the data do not support the hypothesis that release of cytochrome C from the mitochondria is involved in baculovirus-induced apoptosis.

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.

Use of whole genome sequence data to infer baculovirus phylogeny.

Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) and Granulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

Identification and in vivo characterization of the Epiphyas postvittana nucleopolyhedrovirus Ecdysteroid UDP-glucosyltransferase.

The genome of Epiphyas postvittana Nucleopolyhedrovirus (EppoMNPV) contains an ecdysteroid UDP-glucosyltransferase (egt) gene. The egt gene was completely sequenced and surrounding open reading frames identified. EppoMNPV egt is 1479 nucleotides in length encoding a protein with a predicted molecular mass of 55 kDa. Analysis of upstream sequence revealed dual TATA boxes and two CGT upstream activating region motifs. Mapping of the 5' terminus of the egt transcript identified a major transcript produced from an adenine residue 29 nucleotides downstream from the distal TATA box. No transcript was detected from a late promoter motif (GTAAG). Characterization of egt transcripts showed that poly-adenylation occurs at the 3' terminus. EppoMNPV egt transcripts were first detected in infected Epiphyas postvittana larvae by Northern hybridization at 9 h post-infection (hpi) and EGT enzyme activity was detected at 9 hpi in haemolymph from infected larvae. EppoMNPV EGT can conjugate the sugars from both UDP-glucose and UDP-galactose to ecdysone in vitro. Localization assays performed using radiolabelled ecdysone demonstrated that the conjugation of glucose or galactose, from the respective UDP-sugar, led to the prevention of the uptake of ecdysone by SF-21 cells. We propose that EGT functions to prevent the uptake of ecdysone or 20-hydroxyecdysone by metabolite and target cells respectively.

Regulation of ecdysteroid signaling: cloning and characterization of ecdysone oxidase: a novel steroid oxidase from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3alpha-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P)(+)-linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5'-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide.

The Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egt locus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigera larvae. The LT(50) of the egt deletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT(50) was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egt locus, giving a reduction in LT(50) of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.

Regulation of ecdysteroid signalling: molecular cloning, characterization and expression of 3-dehydroecdysone 3 alpha-reductase, a novel eukaryotic member of the short-chain dehydrogenases/reductases superfamily from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalysed conversion into 3-dehydroecdysone (3DE), followed by irreversible reduction by 3DE 3 alpha-reductase to 3-epiecdysone. The 3DE 3 alpha-reductase has been purified and subjected to limited amino acid sequencing. It occurs as two distinct forms, including a probable trimer of subunit molecular mass of approx. 26 kDa. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.2 kb) encoding the 26 kDa protein. Northern blotting showed that the mRNA transcript was expressed in Malpighian tubules during the early stage of the last larval instar. Conceptual translation of the 3DE 3 alpha-reductase cDNA and database searching revealed that the enzyme belongs to the short-chain dehydrogenases/reductases superfamily. Furthermore, the enzyme is a novel eukaryotic 3-dehydrosteroid 3 alpha-reductase member of that family, whereas vertebrate 3-dehydrosteroid 3 alpha-reductases belong to the aldo-keto reductase (AKR) superfamily. Enzymically active recombinant 3DE 3 alpha-reductase has been produced using a baculovirus expression system. Surprisingly, we observed no similarity between this 3DE 3 alpha-reductase and a previously reported 3DE 3 beta-reductase, which acts on the same substrate and belongs to the AKR family.

Identification and expression of two baculovirus gp37 genes.

The gp37 genes of the Mamestra brassicae and Lymantria dispar multicapsid nucleopolyhedroviruses (MbMNPV and LdMNPV) have been identified and characterized. Both genes were similar to other baculovirus gp37 genes and to entomopoxvirus fusolin genes. Phylogenetic analysis showed that baculovirus gp37 genes and entomopoxvirus fusolin genes form two distinct and well-separated clades. There was no evidence of recent gene transfer between the two groups. The gp37 genes also showed a distant similarity to bacterial cellulose- and chitin-binding protein genes, but the significance of this is unclear. MbMNPV and LdMNPV gp37 were both transcribed from consensus baculovirus late transcription start sites. MbMNPV gp37 was additionally transcribed from a putative early transcription start site. Tunicamycin treatment of MbMNPV-infected cells confirmed that MbMNPV GP37 is N-glycosylated. Confocal immunofluorescence microscopy revealed that the protein is located exclusively in the cytoplasm, probably in the endoplasmic reticulum.

Expression and structural characterization of a baculovirus ecdysteroid UDP-glucosyltransferase.

The baculovirus enzyme ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, EGT has been overproduced using a recombinant Autographa californica nucleopolyhedrovirus and an antiserum has been raised against the purified protein. This antiserum was used to visualize the kinetics of expression of EGT by wild-type AcMNPVL-1 and by the overproducing recombinant virus. The inclusion of tunicamycin during these time-course experiments suggested that EGT is glycosylated. This was confirmed by Endo F treatment, which showed that glycosylation increased the apparent subunit molecular mass by approximately 11 kDa. These sugars do not appear to be required for enzyme activity. EGT activity invariantly elutes from gel-filtration columns as a single peak corresponding to a 260 kDa (+/-50 kDa) protein. This suggests that the enzyme is an oligomer of three to five subunits, since the subunit molecular mass is 56 kDa.

Generation of baculovirus expression vectors.

The baculovirus expression system has become an important tool for the expression of heterologous genes because it has several positives attributes. First, high quantities of protein are produced because the target genes are driven by strong viral promoters. Second, most eukaryotic posttranslational modifications are carried out in insect cells in an authentic manner. Thus, proteins expressed with the baculovirus expression system usually have the same activities as the authentic protein. Several approaches have been developed to obtain recombinant baculoviruses easily and nowadays many modified baculoviral DNAs and a huge variety of transfer plasmids are available. Here, we described the rapid generation of recombinant baculoviruses using parental viral DNA that incorporates a lethal deletion and can be selected against. This basic approach should be suitable for the majority of applications.

Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes.

A 3.2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpGV) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs). ORF7L is predicted to encode a 66.7 kDa protein (594 amino acid residues) that is 57% identical (amino acid sequence) to the chiA gene (ORF126) of Autographa californica nucleopolyhedrovirus (AcMNPV), encoding a chitinase. ORF8R is 333 amino acids in length and shows high similarity (between 64% and 67%) with baculovirus cathepsins. The partial ORF, ORF5L, is related to AcMNPV ORF145 of unknown function. Phylogenetic trees were constructed for both chitinase and cathepsin sequences from baculoviruses and other species. In both cases, the baculovirus sequences were monophyletic but with a deep division between the GVs and NPVs, suggesting both genes were present in an ancestral virus prior to the separation of the two genera. However, these studies did not provide definitive evidence for the origin of either protein in baculoviruses. To investigate CpGV cathepsin function, a rescue experiment was performed using a Bombyx mori NPV (BmNPV) mutant (BmCysPD) which lacks a functional cathepsin (cath) gene. Larvae infected with BmCysPD-Cp.cat, a BmCysPD derivative carrying CpGV cath, showed similar symptoms to wild-type BmNPV infected insects, confirming that CpGV cath encodes a functional cathepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infected cells showed that CpGV cath transcription was initiated from a consensus late transcription motif (ATAAG) within the CpGV sequences, indicating that a CpGV late promoter motif was recognized in this NPV system.

Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase.

The baculovirus ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, Autographa californica nucleopolyhedrovirus EGT has been overproduced and purified, and its kinetic properties determined. The enzyme was purified 1100-fold to near-homogeneity using only two major steps, ion-exchange and gel-filtration chromatography. EGT activity was eluted from the gel-filtration column as a single peak corresponding to a 260+/-50 kDa protein, suggesting that the enzyme is an oligomer of three to five subunits, as the subunit molecular mass is approximately 56 kDa. Kinetic analysis showed that EGT has broadly similar specificities for UDP-galactose and UDP-glucose (kcat/Km=1790.8 and 902.1 respectively) when ecdysone is used as the other substrate. On the other hand, it shows marked differences in specificity for the various ecdysteroids tested. Ecdysone seems to be the optimal substrate (kcat/Km=7101.1), whereas 3-dehydroecdysone, an ecdysone precursor in Lepidoptera, is seven times less favourable (kcat/Km=1085.7). Notably, 20-hydroxyecdysone, the active form of the hormone, is conjugated very poorly (kcat/Km=31.6). Analysis of the data revealed that the enzyme mechanism involves the formation of an ecdysteroid-UDP-sugar-enzyme ternary complex. This work represents the most detailed biochemical characterization of an EGT to date.

Complete sequence and transposon mutagenesis of the BamHI J fragment of Cydia pomonella granulosis virus.

The BamHI J fragment of Cydia pomonella granulosis virus was subcloned and subjected to transposon mutagenesis in Escherichia coli using a Tn3 derivative. After screening by restriction endonuclease digestion and polymerase chain reaction (PCR), 44 clones were selected representing insertions every 100 to 300 bp. The complete sequence was compiled and the transposon insertion sites mapped precisely by sequencing. Analysis of the sequence revealed the presence of 7 potential open reading frames (ORFs). The BamHI J fragment was already known to encode IAP and OPDV-E6. Three other ORFs encode products similar to known proteins, viz. an Autographa californica nuclear polyhedrosis virus 8.6 kDa protein, a Lymantria dispar nuclear polyhedrosis virus 34 kDa protein, and vertebrate reovirus omega-1 proteins. The ORF with similarity to omega-1 is also similar to baculovirus p10 proteins. In both cases, the similarity occurs in regions likely to form a coiled-coil structure.

Characterization of the ecdysteroid UDP-glucosyltransferase gene from Mamestra brassicae nucleopolyhedrovirus.

The ecdysteroid UDP-glucosyltransferase (egt) gene of Mamestra brassicae multinucleocapsid nucleopolyhedrovirus (MbMNPV) has been cloned and characterized. MbMNPV egt potentially encodes a protein of 528 amino acids. Analysis of the substrate specificity of the MbMNPV EGT protein showed that it mirrors that of Autographa californica MNPV (AcMNPV) EGT. MbMNPV EGT also appears to be secreted from infected cells. Confirmation that the cloned gene encodes an active EGT was obtained by transient expression assays. Phylogenetic trees of NPVs were generated based on the alignment of baculovirus EGT sequences. These phylogenies support the classification of MbMNPV as a group II NPV that is most closely related to Spodoptera exigua MNPV. Comparison of the EGT-based phylogenies with polyhedrin/granulin-based phylogenies shows that the position of AcMNPV is different in the two trees, possibly indicating that AcMNPV acquired its polyhedrin gene by recombination with another virus.

Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors.

Recombinant baculoviruses were constructed that express the cDNA encoding the prothoracicotropic hormone (PTTH) of Bombyx mori. This hormone stimulates the production of ecdysteroids by the insect's prothoracic glands. Two groups of viruses were constructed, expressing either the entire cDNA encoding prepro-PTTH, or a synthetic chimeric gene encoding a signal peptide fused to the mature PTTH subunit. In both cases, the genes were expressed in wild-type Autographa californica nuclear polyhedrosis virus (AcMNPV) and in vEGTDEL, an ACMNPV mutant that lacks a functional egt gene. The egt gene is required for viral-mediated inactivation of host ecdysteroids. High levels of functional PTTH were produced only by viruses expressing the mature subunit cDNA. This recombinant PTTH resembled the native hormone by all criteria examined. The overproduction of B. mori PTTH induced higher than normal levels of haemolymph ecdysteroids but had no observable effects on the development of infected Spodoptera frugiperda larvae. However, expression of PTTH by AcMNPV was found to inhibit the pathogenicity of the virus. This effect was particularly marked in the case of viruses lacking a functional egt gene.

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.