Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells.

The polydnavirus Toxoneuron nigriceps bracovirus (TnBV) is an obligate symbiont associated with the braconid wasp T. nigriceps, a parasitoid of Heliothis virescens larvae. Previously, to identify polydnavirus genes that allow parasitization by altering the host immune and endocrine systems, expression patterns of TnBV genes from parasitized H. virescens larvae were analysed and cDNAs were obtained. To study the function of the protein from one such cDNA, TnBV1, overexpression of the protein was attempted by using the baculovirus Autographa californica multicapsid nucleopolyhedrovirus. Recovery of stable recombinant virus was unsuccessful, with the exception of recombinants with deletions/mutations within the TnBV1 gene. It was hypothesized that TnBV1 expression was cytotoxic to the Spodoptera frugiperda (Sf21) insect cells that were used to produce the recombinants. Therefore, the Bac-to-Bac system was used to create recombinant baculoviruses maintained in Escherichia coli expressing either TnBV1 (Ac-TnBV1) or an initiator-methionine mutant [Ac-TnBV1(ATG-)]. Microscopy revealed substantial cell death of Sf21 and High Five cells from 48 h post-infection with Ac-TnBV1, but not with the Ac-TnBV1(ATG-) recombinant virus. Ac-TnBV1-infected Sf21 cells, but not those with parental virus infection, showed an increased caspase-3-like protease activity, as well as increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) for breaks in host genomic DNA. Although indicative of apoptosis, blebbing and apoptotic bodies were not observed in infected cells. Transiently expressing TnBV1 alone caused TUNEL staining in High Five cells. These data suggest that TnBV1 expression alone can induce apoptosis-like programmed cell death in two insect cell lines. Injection of Ac-TnBV1 budded virus, compared with parental virus, did not result in an alteration of virulence in H. virescens larvae.

Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner.

The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.

Ancient coevolution of baculoviruses and their insect hosts.

If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary interactions between baculoviruses and their hosts.

Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda cells: a global analysis of host gene regulation during infection, using a differential display approach.

Autographa californica nucleopolyhedrovirus (AcMNPV), the type member of the virus family Baculoviridae, infects pest insects and has been the subject of many studies for its development as a biopesticide. It is also the virus upon which most of the commercial baculovirus protein expression systems are based. AcMNPV infection of cultured host Spodoptera frugiperda (Sf9) cells can induce a number of alterations of host cell properties including altering the cellular cytoskeleton, an arrest of the cell cycle in G(2)/M, and the global shutoff of host protein translation. Additionally, several cellular transcripts have been shown to be down-regulated following AcMNPV infection. In this study, we take a differential display approach to address whether a global down-regulation of Sf9 host transcripts occurs at late times of infection. Additionally, we also use this approach to search for host mRNAs which are up-regulated at early times of infection, and may be important for facilitating baculovirus infection. From these experiments we can confirm a global down-regulation of Sf9 mRNA levels at late times of infection. We also found that up-regulation of individual host gene RNA levels at early times of infection did not occur frequently. One host transcript which was found to be transiently up-regulated as a result of AcMNPV infection was an Sf9 Hsc70 gene. Hsc70 proteins have been shown to play a vital role in the life-cycle of other large DNA viruses, which suggests that this protein is also important for baculovirus infection.

An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin.

Baculovirus occlusion bodies, large proteinaceous structures which contain virions, have recently been engineered to incorporate foreign proteins. The major constituent protein of occlusion bodies from the baculovirus Autographa californica nucleopolyhedrovirus is polyhedrin, and assembly of recombinant occlusion bodies which incorporate a foreign protein depends on an interaction between native polyhedrin and a polyhedrin-foreign protein fusion. This technology has now been applied to the generation of a recombinant baculovirus (ColorBtrus) that produces occlusion bodies incorporating the Bacillus thuringiensis (Bt) insecticidal Cry1Ac toxin protein. ColorBtrus coexpresses native polyhedrin and a fusion protein in which polyhedrin is fused to the Bt toxin, which is in turn fused to green fluorescent protein (GFP). Analysis of ColorBtrus occlusion bodies confirmed that they include both Bt toxin and GFP, yet still incorporate virions. Bioassay of ColorBtrus demonstrated that its speed of action and pathogenicity are strikingly enhanced compared to wild-type virus. ColorBtrus represents a novel, powerful biological insecticide that combines positive attributes of both Bt toxin and baculovirus based systems.

A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni.

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

The genome sequence and evolution of baculoviruses.

Comparative analysis of the complete genome sequences of 13 baculoviruses revealed a core set of 30 genes, 20 of which have known functions. Phylogenetic analyses of these 30 genes yielded a tree with 4 major groups: the genus Granulovirus (GVs), the group I and II lepidopteran nucleopolyhedroviruses (NPVs), and the dipteran NPV, CuniNPV. These major divisions within the family Baculoviridae were also supported by phylogenies based on gene content and gene order. Gene content mapping has revealed the patterns of gene acquisitions and losses that have taken place during baculovirus evolution, and it has highlighted the fluid nature of baculovirus genomes. The identification of shared protein phylogenetic profiles provided evidence for two putative DNA repair systems and for viral proteins specific for infection of lymantrid hosts. Examination of gene order conservation revealed a core gene cluster of four genes, helicase, lef-5, ac96, and 38K(ac98), whose relative positions are conserved in all baculovirus genomes.

Functional and phylogenetic analyses of a putative Drosophila melanogaster UDP-glycosyltransferase gene.

Glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. The recent determination of the complete genome sequence of Drosophila melanogaster has revealed the presence of over 30 putative UDP-glucosyltransferase (UGT) genes in this organism. We report here the molecular cloning and functional characterisation of one of these genes, named DmUgt37a1. The predicted protein comprises 525 amino acids and has about 30% overall amino acid identity with vertebrate members of the UGT family. The phylogenetic relationships of DmUgt37a1 with other members of the UGT family from D. melanogaster are discussed. DmUgt37a1 was expressed in lepidopteran insect cells and the ability of the enzyme to conjugate 38 potential substrates belonging to diverse chemical groups was assessed using UDP-glucose as sugar-donor. However, no activity was detected with any compound under the conditions used and thus, the substrate specificity of the enzyme remains unknown.

Whole genome analysis of the Epiphyas postvittana nucleopolyhedrovirus.

The nucleotide sequence of the Epiphyas postvittana nucleopolyhedrovirus (EppoMNPV) genome has been determined and analysed. The circular dsDNA genome contains 118584 bp, making it the smallest group I NPV sequenced to date. The genome has a G+C content of 40.7% and encodes 136 predicted open reading frames (ORFs), five homologous repeat regions and one unique repeat region. Of the genome, 92.9% encodes predicted ORFs and 2.2% is in repeat regions; the remaining 4.9% of the genome comprises nonrepeat intergenic regions. EppoMNPV encodes homologues of 126 Orgyia pseudotsugata MNPV (OpMNPV) ORFs and 120 Autographa californica MNPV ORFs, with average identities of 64.7 and 53.5%, respectively. Between the four sequenced group I NPVs, 117 ORFs are conserved, whereas 86 ORFs are conserved between all fully sequenced NPVs. A total of 62 ORFs is present in all baculoviruses sequenced to date, with EppoMNPV lacking a homologue of the superoxide dismutase (sod) gene, which has been found in all other fully sequenced baculoviruses. Whole genome phylogenetic analyses of the ten fully sequenced baculoviruses using the sequences of the 62 shared genes, gene content and gene order data sets confirmed that EppoMNPV clusters tightly with OpMNPV in the group I NPVs. The main variation between EppoMNPV and OpMNPV occurs where extra clusters of genes are present in OpMNPV, with sod occurring in one such cluster. EppoMNPV encodes one truncated baculovirus repeated ORF (bro) gene. The only repeated ORFs are the four iap genes. Eight, randomly distributed, unique ORFs were identified on EppoMNPV, none of which show any significant homology to genes in GenBank.

Characterization of a novel silkworm (Bombyx mori) phenol UDP-glucosyltransferase.

Sugar conjugation is a major pathway for the inactivation and excretion of both endogenous and exogenous compounds. We report here the molecular cloning and functional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori, which was named BmUGT1. The complete cDNA clone is 1.6 kb, and the gene is expressed in several tissues of fifth-instar larvae, including fat body, midgut, integument, testis, silk gland and haemocytes. The predicted protein comprises 520 amino acids and has approximately 30% overall amino-acid identity with other members of the UGT family. The most conserved region of the protein is the C-terminal half, which has been implicated in binding the UDP-sugar. BmUGT1 was expressed in insect cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the enzyme. The expressed enzyme had a wide substrate specificity, showing activity with flavonoids, coumarins, terpenoids and simple phenols. These results support a role for the enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant allelochemicals. This work represents the first instance where an insect ugt gene has been associated with a specific enzyme activity.

The complete sequence of the Cydia pomonella granulovirus genome.

The nucleotide sequence of the DNA genome of Cydia pomonella granulovirus (CpGV) was determined and analysed. The genome is composed of 123500 bp and has a G+C content of 45.2%. It contains 143 ORFs of 150 nucleotides or more that show minimal overlap. One-hundred-and-eighteen (82.5%) of these putative genes are homologous to genes previously identified in other baculoviruses. Among them, 73 are homologous to genes of Autographa californica nucleopolyhedrovirus (AcMNPV), whereas 108 and 98 are homologous to genes of Xestia c-nigrum GV (XcGV) and Plutella xylostella GV (PxGV), respectively. These homologues show on average 37.4% overall amino acid sequence identity to those from AcMNPV and 45% to those from XcGV and PxGV. The CpGV gene content was compared to that of other baculoviruses. Several genes reported to have major roles in baculovirus biology were not found in the CpGV genome, such as gp64, the major budded virus glycoprotein gene in some nucleopolyhedroviruses, and lef-7, involved in DNA replication. However, the CpGV genome encodes the large and small subunits of ribonucleotide reductase, three inhibitor of apoptosis (iap) homologues and two protein tyrosine phosphatases. The CpGV, PxGV and XcGV genomes present a noticeably high level of conservation of gene order and orientation. A striking feature of the CpGV genome is the absence of typical homologous repeat sequences. However, it contains one major repeat region and 13 copies of a single 73-77 bp imperfect palindrome.