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.

Bracoviruses contain a large multigene family coding for protein tyrosine phosphatases.

The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The virions produced in the wasp ovaries are injected into host lepidopteran larvae, where virus genes are expressed, allowing successful development of the parasite by inducing host immune suppression and developmental arrest. Bracovirus-bearing wasps have a common phylogenetic origin, and contemporary bracoviruses are hypothesized to have been inherited by chromosomal transmission from a virus that originally integrated into the genome of the common ancestor wasp living 73.7 +/- 10 million years ago. However, so far no conserved genes have been described among different braconid wasp subfamilies. Here we show that a gene family is present in bracoviruses of different braconid wasp subfamilies (Cotesia congregata, Microgastrinae, and Toxoneuron nigriceps, Cardiochilinae) which likely corresponds to an ancient component of the bracovirus genome that might have been present in the ancestral virus. The genes encode proteins belonging to the protein tyrosine phosphatase family, known to play a key role in the control of signal transduction pathways. Bracovirus protein tyrosine phosphatase genes were shown to be expressed in different tissues of parasitized hosts, and two protein tyrosine phosphatases were produced with recombinant baculoviruses and tested for their biochemical activity. One protein tyrosine phosphatase is a functional phosphatase. These results strengthen the hypothesis that protein tyrosine phosphatases are involved in virally induced alterations of host physiology during parasitism.

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.

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.

Assessment of foreign protein production by recombinant Heliothis (Helicoverpa) armigera entomopoxviruses in Spodoptera frugiperda cells.

This report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.