The bertha armyworm (Mamestra configurata) is a major pest of canola and other oilseed crops. A promising control agent for this species is the baculovirus Mamestra configurata nucleopolyhedrovirus (MacoNPV). Baculoviruses are insect-specific viruses. Infections initiate in the host midgut following ingestion of virus particles called occlusion bodies. For a productive infection to occur, the occlusion bodies must dissolve to release the infectious occlusion-derived virions. These virions must pass through the peritrophic matrix, a protein-chitin meshwork that lines the midgut of most insects and provides protection against abrasion and pathogen invasion. The mechanism by which the baculovirus virions transit the peritrophic matrix is unknown. Following the initial infection of midgut cells, a second virion phenotype, the budded virus, is released from infected cells and establishes a systemic infection within the insect. The 11K group of genes, which are conserved among baculovirus species and other insect-infecting viruses, encode proteins with a predicted chitin-binding domain. The degree of conservation of these genes among insect-infecting viruses suggests that they may play a role in insect infectivity. It is possible that the gene products could be involved in an interaction between the baculovirus occlusion-derived virions and the peritrophic matrix or the chitin-secreting cells of the midgut epithelium, and therefore may be involved in initial oral infectivity. The two 11K genes from MacoNPV (ORF 118 and ORF 164), and their homologues in a second species of baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV [ORF145 and ORF150]) were expressed in a baculovirus expression system. The ability of the proteins, Maco118, Maco164, Ac145, and Ac150, to bind to chitin was assessed in vitro using chitin-coated beads. Each of the four proteins binds to chitin, and hydrophobic interactions mediate the binding. Other binding mechanisms are likely involved, but were not determined in this project. To determine the function of these proteins, a series of gene knockout and repair constructs was produced for AcMNPV ORF 145 and ORF 150 using an established bacmid system. An analysis of the knockout and repair constructs using quantitative real-time polymerase chain reaction showed that deletion of either ORF 145 or ORF 150 had no effect on the rate of budded virus production or viral DNA replication. Oral and injection bioassays were performed in Trichoplusia ni larvae to determine if there were differences in infectivity between the knockout, repair, and wild type constructs. Injection assays, in which budded virus from each construct was injected directly into the insect haemocoel, therefore bypassing the midgut and peritrophic matrix, indicated that there was no statistical difference in infectivity between the knockout, repair, and wild type constructs at a dose of 15 TCID50 U per larva. Oral bioassays, in which larvae were fed occlusion bodies from each virus construct, indicated that there was no statistical difference in mortality rates between the knockout, repair, and wild type constructs. The results from this study indicate that although the baculovirus 11K genes are highly conserved among baculovirus species, and the 11K gene products from MacoNPV and AcMNPV interact with chitin, they are not required for oral infectivity in T. ni larvae, and likely serve another function in the baculovirus infection cycle.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.

Master of Science / Division of Biology / Ana Lorena Passarelli / Baculoviruses are a large, diverse, and an ecologically-important group of entomopathogens. The ac78 gene of the prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is one of the 38 genes conserved among all baculoviruses sequenced to date. Previous studies show that Ac78 is essential for optimal production of occlusion-derived virions (ODVs) and budded virions (BVs), which are two virion types produced during baculovirus infection. However, the biochemical mechanism by which Ac78 is involved in these processes remains unknown. The AcMNPV sulfhydryl oxidase ac92 is a conserved gene, and its product, Ac92, is ODV and BV envelope-associated. Recently, the Ac78 and Ac92 homologs in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) were reported to interact and co-localize to the site of BV and ODV formation. To investigate the relationship between Ac78 and Ac92, we determined their localization in the presence and absence of AcMNPV infection, performed co-immunoprecipitations to assess interaction relationships, and provided an updated report of Ac78 and Ac92 homology with other proteins. We concluded that in the absence of viral infection, Ac78 and Ac92 localized perinuclearly in the cytoplasm and that localization of Ac92 was not affected by Ac78. During AcMNPV infection, Ac78 and Ac92 co-localized within the nucleus and surrounding virus replication and assembly sites (ring zone). Co-immunoprecipitation experiments showed that at least two differentially-tagged Ac78 proteins were part of a complex in the presence of other AcMNPV proteins. Ac78 did not associate with Ac92 during AcMNPV infection. Our characterization of the relationship between Ac78 and the AcMNPV sulfhydryl oxidase is a preliminary step in a broader effort to elucidate important biochemical pathways underlying the poorly described structural changes in capsid proteins and other proteins involved in virion stability, folding, and infectivity. In a separate project, the same approach was applied in a different virus system to determine the relationship between the small accessory protein C and the measles virus (MeV) replication complex. Co-immunoprecipitation experiments showed that during MeV infection, C associated with large protein (L) and phosphoprotein (P), which comprise the MeV replication complex, and nucleoprotein (N), which encapsidates the RNA genome. Expression constructs for full-length MeV L were generated, and L was successfully expressed following transfection. Subsequent co-immunoprecipitation experiments showed that C did not precipitate with L, P, nor N when transfected in isolation from MeV infection, indicating that another factor resulting from MeV infection is necessary for the association of C with the MeV replication complex. The results of this investigation are an important step in elucidating a biochemical mechanism underlying the function of C as a quality control factor in MeV replication. MeV has been attenuated and is a highly effective vaccine against pathogenic MeV and an active subject of clinical research as an oncolytic agent for treating a number of human cancers. Taken together, the investigations of Ac78 and C and their respective relationships with the AcMNPV sulfhydryl oxidase and the MeV replication complex adds knowledge of biochemical mechanisms underlying the important functions of small accessory proteins containing less than 200 amino acids as mediators in viral replication processes of two different viral systems.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis. / Land and Food Systems, Faculty of / Graduate

No description available.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Apoptosis is an extremely conserved process among metazoans. This dissertation will describe apoptotic regulation in two orders of insects, Lepidoptera and Diptera. In the lepidopteran host Trichoplusia ni, we describe phenotypes of infection with the baculovirus AcMNPV lacking the caspase inhibitor gene P35. In the lepidopteran host Spodoptera frugiperda, infection with this mutant virus results in apoptosis, which dramatically hinders spread of the virus in the host. In T. ni, however, infection with this mutant virus is similar to wild-type with normal spread, but the end result of liquefaction does not occur. Experiments indicated that infection of T. ni cells with the P35 mutant virus (P35Δ) resulted in caspase activation, and the P35Δ virus lacked the ability to inhibit these active caspases. With the P35Δ virus a slower entry phenotype was observed, but when the P35Δ virus was grown in the presence of a caspase inhibitor the entry phenotype was rescued. This indicated that caspases have detrimental effects on budded virions, and illustrated that P35 is needed to make robust virions. With regards to Diptera, apoptosis-regulatory genes were annotated in the yellow fever mosquito, Aedes aegypti. The genes annotated included multiple caspases and caspase regulators. Phylogenetic relationships were determined among the caspases from Anopheles gambiae, Ae. aegypti and Drosophila melanogaster, expression patterns were determined for all the annotated genes in Ae. aegypti, and one of the genes, an IAP antagonist named IMP, was functionally characterized. Further characterization of the phylogenetic relationships of caspases from fifteen dipteran species was performed by obtaining gene models for caspases of recently sequenced genomes for twelve Drosophila species and three mosquito species. Furthermore, several Drosophila and mosquito species were found to contain caspase genes with substitutions in critical active site residues. These genes were proposed to encode caspase-like decoy molecules. While these have been found in humans and nematodes, this is the first report for these molecules in insects. One of the caspase-like decoy molecules was found to increase the activity of its paralog caspase.

Le lépidoptère Spodoptera frugiperda est un ravageur important des cultures mais aussi l'organisme source de la lignée cellulaire Sf9, utilisée pour la production de protéines recombinantes dans le système d'expression baculovirus/cellule d'insecte. Dans la première partie de ce travail, nous avons étudié la transcription chez le baculovirus AcMNPV aux temps précoces de l'infection. Nous montrons tout d'abord que les régions homologues (hrs) portées par le génome de ce virus contiennent de nombreux motifs de reconnaissance pour des facteurs de transcription de type AP1/CREB, et que ces sites fixent spécifiquement des protéines de la cellule-hôte, Sf9. Par ailleurs, dans le contexte de l'infection, ces sites sont nécessaires à la transactivation médiée par le facteur viral très précoce IE1 qui se fixe lui aussi sur les hrs. Cette étude montre pour la première fois l'implication de facteurs cellulaires dans ce mécanisme de transactivation virale. La progression de ces travaux a cependant été freinée par le peu de données disponibles sur les gènes exprimés par Sf9. Pour en faciliter l'accès, nous avons donc construit une banque d'ADNc, dont l'exploitation fait l'objet de la deuxième partie de ce mémoire. Son criblage a permis d'obtenir la séquence complète de l'ADN complémentaire du gène hsp90 chez S. frugiperda, et d'en déterminer les principales caractéristiques. Par ailleurs, le séquençage à grande échelle de la banque a conduit à la constitution d'une banque d'ESTs, permettant l'identification de la quasi-totalité des gènes de protéines ribosomales. L'analyse de leur séquence a révélé l'existence de particularités qui semblent restreintes aux insectes et aux lépidoptères, un résultat inattendu compte tenu de la grande conservation de ces gènes entre espèces.

No description available.