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Title: The role of ODV structural proteins in baculovirus replication
Author: Chambers, Adam C.
Awarding Body: Oxford Brookes University
Current Institution: Oxford Brookes University
Date of Award: 2012
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Baculoviruses are arthropod-specific viruses with a circular, double-stranded DNA genome (80-180 kb). Two structural forms are produced during virus replication, comprising budded virus (BV) and occlusion-derived virus (ODV). The BV is produced from 12 hours post infection (hpi) and spreads the infection from tissue to tissue within the host. The ODV is formed 20 hpi and enveloped within occlusion bodies (OBs). The aim of this project was to elucidate the mechanisms involved in ODV production by deleting putative genes involved in oDV, but not BV production. A secondary aim of the project was to determine whether removing these genes improved the quantity/quality of recombinant proteins produced by baculovirus expression vectors. Genes for ODV structural proteins were selected and individual gene deletion mutants were generated. Three of these (Δ orf79 , Δodv-e28, and Δodv-ec43) unexpectedly prevented virus replication in insect cell culture. orf79 and odv-ec43 were demonstrated to be essential genes as viability could be restored after a rescue assay with the target gene, whereas the loss of infectivity in t-.odv-e28 probably resulted from an effect on helicase, an essential neighbouring gene. j Three deletion mutant viruses (AcΔcg30, AcΔodv-e66 and AcΔ.odv-e56) that were viable in insect cell culture were studied for any effect on ODV production and OB formation using bioassays and electron microscopy imaging. Deletion of odv-e66 and odv-e56 both negatively affected oral infectivity of OBs, whereas the deletion of cg30 increased infectivity. The electron microscopy imaging of the OBs of these deletion mutant viruses identified abnormalities for AcΔcg30POl'+ and ActΔodv-e56po1+. AcΔcg30P01+ OBs seemed to be degraded due to deformities in the polyhedron envelope (PE) while AcΔodv-e56po,+ OBs were surrounded by another protein structure but had no apparent difference in structure or ODV packaging compared to the parental virus. The formation of BV by the pif3 deletion mutant virus was reduced, although plaques were still formed in titrations of infectivity. BV production could only be restored by a rescue assay with pif3, suggesting PIF3 may have another function that is yet to be fully characterized. The deletion of orf118/pif1 and pif2 were analysed for their effect on recombinant protein production expressed from the polh promoter, using activity assays. Both the deletion of orf118/pif1 and pif2 caused a reduction in the intracellular protein, beta-galactosidase. However, the deletion of pif2 did not impact the expression of urokinase, an extracellular recombinant protein. This project also investigated through mutagenesis the possible role of the PIF1 RGD motifs in oral infection of larvae. The initial findings suggest that the PIF1 RGD motifs functional role is not very clear and indicate that PIF1 RGD motifs are not involved in integrin binding but could mediate other interactions during viral entry into the midgut cells. While the mechanism involved in the production of ODV was not elucidated, a number of interesting observations have been made about the targeted structural components of the ODV that were investigated, which could aid the understanding of the major transition from BV to ODV production during the baculovirus infection
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available