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Title: Taking a closer look : exploring the functional roles of P10 in baculovirus-infected cells
Author: Graves, Leo P.
ISNI:       0000 0004 7430 818X
Awarding Body: Oxford Brookes University
Current Institution: Oxford Brookes University
Date of Award: 2016
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P10 is a small, highly expressed baculovirus protein whose function continues to be a mystery. Believed to be non-essential during virus infection, P10 is associated with a number of intracellular structures with suggested roles in polyhedron formation, nuclear stability and lysis. The work presented here aimed to elucidate the functional role of this protein during virus infection of cell cultures. To investigate the role of P10 during infection, a variety of Autographa californica nucleopolyhedrovirus (AcMNPV) mutants were constructed by a process of co-transfection and plaque-purification. These recombinant viruses included; a p10 deletion and associated rescue virus that confirmed that P10 is essential for nuclear lysis and the formation of an intact polyhedral envelope. Additional virus recombinants to investigate the host-cell specificity of P10 were explored with the replacement of the AcMNPV P10 coding region with that from Spodoptera frugiperda (Sf) NPV (Ac_sfp10). The resulting low expression of SfMNPV P10 resulted in the absence of a P10 perinuclear cage, nuclear lysis and induced cytoxic effects. To determine whether reduced P10 synthesis was a factor in P10 function, a set of nine AcMNPV promoter deletions, made upstream from the ATG translation site of the p10 coding region, were constructed. Analysis of these viruses showed that a decrease in P10 expression correlated with a reduction in promoter length and a concomitant decrease of nuclear lysis with reduced P10 expression. This thesis describes the first use of serial block-face scanning electron microscopy (SBF-SEM) for the study of P10 structure formation in AcMNPV-infected TN-368 cells, identifying the independent formation of cytoplasmic and nuclear P10 fibrous bodies. The use of 3D modelling of P10 highlighted an expanding nuclear “worm-like” feature and dynamic cytoplasmic structures. The cytoplasmic structures developed from thin angular fibrils that condensed to thicker fibrous structures, forming a peri-nuclear cage that remodelled over time to form a large polarised mass. The high-resolution 3D characterisation of P10 structures using SBF-SEM provided previously unparalleled data on P10 and suggests a possible mechanism in nuclear stability and then lysis. During these studies an intimate association was found between P10, electron dense spacers (EDS) and occlusion bodies. To determine whether P10 was essential for EDS formation and calyx formation, recombinant viruses absent of the p10 gene were observed using electron microscopy. These viruses confirmed EDS continues to form in the absence of P10, however, EDSs fail to associate with occlusion bodies, which result in a fragmented calyx. This indicates EDS may require the presence of P10 to associate with polyhedra and the production of an intact polyhedral envelope. This was supported from SBF-SEM of AcMNPV-infected cells that showed EDS completely encased the occlusion bodies, providing a mechanism for the role of EDS in calyx formation. This study provides new insights in to the structural features of P10 and its association with other virus structures, and establishes the functional requirement for p10 to be expressed to high levels. These results will contribute to the long running quest to understand this multifunctional protein.
Supervisor: King, Linda ; Possee, Robert ; Irons, Sarah ; Hughes, Louise Sponsor: Oxford Brookes University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral