Baculovirus very late expression was examined in insect cells infected with AcMNPV or mutants lacking FP-25 or FP-25 and p35 (AcdefrT). The combination of these two mutations in AcdefrT results in the appearance of the few polyhedra phenotype in TN-368 cells and enhanced budded virus production. This is accompanied by extensive plasma membrane blebbing consistent with the apoptotic response. TN-368 cells are normally resistant to apoptosis after infection with a virus lacking p35, but this appears to be overcome if FP-25 is defective. Plasma membrane blebbing is absent in cells infected with AcdefrTp35r , a virus was a reconstituted p35. However, previous studies showed that while polyhedrin gene expression was lower in cells infected with FP-25 mutants, levels of pi0 gene expression remained unaffected. The major aim of this thesis, therefore, was to determine the role(s) of sequences upstream and downstream of both very late gene promoters in regulating the reported earlier expression ofpi0 compared with polyhedrin. To determine how the formation of ultra structures by the very late proteins was affected by the dual mutations in AcdefrT, conventional light microscopy and time lapse photography were used to analyse polyhedra production and immunofluorescence confocal laser microscopy was used to assess PIO. Polyhedra production was reduced in AcdefrT- and AcdefrTp35r-infected TN368 cells in comparison to AcMNPV-infected cells. Apoptosis was visible in AcdefrT-infected TN-368 cells from 12 hpi, with the apparent release of apoptotic bodies apparent from 33.5 hpi. There was little difference in PIO localisation or formation in AcMNPV-, AcdefrT- or AcdefrTp35r-infected TN368 cells, despite the expectation that the protein might have been lost within apoptotic bodies. The promoters of polh and piO were studied in AcMNPV-, AcdefrT- and AcdefrTp35r-infected cells. Previous reports demonstrate that piO is expression earlier in infection than polh; however, this was not demonstrated using Q-PCR in TN-368 AcMNPV-infected cells. There was a reduction inpolh expression in AcdefrT- and AcdefrTp35r-infected TN-368 cells in comparison to AcMNPV infected cells. However, there was no difference in expression ofpi0 in each of the three virus-infected cells. To elucidate the region of the polh promoter affected by the FP-25 mutation, hybrid promoters were constructed between polh and piO. These promoters, two native (POIlTAA(]p°lh and pIOTAA(]pIO) and two hybrid (polllTAA(]P1O and PlOTAA(]p°lh) were inserted into the egt locus of AcMNPV, AcdefrT and AcdefrTp35r • When inserted into the same virus background both hybrid promoters acted the same. When inserted into AcMNPV and AcdefrTp35r both hybrid promoters acted as the native polh promoter. However, when inserted into AcdefrT both hybrid promoters acted as the native pi0 promoter. It was concluded that P35 must have a role in the transcription of the very late genes. The polh promoter was studied further, focusing on the essential element, the TAAG motif. The TAAG motif plus 2 upstream and 2 downstream nucleotides were mutated base-by-base to reveal essential nucleotides. Mutating the TAAG motif abolished transcription from the promoter. However upon mutating surrounding nucleotides gave little effect on gene expression in comparison to the native polh promoter. The TAAG motif was studied further by placing it into the entomopoxvirus spheroidin promoter. Alongside the native spheroidin promoter, mutant spheroidin promoters containing the TAAG motif were inserted into the polh locus ofAcMNPV. There was greater expression from the TAAG containing spheroidin promoters in comparison to the native polh promoter.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:490509 |
| Date | January 2007 |
| Creators | Pritchard, Carolyn Victoria |
| Publisher | Oxford Brookes University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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