As an Adenovirus infects a host cell a multitude of molecular interactions occur, some driven by the virus and some driven by the cell it is infecting. Many of these areas of Adenovirus biology have been intensely studied over the last half century, however, many questions remain unanswered. The aim of this study was to investigate, more closely, a long studied molecular interaction, namely the role of the tumour suppressor p53 in the Adenovirus life cycle, and also to investigate the related, but much less studied, interaction between Adenoviruses and the host cell DNA repair machinery.
Controversy surrounds the role of p53 in the Adenovirus life cycle, with current dogma favouring the view that p53 is inactivated, as it presumably presents an obstacle to a productive infection. In Chapter 3, a standardised infection protocol was developed to examine this area of Adenovirus biology more closely. This was followed with an array of cell viability and western blotting analyses that not only showed p53 was not an antagonist of the Adenovirus life cycle, but in some cases p53 acted as a protagonist. Isogenic cell lines were used to reinforce this point. Following this, data were provided that virus DNA replication was linked to the ability of an Adenovirus to kill cells. Furthermore, p53 was shown by immunofluorescence to be present in infected cells at a time that corresponded with virus DNA replication, albeit at low levels. By adding p53 back into cells, it was shown that the number of Adenovirus progeny could be stimulated to levels produced in genetically wild type TP53 cells. A selection of promoter/reporter assays and infection/transfection assays then showed how p53 might be aiding the virus life cycle. These data showed that low levels of p53 cooperated with the Adenovirus transactivator, E1A, to promote late gene expression, and this translated into a modest increase in virus late antigens in infected cells. Taken together these data show that, contrary to current dogma, p53 generally aids an Adenovirus infection and it may do this through promoting virus late gene expression.
Recent data have emerged suggesting Adenoviruses must disable the host DNA double-strand break machinery to achieve a productive infection. As this area of Adenovirus biology is in its infancy, and as p53 has recently been identified as an integral component of these DNA repair processes, the contributions of the host cell repair machinery to Adenovirus biology were examined in Chapters 4 and 5. In Chapter 4, western blotting showed that upon Adenovirus infection, a key component of the homologous recombination repair machinery, Rad51, was markedly up-regulated. This up-regulation occurred independently of other key repair proteins, and was found to be a generalised feature of an Adenovirus infection. Surprisingly, p53 did not appear to be involved in this up-regulation, and neither were several other nodal host regulatory proteins. The up-regulation was then linked to Adenovirus DNA replication using a temperature-sensitive mutant Adenovirus, ts125. In Chapter 5, functional analysis of this up-regulated protein showed that Rad51 colocalised with Adenovirus replication centres. This colocalisation coincided with a time when virus DNA replication was occurring. Furthermore, transient over-expression of Rad51 drastically increased the amount of virus progeny produced. This effect was reproduced in two very different cell types and with a selection of attenuated mutant viruses. Finally, several models were proposed that might account for this newfound effect of Rad51 on the Adenovirus life cycle.
The data presented in this thesis shows that Adenovirus not only interacts with key molecular machinery within the host cell, but also manipulates this machinery to its own end. These data add additional layers of complexity to current knowledge of the virus/host cell relationship, and thus reveal new avenues of research for future work.
| Identifer | oai:union.ndltd.org:ADTP/217721 |
| Date | January 2007 |
| Creators | Russell, Iain Alasdair, n/a |
| Publisher | University of Otago. Dunedin School of Medicine |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Iain Alasdair Russell |
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