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Title: Studies of adeno-associated virus-induced DNA damage responses
Author: Garner, Elizabeth Anne
ISNI:       0000 0004 2668 9221
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2008
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Adeno-associated virus (AAV) infection triggers a DNA damage response in the cell. This response is not induced by viral proteins but by the structure of AAV ssDNA being recognized by the cell as damaged DNA. The consequence of this is the killing of cells lacking p53 activity. We have observed that cells that lack p21 or pRb activity are also sensitive to AAV-induced cell death. We report that cells respond to AAV infection by activating two DNA damage-signaling cascades. The first activates the p84N5 protein, which in turn activates caspase-6, leading to cell death. The second cascade activates the p53-21-pRb pathway, which inhibits activation of the p84N5 protein and thus prevents cell death. The result of the antagonistic interaction between these two pathways is that cells that do not exhibit functional p53-p21-pRb signaling undergo apoptosis as a consequence of AAV infection. Cells with a functional p53- 21-pRb pathway are refractory to AAV-induced cell death. These results show that p53, although a pro-apoptotic protein, together with pRb and p21 proteins, is a member of an anti-apoptotic cellular mechanism. As such, these experiments reveal features that may be exploited to specifically kill cells that lack the p53-p21-pRb pathway, such as cancer cells. We also consider the role of DNA damage signaling in the restriction of AAV replication. We report that helper viruses may inhibit ATM, ATR and DNA-PK signaling and that this may be advantageous for AAV replication. Inhibition of ATM and DNA-PK in tissue culture cell lines led to an increase in AAV replication. We show that ATM, ATR and DNA-PK may be responsible for the hyperphosphorylation of replication protein A (RPA) in response to AAV infection. We speculate that hyperphosphorylated RPA may be a fundamental restriction factor for viral replication and delineate novel experimental tools to test such a hypothesis. The implications of these observations for oncolytic therapy are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available