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The relationship of adenoviruses to the DNA damage response protein BLM & Understanding the modulation of DNA repair pathways by the human oncogenic virus KSHV

Ryan, Ellis (2011)
M.Res. thesis, University of Birmingham.

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Project 1:
Adenoviruses (Ad) are able to interact with a number of proteins involved in the cellular response to DNA damage, in order to prevent concatamerisation of the viral genome and inhibition of viral replication. It is becoming increasingly apparent that the adenoviral E3 ligase complex (comprised of E1B-55K and E4 adenoviral proteins) is able to target a number of cellular DNA damage response proteins for proteasomal degradation. Here we show that the RecQ helicase, BLM, is degraded during infection with adenovirus serotypes 4, 5 and 12. In addition, we were able to show, through the use of mutant viruses, that the degradation of BLM is dependent on E1B-55K and E4 activity during Ad5 infection, a mechanism which is suggested to occur during both Ad4 and Ad12 infection. Using co-immunoprecipitation and GST pull-down assays, we were able to confirm E1B-55K and BLM interactions in vivo and in vitro during Ad5 and Ad12 infection, whilst additionally suggesting that E1B-55K binds to BLM via a region similar to the known E1B-55K binding site on p53. Finally, we found that the degradation of BLM during adenovirus infection was dependent on proteasome activity, establishing that a component of the E3 ligase complex, Cullin 4B, may be responsible for the degradation of BLM during adenovirus infection. These observations show that another component involved in DNA end resectioning, besides MRN, is a target for viral degradation by the adenovirus family.

Project 2:
Kaposi’s sarcoma associated herpesvirus (KSHV) is a double-stranded DNA virus belonging to the herpesvirus subfamily. Complex cellular defence mechanisms limit the potential for viruses, including KSHV, to replicate in the host. Such defences include the interferon response that can restrict virus replication and contain spread. It is becoming increasingly apparent that in addition to these immune defences, the cellular response to DNA damage also functions as an important anti-viral mechanism. Here we present the first report showing that de novo KSHV infection can activate components of the cellular DNA damage response; namely phosphorylation of ataxia-telangiectasia mutated (ATM), NBS1, Chk1, replication protein A (RPA) and H2AX. In addition, we show that KSHV reactivation to evoke lytic replication also activates components of the DNA damage response; namely phosphorylation of ATM, Chk2, RPA and H2AX. The inhibition of the ATM/ataxia-telangiectasia mutated and RAD3-related (ATR) signalling pathways may be important for KSHV infection efficiency, since abrogation of these signalling pathways improves KSHV infectivity. Curiously, abrogation of the ATM/ATR signalling pathways during KSHV reactivation impeded reactivation, suggesting a complex relationship between KSHV and the cellular DNA damage response. Finally, we have shown that KSHV is able to both inhibit, and induce the cellular DNA damage response, depending on the context, in response to DNA damaging agents, bringing another level of complexity to the relationship. Taken together, these data reveal a complex interaction between KSHV and the DNA damage response that requires further study to delineate the mechanism and understand the consequences for pathogenesis.

Type of Work:M.Res. thesis.
Supervisor(s):Grand, Roger and Stewart, Grant and Blackbourn, David J.
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Cancer Sciences
Subjects:QR355 Virology
R Medicine (General)
RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Institution:University of Birmingham
ID Code:3078
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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