Forrester, Natalie Alison (2012)
Ph.D. thesis, University of Birmingham.
It is well established that adenoviruses inactivate the host cell DNA damage response to enhance viral DNA replication. This is achieved, in part, by the ability of viral E1B55K and E4ORF6 proteins to hijack host cell cullin-containing E3 ubiquitin ligase complexes and target key cellular proteins such as Mre11, p53 and DNA ligase IV for degradation via the ubiquitin-proteasome pathway. To assess the generality of this viral response, studies were undertaken using a panel of representative serotypes from adenovirus species A to E to determine how they interact with the host cell DNA damage response. Notably, serotypes from species B and D were unable to promote the degradation and/or relocalization of Mre11 and p53, although DNA ligase IV degradation was fundamentally conserved. Furthermore, species B and D serotypes induced the sustained overexpression of transcriptionally inactive p53, and induced both ATM and ATR kinase activity. As these events would typically be viewed as detrimental to virus survival, these data suggest that different adenovirus serotypes have evolved novel strategies in order to subvert the cellular DNA damage response during infection.
Adenoviruses have long been utilised as useful tools for identifying, and characterizing, the function of fundamental cellular proteins such as tumour suppressors and those involved in the DNA damage response. Therefore, studies were also carried out to identify novel Ad12E1B54K-interacting proteins through mass spectrometric analysis, and subsequently to examine the functional significance of these interactions. Several putative novel Ad12E1B54K-interacting proteins were identified using this approach, one being the transcriptional intermediary factor 1γ (TIF1γ), a transcriptional regulator that has recently been identified as a tumour suppressor. Further studies determined that TIF1γ was relocalized to nuclear tracks in an E4ORF3-dependent manner early during adenovirus infection, and was subsequently degraded in a ubiquitin-mediated proteasome-dependent manner. Uniquely, TIF1γ degradation was shown to be E1B55K/E4ORF6-independent and E4ORF3-dependent. Data presented in this thesis also suggest that E4ORF3 does not utilise host cell cullin-based E3 ubiquitin ligases in order to promote TIF1γ degradation. The ability of E4ORF3 to target cellular substrates for degradation represents a novel way in which adenoviruses are able to target cellular substrates. Significantly, TIF1γ degradation was conserved during infection with Ad serotypes from species A to C which may highlight its importance for productive viral infection. It also appears that TIF1γ may have an as yet unidentified role in the DNA damage response since it was also found to interact with components of the ATR kinase pathway.
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