Investigating the role of Adenovirus E1b-55k, E4orF3 and E4orF6 proteins during infection

Albalawi, Abeer (2021). Investigating the role of Adenovirus E1b-55k, E4orF3 and E4orF6 proteins during infection. University of Birmingham. Ph.D.

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Abstract

Viruses have evolved to utilise host cell factors that promote viral replication and inactivate host cell factors that inhibit viral replication. For adenovirus, early region proteins modulate the functional activity of pro-viral and anti-viral host cell proteins in order to promote viral replication. In this regard, adenovirus early region proteins, E1B-55K, E4orf3 and E4orf6 engage with the cellular Ubiquitin-Proteasome System, both independently and cooperatively to inactivate anti-viral host cell proteins. The principal aims of this thesis were to: investigate further how adenovirus E1B-55K, E4orf3 and E4orf6 proteins function in isolation to modulate host cell factors through engagement with the Ubiquitin-Proteasome System; and consider the role of the cellular chromatin remodelling complex, FACT in adenovirus infection.
In order to study their functions in isolation we have developed, and characterized, clonal U2OS cell lines that express human Ad5 and Ad12 early region proteins in a tetracycline-responsive manner. Using site-directed mutagenesis coupled with pharmacological inhibition of the proteasome and Cullin Ring Ligases we determined that Ad5 E4orf6 is subject to ubiquitylation on K95 and K171 and undergoes proteasome-dependent degradation in a Cullin Ring Ligase-dependent manner. Using quantitative mass spectrometry we also established that Ad12 E4orf6, but not Ad5 E4orf6, engages with Cullin Ring Ligases to promote the proteasome-mediated degradation of core histone proteins. In this regard, both adenovirus 5 and adenovirus 12 were shown to promote the loss of core histones during infection but the mechanisms underlying this process require further investigation. Using a combination of immunoflouresence, GFP pull-down and RNA interference allied with Western blot analyses work presented here also established that FACT complex components, SPT16 and SSRP1 associated with the RPA complex at viral replication centres during infection and that ablation of FACT gene expression affected adenovirus early region gene product expression during infection.
In consideration of previous published work and the data presented here, this study indicates that adenovirus utilises the Ubiquitin Proteasome System at multiple levels to regulate both host cell protein and adenovirus early region protein function during infection and suggests that cellular histones limit adenovirus replication. Moreover, our data suggests that the histone chaperone protein complex, FACT has pro-viral activities during adenovirus infection. Taken together, our studies have identified potential new functions for adenovirus early region proteins and have developed useful resources for studying viral protein-host protein interactions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):