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Protein interactions of membrane protein U24 from Roseolovirus and implications for its function Sang, Yurou


This dissertation describes the investigation of the interactions between the tail-anchored membrane protein U24 from Human Herpesvirus type 6A (HHV-6A) and type 7 (HHV-7) and its potential binding partners. The roles that these interactions play in U24s’ function will be presented. It has been suggested that U24 from HHV-6A (U24-6A) may trigger an autoimmune reaction in multiple sclerosis (MS), through its molecular mimicry of myelin basic protein (MBP). Both versions of U24 have been implicated in endocytic recycling via specific binding partners, namely WW domains. The first part of this thesis is a review of the foundations that this thesis is based on, from the description of Roseoloviruses, associated diseases to molecular characterizations. The two main techniques used in the studies will be described as well. Prior to commencing any structural or interaction studies, a protocol is devised to express and purify recombinant U24 from HHV-7 (U24-7), as well as preliminary studies to prepare samples suitable for structure determination by nuclear magnetic resonance (NMR) spectroscopy. U24-6A was shown to be a mimic of MBP and it was suggested that it could be implicated in MS by competing with MBP for its interactions, such as the interactions with Fyn-SH3 domain. The interactions between U24-6A and Fyn-SH3 domain were therefore probed and found to be weak, calling into question this mimicry hypothesis. Because of the weak binding with Fyn-SH3 domain, alternative functions and binding partners were then explored. WW domains were chosen because their binding ligand, the PPxY (PY) motif, is present in U24 and was identified to be essential for U24’s function. In the next part of this thesis, the investigation of the interactions between U24s and WW domains in Nedd4, which is a key component required for endocytosis, are described. U24-7 and phosphorylated U24-6A were found to bind strongly to Nedd4-WW domains, suggesting the negative charge upstream from PY motif in U24 is important for high affinity interactions. Non-canonical Smurf2 WW domains were explored as well. Finally, the results presented in this thesis will be discussed in the context of the function of U24.

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