AbstractMany viruses initiate infection through a multistep process involving host cell membrane proteins. Hepatitis C virus (HCV) is an important human pathogen that infects more than 185 million people worldwide and results in progressive liver disease. Recent advances have identified an essential role for tetraspanin CD81 and tight junction protein Claudin-1 in HCV entry into hepatocytes in the liver. CD81 associates with Claudin-1 and this complex is necessary for virus internalisation; defining the full length interface of this membrane protein interaction is therefore important for the design of future anti-viral therapies. Structural information is lacking for CD81: indeed, there is no high resolution structure for any full-length tetraspanin. This thesis describes an analysis of the protein-protein interaction interface between CD81 and Claudin-1 full-length proteins using a split-ubiquitin yeast assay. Also, using recombinant protein production of CD81, this thesis describes work towards successful crystallisation trials of a full length tetraspanin.
CD81 homotypic and heterotypic interactions with Claudin-1 were analysed in a high-throughput format in yeast, showing that this interaction is specific and does not require other mammalian cell factors. This work demonstrates that the CD81 large extracellular loop and its first transmembrane domain are involved in the CD81-Claudin-1 interaction: a novel full length molecular model predicted interacting amino acid residues that were confirmed in vivo using yeast assays. Thermal stability assays used to investigate recombinant membrane protein found that both detergent and buffer components are vital for the stability of recombinant CD81, which shows increased thermostability in the presence of cholesteryl hemisuccinate. Using the improved protein solution environment found here, and the increased understanding of the tetraspanin interaction interface; this work paves the way for CD81 structural characterisation alone or in combination with Claudin-1.
|Date of Award||30 Jan 2015|
|Supervisor||Roslyn Bill (Supervisor)|
- tight junction protein
- site-directed mutagenesis
- P. pastoris
- recombinant protein production