Understanding the mechanism of Adenovirus endosomal escape

Thesis

Adenoviruses (AdV) are non-enveloped dsDNA viruses that cause respiratory and gastrointestinal diseases and are commonly used as vaccine and gene therapy vectors. In order to replicate inside a host cell, AdV must first penetrate the endosomal membrane and gain access to the cytosol. A highly conserved amphiphilic alpha helix within AdV protein VI (pVI) has been proposed as the membrane-lytic factor that is necessary for endosomal escape. However, the precise mechanism of endosomal membrane disruption is uncertain. This investigation aimed to characterise pVI and investigate the contribution of different domains using several biophysical techniques and electron microscopy. Firstly, a purification protocol was developed for recombinant wild type pVI and two loss of function mutants. The three pVI proteins, along with a synthetic peptide composed of the suggested membrane-lytic domain of pVI, were then examined using circular dichroism and analytical ultracentrifugation in order to further understand their secondary structure and oligomeric state. The effect of pVI on liposomes was then investigated using dynamic light scattering and a dye release assay and visualised using electron cryo-microscopy (cryoEM). Finally, electron cryo-tomography (cryoET) was used to examine membrane disruption events in 3D. The activity of pVI constructs upon liposomes was observed to be highly dependent on the membrane composition, with phosphatidylethanolamine reducing the activity of pVI.

Authors: Sawtell, K

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: protein structure; protein characterisation; liposomes; viral entry; protein purification; endocytosis; electron microscopy; adenovirus
• Subjects: Structural Biology; Virology; Cell Biology