Probing structure, stability and dynamics of viruses by mass spectrometry

Abstract

Our capacity to deal with many viral infections has vastly improved, largely through the development of successful vaccines and antivirals. However, for some viruses and through the emergence of new strains and more resistant strains, this has proved more challenging. By continuing to explore the fundamental processes underlying viruses and their interaction with their host, it may be possible to treat or prevent these more challenging viral infections. In this thesis I probe the structure, stability and dynamics of viruses by mass spectrometry (MS), more specifically I explore these aspects for a self-assembling virus-like particle (VLP), the P particle, that has the potential as a chimeric vaccine and for the hepatitis B virus (HBV) and the HBV related e-antigen. The stability and dynamics of the P particle, important properties that would have implications in its use as a therapeutic vaccine, were investigated. We characterised the P particle under reducing and non-reducing conditions, due to the presence of inter-subunit disulphide bonds, and identified previously uncharacterised P domain complexes. By changing the solution phase pH and ionic strength we showed how the equilibrium between these complexes could be manipulated. Using ion mobility mass spectrometry (IMMS) in combination with molecular modeling we were able to propose probable structures for these P domain complexes. Our results demonstrate the interchangeable nature and dynamic relationship of all P domain complexes and confirm their binding activity to the host receptors. The interaction of the HBV capsid with two antibodies, Fab E1 and Fab 3120, that bind through distinct epitopes was studied. We used an approach of hydrogen deuterium exchange-mass spectrometry (HDX-MS) to examine the dynamics of the interaction, identifying a reduction in deuterium incorporation both at the binding epitope on HBV and regions distal to the epitope. Overall our results suggest a global rigidification of the capsid and suppression of its breathing motions. We employed an alternative methodology to further characterise the interaction, this time employing two complementary ESI-based techniques, native MS and gas-phase electrophoretic mobility molecular analysis (GEMMA). We demonstrate their capability to monitor the concentration-dependant antibody binding process and their ability to resolve different modes of antibody binding to the T = 3 and T = 4 capsid morphologies, consistent with the cryo-EM model of binding. These data indicate a rapid means of characterisation of such complexes. We further applied the HDX-MS methodology to study the structural properties of the HBV core-antigen (cAg) and the e-antigen (eAg) (an immune regulator). Despite almost complete sequence identity, the dimeric proteins adopt distinct quaternary structures, the likely basis of their differing properties. HDX-MS detected many regions that differed substantially in their HDX dynamics. Comparison of the HDX of unassembled cAg with that in assembled capsids indicated increased resistance to exchange in the region of the inter-dimer contacts. We also demonstrated that eAg undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a cAg-like structure in many, albeit not all, regions. Together, these results demonstrate the highly dynamic nature of these similar proteins. The work detailed in this thesis allows for a more detailed insight into the structure, stability and dynamics of both the norovirus-derived P particle, and HBV. These results may indeed have an impact of on their respective roles as a vaccine platform and in the treatment of chronic HBV infection.