The enterovirus 71 particle: An evolutionary approach to investigate structure and function with implications for drug and vaccine development.

The capsid plays many important roles in the virus life cycle, including host cell recognition, cell entry, uncoating and protection of viral RNA. In this thesis, artificial selection, use of inhibitory compounds and structural analysis have been combined to further understand aspects of these multiple roles. The VP1 pocket is a hydrophobic cavity in the capsid that harbours a fatty acid, known as the pocket factor. The presence of this fatty acid can increase capsid stability and its release is required for the virus to uncoat. Certain compounds are able to bind to the pocket and displace the natural fatty acid (causing a further increase in capsid stability) and can inhibit infection by making the virus so stable that it is unable to uncoat. Novel compounds, termed NLD and ALD, have been designed in silico based upon the previous best EV71 pocket binding inhibitor GPP3. These were shown to inhibit EV71 infection in cells, with NLD being more than one order of magnitude more potent than the previous best EV71 pocket-binding inhibitor. Resistance towards these compounds was studied to reveal a double mutation in VP1 (I113M and V123L). Mapping the mutations to the EV71 crystal structure revealed that they were located in the VP1 pocket, and modelling predicted that they would prevent NLD and the natural pocket factor from binding. Further characterisation of the resistant isolate revealed that the mutations were thermally- and genetically-unstable. Further investigation on thermal stability involved selection of a thermally-stable virus. This generated viruses with a double mutation in the VP1 pocket (V179A and L183V). These mutations were predicted to increase the size of the VP1 pocket, and were shown to affect the way the virus interacts with the natural pocket factor. The effects of a variety of different pocket factors on the heat stability of WT EV71, the thermostable mutant, and the inhibitor-resistant mutant were analysed. In addition, work was conducted to assess the effect of pH on uncoating. For uncoating it is known that EV71 must be incubated at a low pH in the presence of its major receptor SCARB2. To investigate the effects of this, EV71 was incubated at a low pH during infection. This was shown to reduce the virus titre, and repeated exposure to this condition selected for a VP1 mutation (N104S). This residue is predicted to be involved in binding to SCARB2, and the mutant virus was shown to differ in susceptibility to compounds that affect entry and uncoating.