Picornavirus entry: membrane permeability induced by capsid protein VP4

Non-enveloped viruses such as picornaviruses must penetrate the host cell membrane without the advantage of membrane fusion. This process is thought to involve membrane permeabilisation but the mechanism remains poorly understood. During picornavirus cell entry, capsid protein VP4 is released from the virus and is implicated in the delivery of the viral genome into the cytoplasm. The studies described in this thesis were undertaken to improve our understanding of the role of VP4 in cell entry. The approach to investigate this used recombinant VP4 and liposome model membranes. Recombinant VP4 was shown to induce membrane permeability with characteristics similar to that induced by both model pore-forming peptides and infectious virus particles and was influenced by pH, membrane composition and VP4 myristoylation. Chemical crosslinking and dextran release studies demonstrated that VP4 formed a multimeric size-selective membrane pore. The VP4 pore complex was visualised by transmission electron microscopy, which confirmed the multimeric nature of the pore and showed a lumen diameter in agreement with the dextran release studies and consistent with the dimensions required for the passage of viral RNA. The structure of VP4 reconstituted in membrane-mimetic detergent micelles was analysed by circular dichroism spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. This showed VP4 contained predominantly alpha helical content and the acquired 2D NMR spectrum was typical of a membrane protein. The membrane permeability induced by synthetic peptides showed that VP4 activity maps to the N-terminal half of VP4. In addition, VP4 activity was enhanced by the presence of a peptide corresponding to the N-terminus of VP1, an additional capsid protein also implicated in cell entry. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.