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Picornavirus Entry: Membrane Permeability Induced by Capsid Protein VP4

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Picornavirus entry: membrane permeability induced by capsid protein VP4 Anusha Panjwani Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Molecular and Cellular Biology September, 2013 1 The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Anusha Panjwani to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. © 2013 The University of Leeds and Anusha Panjwani 2 Acknowledgements First and foremost I’d like to thank my supervisor Toby Tuthill for all his support, advice, encouragement and patience throughout the course of my PhD. Thank you for being there to answer my never-ending questions, being my sounding board for ideas, for your confidence in me and always seeing the positive side to things! I would like to thank my University of Leeds supervisor, Nic Stonehouse for her encouragement and support through the course of my PhD. Terry Jackson for his input and pertinent advice through my PhD will be remembered. Professor Dave Rowlands for great project and career advice, meals and walks, that made the journey that much more interesting. Thanks to all members of the Picornavirus structure group and everyone at Pirbright, in particular Joseph, Kyle, Lisa, Lidia, Beatrice for giving me so many fond memories and helping me maintain my sanity especially towards the end of my PhD! Thank you to Josie for being an amazing friend and housemate. A big thank you to Professor Jim Hogle and Doreen Hogle for making my visit to Harvard Medical School comfortable, memorable and productive! Thank you to Professor James Chou for giving me the opportunity to work in his lab and my friends and colleagues in Boston Mike, Bill, Mukesh, Sven, Marcello, Florian, Tanxing, Maria for all those useful discussions in the lab and at Starbucks. My loving thoughts for my parents and my brother for their understanding, encouragement, unconditional warmth and love. Thank you! 3 Abstract 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. 4 Table of contents Acknowledgements ............................................................................................................. 3 Abstract .............................................................................................................................. 4 Table of contents ................................................................................................................ 5 Table of figures ................................................................................................................. 10 Table of tables .................................................................................................................. 13 Abbreviations ................................................................................................................... 14 Chapter 1 Introduction ...................................................................................................... 21 1.1 Introduction ................................................................................................................ 22 1.1.1 Clinical features and significance of human rhinovirus, poliovirus and foot-and- mouth disease virus ............................................................................................................ 22 1.1.2 Classification of Picornaviruses ........................................................................... 24 1.2 Genome organisation.................................................................................................. 26 1.3 Polyprotein translation and processing ...................................................................... 28 1.3.1 Viral proteins ....................................................................................................... 29 1.4 Genome replication .................................................................................................... 34 1.5 Picornavirus assembly and structure .......................................................................... 38 1.5.1 Virus Assembly .................................................................................................... 38 1.5.2 Virus structure..................................................................................................... 41 1.6 Virus cell entry ............................................................................................................ 45 1.6.1 Membrane penetration by enveloped viruses ................................................... 46 1.6.2 Membrane penetration by non-enveloped viruses ............................................ 49 1.6.3 Non-enveloped virus capsid proteins facilitating membrane penetration......... 50 1.6.4 Picornavirus entry ............................................................................................... 53 1.7 Aims............................................................................................................................. 65 Chapter 2 Materials and Methods ..................................................................................... 66 2.1 General Materials ....................................................................................................... 67 2.1.1 Bacterial strains ................................................................................................... 67 2.1.2 Cell lines .............................................................................................................. 67 5 2.1.3 Expression vectors .............................................................................................. 67 2.1.4 Antibodies ........................................................................................................... 67 2.1.5 Detergents........................................................................................................... 67 2.1.6 Lipids ................................................................................................................... 68 2.2 Manipulation of recombinant DNA ............................................................................. 68 2.2.1 DNA oligonucleotide primers .............................................................................. 68 2.2.2 Polymerase chain reaction (PCR) ........................................................................ 68 2.2.3 DNA sequencing .................................................................................................. 69 2.2.4 Restriction digestion of DNA ............................................................................... 69 2.2.5 Agarose gel electrophoresis ................................................................................ 70 2.2.6 Gel extraction of DNA ......................................................................................... 70 2.2.7 DNA ligations ....................................................................................................... 70 2.2.8 Transformation of chemically competent bacteria ............................................ 70 2.2.9 Bacterial cultures ................................................................................................ 71 2.2.10 Purification of bacterial DNA .............................................................................
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