Vaccinia virus protein A40 is an immunomodulator Susan Anne Jarmin IMPERIAL COLLEGE LONDON Department of Virology A thesis submitted for the degree of PhD 2009 1 Declaration The work detailed herein is the work of the candidate except where clearly indicated. The in vivo animal injections were undertaken by Dr. Ron A-J Chen. The leukocyte infiltration flow cytometry analysis and cytotoxicity assay on extracted cells detailed herein were undertaken in a collaborative manner with Dr. Nathalie Jacobs. The polyclonal anti-A40 antibody was generated by Harlan Sera-Lab (Leicestershire, England). ------------------------------------------------------------------------ Susan A. Jarmin 2 Acknowledgements Sincere thanks go to Professor Geoffrey L. Smith for his support, guidance and encouragement. It has been a pleasure and a privilege to undertake the work detailed in this thesis. I am immensely grateful to have been given the opportunity to work in his lab, in an environment that was so inspiring and supportive. I was fortunate to have the guidance and expertise of my co-supervisor Dr. Nathalie Jacobs for the first half of my studies. I am grateful for her mentoring and the supply of Belgian chocolate. To Dr Ron Chen, a big thank you for all the help and encouragement and probably wasted Chinese lessons. To all members of the GLS lab, past and present, thank you for making the experience enjoyable and rewarding. To my fellow PhD students, Adi and Rory, thank you for your support, friendship, laughs and help over the last three years; Laura for her friendship and support in keeping me relatively sane this last year; Kim, those support and unwavering supply of an ear to listen to me and a gentle reminder to relax have often kept me sane. Claire and Mike, your camaraderie and dancing were greatly appreciated. A big thank you is also due to Dr Mike Skinner and Dr Stephen Laidlaw for all their support and guidance over the years. Finally, thanks to my family, for their support, encouragement and a source of inspiration and unquestioning love. To my parents, a huge thank you for their support, guidance and unwavering patience. To my sister, Sarah, thank you for all your help and support and for being bribed with JellyTots. To my brother, for being a patient listener and pillar of support. All your love and support has been greatly appreciated and I thank you for the gentle nudging along the way. 3 Abstract Vaccinia virus (VACV) strain Western Reserve gene A40R encodes a type II membrane glycoprotein with a C-type lectin-like domain at the C terminus. The A40 protein is not incorporated into virions, is nonessential for virus replication in cell culture and does not affect virus virulence in a murine intranasal model of infection. However, A40 does affect the outcome of infection in an intradermal infection model in which the virus lacking gene A40R produced smaller lesions and alterations in the host immune response. A40 has amino acid similarity to C-type lectins, such as NKG2A and DC- SIGN. This observation together with its location on the infected cell surface and its ability to bind to the surface of cells of the immune system is consistent with A40 functioning as an immunomodulator. It is possible that A40 might function by mimicking native host lectins or by modulating recognition of VACV-infected cells by cells of the immune system. To investigate the mechanism by which A40 affects the outcome of infection in vivo, a cloning and experimental strategy was devised to search for its ligand(s) and try to determine its structure in collaboration with protein crystallographers. To achieve these goals, the recombinant A40 protein has been produced in E. coli, mammalian cells, and from insect cells infected with recombinant baculoviruses. Bacterially expressed recombinant A40 was used to generate an antibody specific to A40 and this was then purified, characterised and used to further the characterisation of A40. The potential for A40 to interact with a ligand on the surface of another cell was investigated by a cell binding assay using recombinant A40 protein. This protein was produced in a mammalian system and was found to bind to the surface of immune cells but not to epithelial cells. In cytotoxicity assays, the absence or over-expression of A40 was found to modulate the ability of NK cells to kill VACV infected cells. 4 Contents Declaration ................................................................................................................................................. 2 Acknowledgements .................................................................................................................................... 3 Abstract ...................................................................................................................................................... 4 Contents ..................................................................................................................................................... 5 Table of figures ........................................................................................................................................ 12 Table of tables .......................................................................................................................................... 13 Abbreviations .......................................................................................................................................... 14 Chapter 1. Introduction ..................................................................................................................... 29 1.1 Introduction to poxviruses and their historical significance....................................................... 29 1.2 Genome structure of OPVs ......................................................................................................... 31 1.3 Virion structure ........................................................................................................................... 32 1.4 VACV life cycle ........................................................................................................................... 33 1.4.1 Virus binding ..................................................................................................................... 33 1.4.2 Virus entry ......................................................................................................................... 33 1.4.3 Gene Expression ................................................................................................................ 34 1.4.4 DNA replication ................................................................................................................ 37 1.4.5 Virus assembly, maturation & release ............................................................................... 37 1.5 Effect of VACV on host cells ....................................................................................................... 38 1.6 Use of VACV in medical research ............................................................................................... 41 1.6.1 VACV-based vaccines ...................................................................................................... 41 1.6.2 VACV-based oncotherapy................................................................................................. 42 1.7 Innate immune response to VACV infection ................................................................................ 43 1.7.1 Complement ...................................................................................................................... 44 1.8 Innate immune cells .................................................................................................................... 45 1.8.1 Neutrophils ........................................................................................................................ 45 1.8.2 Macrophages ..................................................................................................................... 46 5 1.8.3 Natural killer cells ............................................................................................................. 47 1.8.4 NKT cells .......................................................................................................................... 54 1.8.5 Importance of C-type lectins in immune response ............................................................ 54 1.8.6 DCs .................................................................................................................................... 60 1.8.7 T cell receptor γδ cells ....................................................................................................... 61 1.9 Pattern recognition receptors (PRRs) ......................................................................................... 61 1.9.1 Toll-like receptors (TLRs) ................................................................................................. 62 1.9.2 Soluble PRRs ..................................................................................................................... 66 1.9.3 Soluble mediators of immune response ............................................................................. 68 1.10 Apoptosis ................................................................................................................................ 74 1.11 Adaptive immune responses to VACV infection ....................................................................