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Proquest Dissertations Conformational Changes of Polyomavirus during Cell Entry by Marjan Dolatshahi Department of Anatomy and Cell Biology McGill University Montreal, Quebec, Canada Submitted in February2008 A thesis submitted to McGill University in partial fulfillment of the role of the requirements of the degree of Master of Science Copyright © Marjan Dolatshahi 2008 Library and Archives Biblioth&que et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'6dition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-66908-2 Our file Notre r6f6rence ISBN: 978-0-494-66908-2 NOTICE: AVIS: The author has granted a non- L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduce, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lntemet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimis ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie priv6e, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1*1 Canada Acknowledgements First of all, I would like to thank my supervisor, Dr. Isabelle Rouiller, for all her support, patience, understanding, and generosity during my masters program. She never hesitated to offer any opportunity to improve my scientific knowledge and experience. More importantly, she believed in my potential and amplified my enthusiasm for learning through her persistent encouragement and scientific feedback. Moreover, I am really thankful to her for providing me with financial support during my studies. I am grateful to my advisory committee members, Dr. Hojatollah Vali and Dr. John Presley who always provided me with constructive criticism and scientific support. I would also like to thank Dr. Hojatollah Vali for not only being a dedicated teacher, but also a very motivating friend for me. He introduced me to the research field and his intellectual guidance and great advice helped me to maintain the right direction in my research. I can not express my gratitude to him by words. Special thanks go to Dr. Paulo Amati and Dr. Robert Liddington for providing the viruses. Many thanks go to Dr. Tim Baker, Dr. Wendy Ochoa and Dr. Norm Olson, Dr. Dorit Hanein and Dr. Robert Liddington for their support in data collection. Thanks to Dr. Tim Baker, Dr. Xiaodong Yan, Dr. Robert Sinkovits and all the RobEM team for their support in using RobEM. Many special thanks go to Dr. John Bergeron who provided me with plenty of useful opportunities in his lab when I took my first steps in research. My sincere thanks go to Ali Fazel for being such a patient teacher and helped me find my way in the beginning of my research experience. I am really grateful to Dr. Eugene Daniels for the opportunity of working as a teaching assistant in Anatomy labs, which was a highly enriching experience. Also, I would like to thank all the staff and professors in the Anatomy lab especially Dr. Ayman Behiery for his constant encouragement and guidance. Thanks to the staff of the Departments of Anatomy and Cell Biology who contributed their time and expertise to help me particularly Sandra Botbol, Prabha Ramamurthy, Nancy Nelson, and Abdullah A1 Masud. Many thanks go to all the staff of the Facility of Electron Microscopy Research, especially Dr. Kelly Sears and Jeannie Mui, who assisted me with electron microscopy experiments. ii A special thank you goes to Avi Biswas for providing me with a computer program that accelerated my research program. I am deeply thankful to Martin Fleming for his great immediate help in solving all my computer problems. I am really thankful to my dear friend Fereshteh Azari for her enjoyable company and genuine friendship through my studies. I would like to express my gratitude to my caring sister, Masi, and my kind brother in- law, Sasan, who were standing behind me all the way. I do not know how to thank them for their unconditional love and support both emotionally and financially. Finally I would like to thank my parents who contributed their lives to my success, for flooding my heart with hope and pushing me forward in my weakness with their never- ending love. I would like to extend my thankfulness to my other family members who always cherished me with their care and affection. iv Abstract Similar to other non-enveloped viruses, the mechanism of cell entry for polyomaviruses is poorly understood. The polyomavirus capsid is an icosahedron composed of 72 pentamers of the major capsid protein VP1. There is one copy of minor capsid proteins, VP2 or VP3, at the center of each pentamer. According to previous studies, polyomavirus cell entry is a multi-step process which includes: 1) VP1 binding to sialic acid (SA) on the surface of host cells, 2) interaction of VP1 with a4pl integrin and 3) subsequent cell penetration. Biochemical studies have shown that SA alters polyomavirus protease sensitivity, suggesting a conformational change. The aim of this study was to determine these conformational changes at the molecular level. Therefore, we used single particle cryo-electron microscopy to construct 3D maps of wild type (WT) murine polyomavirus, WT bound to SA, a mutant with a disrupted integrin binding site, and the mutant bound to SA. Our results reveal that in both WT and mutant viruses, a significant conformational change happens after binding with SA which is seen as an additional ring of density inside the virus. Moreover some negative densities are seen in the difference map of WT and WT bound with SA, which suggests movement of some viral proteins after binding with SA. IV Resume Tout comme les autre virus sans enveloppe, le mecanisme d'entree de polyomavirus dans la cellule est mal compris. La capside de polyomavirus est un icosaedre compose de 72 pentameres de la proteine principale de la capside VP1. II y a une seul copie de proteines mineures de la capside VP2 ou VP3 au centre de chaque pentamere. D'apres des etudes precedentes, l'entree de polyomavirus dans la cellule est consistee de plusieurs etapes incluant: 1) l'attachement de VP1 a l'acide sialique (AS) a la surface des cellules hotes, 2) 1'interaction de VP1 avec l'integrine a4(31, et 3) l'entree dans la cellule. Des etudes biochimiques precedentes ont montrees que AS altere la sensibilite de polyomavirus a la digestion proteasique suggerant un changement de conformation. L'objectif de cette etude est de determiner les changements de conformation au niveau moleculaire. Pour ce faire, nous avons utilise la cryo-microscope electronique et la technique de particule isolee pour calculer les cartes tridimensionnelles du virus polyomavirus murin sauvage, du virus sauvage lie a AS, d'un mutant avec une mutation dans la site d'attachement a l'integrine, et du mutant lie a AS. Nos resultats montrent que le virus sauvage et le mutant subissent tous les deux un changement de conformation signifiant apres l'attachement de AS. Le changement est visible comme un anneau de densite a l'interieur du virus. De plus, des densites negatives sont vues dans la carte de difference entre sauvage et sauvage+AS, ce qui suggere le mouvement de certaines proteines virales apres la liaison a AS. v List of Abbreviations aa: amino acid BKV: BK virus CC: Correlation Coefficient CCD: Charge Couple Device Cryo-EM: Cryo-electron microscopy CTF: Contrast Transfer Function D138N: point mutation in amino acid 138 (aspartic acid is replaced by asparagine) DFT: Discrete Fourier Transform EM: Electron microscopy ER: Endoplasmic Reticulum ERAD: Endoplasmic Reticulum Associated Protein Degradation FFT: Fast Fourier Transform FSC: Fourier Shell Correlation FT: Fourier Transform Gly: Glycine HIV: Human Immunodeficiency Virus JCV: JC virus kbp: Kilobasepair LDV: Leucine, Aspartic acid, Valine LT: Large Tumor Mutant+ NANA: D138N mutant polyomavirus bound with a 2,3 N-acetyl neuraminic acid NANA: a 2,3 N-acetyl neuraminic acid NMR: Nuclear Magnetic Resonance PARP: Poly ADP-ribose Polymerase SA: Sialic Acid SL: Sialyl Lactose SNR: Signal to Noise Ratio SV40: Simian Vacuolating Virus 40 vi 3D: Three Dimensional 2D: Two Dimensional TMV: Tobacco Mosaic Virus VLP: Virus-Like Particles VP 1: Viral Protein 1 VP2: Viral Protein 2 VP3: Viral Protein 3 VP2/3: The common sequence between VP2 and VP3 WT: Wild Type WT + NANA: Wild Type polyomavirus bound with a 2,3 N-acetyl neuraminic acid vii Table of Contents Title page i Acknowledgements ii Abstract iv Resume v List of Abbreviations vi Table of contents viii List of Tables and Figures x 1.
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