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Structure, Function and Intracellular Dynamics of Alphavirus Replication Complexes

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Structure, Function and Intracellular Dynamics of Alphavirus Replication Complexes Institute of Biotechnology Department of Biological and Environmental Sciences Division of Microbiology Faculty of Biosciences University of Helsinki and Viikki Graduate School in Biosciences Structure, function and intracellular dynamics of alphavirus replication complexes Giuseppe Balistreri ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public examination in Room 2041 at Biocenter Viikki (Viikinkaari 5), Helsinki, on 4th August 2010. Helsinki, 2010 To my astonishing beautiful wife Laura, the Sun in my life, and my little princesses Emilia, Sara and Kiira, who make my days such an unstoppable explosion of joy! Supervisor Professor Emeritus Leevi Kääriäinen and Docent Tero Ahola Institute of Biotechnology, University of Helsinki Viikinkaari 9, 00790 Helsinki, Finland Reviewers Professor Pirjo Laakkonen Molecular Cancer Biology Research Program, Institute of Biomedicine, University of Helsinki, Helsinki, Finland and A.I. Virtanen Institute for Molecular Medicine, University of Kuopio, Kuopio, Finland Docent Vesa Olkkonen Minerva Foundation Institute for Medical Research Biomedicum 2U, Tukholmankatu 8 00290 Helsinki, Finland Opponent Professor Kai Simons Max-Plank-Institute of Molecular Cell Biology and Genetics Pfotenhauerstrasse 108, 01307 Dresden, Germany ISBN 978-952-10-6377-0 (pbk.) ISBN 978-952-10-6378-7 (PDF) Helsinki University Printing House Helsinki 2010 Abstract Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these ‘exotic’ compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as “spherules”. These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus- induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme. Acknowledgments I would like to express my deep gratitude to all the wonderful persons I have come across during these years at the Institute of Biotechnology. I will start with my beloved colleagues and friends in the SFV group, with whom I have had the privilege to work. To Pirjo Spuul, Andrey Golubtsov, and Maarit Neuvonen: there is not much space, you see, in this thesis book, but I know you will squeeze out of this THANK YOU all the wonderful time we have had together and will continue to have. I truly love you all! I would like to extend my gratitude to the rest of the former and present members of the group, including Julia Magden, Anne Salonen, Peter Sarin, Pia Salomaa, Nana Nordman, Airi Sinkko, Javier Caldentey, Leena Pohiola, and the new members Kirsi Hellström, Katri Kallio, Yaseen Syed-Basha and Antti Ahde. I thank Dr. Tero Ahola for giving me the opportunity to finalize my research in his group and for his help and support during these years. I am particularly grateful to the former and current directors of the Institute of Biotechnology, Professor Mart Saarma and Professor Tomi Mäkelä, for being always so supportive and for creating such a productive working environment. I am also thankful to Professor Timo Korhonen, head of the Division of Microbiology, for his positive and encouraging attitude towards my studies. I am grateful to Ritva Virkola and Anita Tienhaara for guiding me through the practical arrangements on the completion of my graduate studies. Part of the merit for the completion of this PhD thesis is also of the Viikki Graduate School in Biosciences, for the top- level education provided, and the excellent work of the former and current coordinators Eeva Sievi and Sandra Falk. A special thank goes to the members of my “follow-up” group, Professor Marc Bauman and Jaana Bamford, for their constructive critics towards my work and for useful advice. For making the reviewing process of this thesis one of the most educative experiences of my PhD training, I would like to express my gratitude to Professor Pirjo Laakkonen and Docent Vesa Olkkonen. I will make treasure of the fruitful discussions with all the group leaders and professors on the campus, especially Dr. Johan Peränen, Claudio Rivera, Pekka Lappalainen, Jussi Jantti, Sirkka Keränen, Ulla Pirvola, Maria Vartiainen, Leonard Kirugh, Sarah Butcher and Dennis Damford. A big, big hug to all my colleagues and friends in the 5th and 4th floor, I already miss you! It has been a pleasure to collaborate with Dr. Eija Jokitalo and all the members of the fantastic EM unit, particularly Helena Vihinen, Mervi Lindman.and Aria Strandell. As to how did I end up in Finland? This was evidently a conspiracy between my wife Laura and Professor Marja Makarov, who reasoned the way to keep me here for a long, long time: they brought me at the office door of Professor Leevi Kääriäinen…the rest is in this thesis! It has been a long way but it feels as we started yesterday. I would like to thank you both for all you have done for me; I hope I have deserved it. Working with you Leevi is a trilling experience. I have found in your contagious enthusiasm and perfectionism an endless source of motivation. I wish you a bright future and I am sure we will have the opportunity to soon work together again. Finally, I would like to thank all the members of my big and wonderful family, who are always supporting me and helping in moving forward. 7 Helsinki, 2010 2 Contents Abstract 4 Acknowledgements 5 List of original publications 8 Abbreviations 9 1 Introduction 10 1.1 +RNA virus replication in association with cellular membranes 10 1.2 Alphaviruses 14 1.3 SFV replication cycle 15 1.4 General composition of alphavirus replication complex 18 1.5 The spherules of alphavirus: ‘mini-organelles’ for viral RNA replication 21 1.6 General principles of endocytosis 22 Endocytosis assisted by clathrin 23 Caveolae mediated endocytosis: caveolins, cavins and flotillins 24 Macropinocytosis and phagocytosis 25 Other types of endocytosis 26 2. Aims of the study 27 3. Methods 28 4. Results 31 4.1 Approaching the high resolution 3D-structure of alphavirus replication complex: from conventional EM to correlative cryo-electron tomography (Unpublished) 31 4.2 Dynamics of SFV replication complexes (I, II) 36 4.2.1 Spherules arise at the plasma membrane (I) 36 4.2.2 Internalization of newly formed spherules from the PM (I, II) 40 4.2.3. The signal for spherule internalization resides in the replicase component nsP3 (II) 45 4.2.4 Intracellular transport of SFV spherules: biogenesis of CPV-I (I) 46 4.3 SFV infection induces down-regulation of transferrin receptor and redistribution of EEA-1 (unpublished) 47 4.4 Addressing the function of nsP2 with ts-mutants (III) 49 Discussion 53 Structure of SFV spherules 53 Spherule formation 55 RNA replication and the multifunctional protein nsP2 60 The viroplasms of SFV: spherules and CPVs 62 Comparison with viroplasms induced by other +RNA viruses 65 Internalization of spherules from the PM: spherules as unconventional endocytic cargoes 68 References 72 List of original publications This thesis is based on the following articles, which are referred to in the text by their roman numerals: I.
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