INSTITUTO DE BIOLOGÍA MOLECULAR Y CELULAR (IBMC) UNIVERSIDAD MIGUEL HERNÁNDEZ DE ELCHE BIOPHYSICAL CHARACTERIZATION OF MEMBRANE- ACTIVE REGIONS OF STRUCTURAL AND NON- STRUCTURAL PROTEINS FROM DENGUE VIRUS CARACTERIZACIÓN BIOFÍSICA DE LAS REGIONES MEMBRANO-ACTIVAS DE PROTEÍNAS ESTRUCTURALES Y NO ESTRUCTURALES DEL VIRUS DEL DENGUE Henrique de Castro Nemésio PhD Thesis / Tesis Doctoral 2014 2 D. Antonio Ferrer Montiel, Catedrático de Bioquímica y Biología Molecular y Celular y Director del Instituto de Biología Molecular y Celular de la Universidad Miguel Hernández de Elche, DA SU CONFORMIDAD a la lectura de tesis doctoral titulada: “Biophysical characterization of membrane-active regions of structural and non- structural proteins from Dengue Virus” / ” Caracterización biofísica de las regiones membrano-activas de proteínas estructurales y no estructurales del virus del Dengue”, para optar al grado de Doctor en Ciencias presentada por D. Henrique de Castro Nemésio. Para que conste y surta los efectos oportunos, firma el presente certificado en Elche, a 25 de Junio de 2014. Fdo. Prof. Antonio Ferrer Montiel 3 4 Don José Villalaín Boullón, Doctor en Ciencias, Investigador del Instituto de Biología Molecular y Celular y Catedrático de Bioquímica y Biología Molecular de la Universidad Miguel Hernández de Elche, CERTIFICA: Que el trabajo de investigación que conduce a la obtención del grado de doctor titulado: “Biophysical characterization of membrane-active regions of structural and non-structural proteins from Dengue Virus” / ” Caracterización biofísica de las regiones membrano-activas de proteínas estructurales y no estructurales del virus del Dengue”, del que es autor D. Henrique de Castro Nemésio, ha sido realizado bajo su dirección en el Instituto de Biología Molecular y Celular de la Universidad Miguel Hernández de Elche. Para que conste y surta los efectos oportunos, firma el presente certificado en Elche, a 25 de Junio de 2014 Fdo. Prof. Dr. José Villalaín Boullón 5 6 ACKNOWLEDGMENTS One seldom has the opportunity to acknowledge the importance of someone or a group of people in our lives. I am about to finish an important stage and it wouldn’t have been so without the help and support of many people of which I will name and thank in no particular order: I would like to thank Professores Manuel Prieto and Miguel Castanho for having shown me the way to Profesor José Villalaín’s laboratory, where I conducted the research required to produce this manuscript. To Profesor José Villalaín, my tutor and advisor, it would be an understatement to say that this thesis would not exist without him. He trusted and took me under his protection in his laboratory, even knowing I had little to no laboratory experience. I knew I could count on him from day one when I arrived in the middle of “Semana Santa” in Spain and he picked me up from Alicante’s train station and drove me to (an already arranged and booked for me) rented flat in Elche. I thank you for everything. To Profesores Antonio Ferrer and José Manuel González Ros, directors of IBMC, the former as of today and the latter when I first arrived in Spain, for having given me the opportunity to be a part of this great IBMC family. To Francis, I believe a thousand thanks would not suffice. She was my tutor in every aspect of the laboratory, much like a big sister that tells you how to do things the proper way and not mess up. She taught both the morose and often ineffective official techniques and their faster and effective unofficial counterparts. Owing to her, my Spanish was thrust forward at incommensurable speed and would never be the same from then on (no more OK’s were heard in JVB lab). I will miss the coffee breaks, congresses, parties and everything that were part of our joint years. To everyone at “Secretaria”, especially to May and Javier for their never-ending help and knowledge of the ins and outs of the bureaucratic machinery. To the first friends and companions I had in Elche, Cheo, Silvia and Sakthi. I owe you a lot ever since we first met in the institute. One might say that strangers in a strange land walk hand in hand. 7 To all the Cubans, Dani, Jenny, Regla, Raiselys and Yanelis, I thank you for your friendship and all that stemmed from it, stories, jokes, laughs, dinners and Salsa classes. To Alicia and Pablo, a striking example of those cases where people that are right in front of you the whole time turn out to be really important. All the late night events, jokes, discussions and conversations will be sorely missed and always remembered. To Estefanía, Lourdes, Marcela and Rocío for all the conversations and much needed work pauses and after-work events, from concerts to weekend excursions. To all my friends in my country, they know who they are; I believe names are not required. Your friendship is invaluable. To my parents Jorge and Maria Helena, how can you really thank your parents? All their support and trust all along the way has led me to this very point. All my decisions (provided they would be sort of good decisions) were always met with confidence and approval even if it meant leaving their roof (before any facial hair) was visible or meeting sporadically and for short periods of time. It is quite obvious you are essential in my life. The same goes to Clara, my sister. Both her frankness and honesty well beyond her years and her perseverance are qualities I admire and try to use them on a daily basis. I am lucky to call you my sister. To Ana, your patience and unfaltering trust and love push me forward. I hope we can be side by side for quite some time; it is just a matter of synchronization. I wholeheartedly thank you all for being a part of my life. 8 ABBREVIATIONS (v) or (ds) RNA (viral) or (double stranded) Ribonucleic acid BMP Bis (monomyristoyl) phosphate BPI L-D-Phosphatidylinositol-4-phosphate from bovine brain BPS Brain L-α-phosphatidylserine CF Carboxyfluorescein Chol Cholesterol CL Cardiolipin CSF Cerebrospinal fluid DENV Dengue virus DENV1-4 Dengue virus serotype 1-4 DF Dengue fever DHF Dengue haemorrhagic fever DMPA 1, 2-dimyristoyl-sn-glycero-3-phosphate DMPC 1, 2-dimyristoyl-sn-glycero-3-phosphocholine DMPG 1, 2-dimyristoyl-sn-glycero-3-[Phospho-rac-(1-glycerol)] DMPS 1, 2-dimyristoyl-sn-glycero-3-[Phospho-L-serine] DMV Double membrane vesicles DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DPH 1, 6-diphenyl-1, 3, 5-hexatriene DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DSC Differential scanning calorimetry DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine DSS Dengue shock syndrome EGF Epidermal growth factor EPA Egg L-α-phosphatidic acid EPC Egg L-α-phosphatidylcholine ER Endoplasmic reticulum ESM Egg sphingomyelin ESR Electro spin resonance FPE Fluorescein phosphatidylethanolamine FTIR/IR Fourier transform infra-red GUV Giant unilamellar vesicles HCV Hepatitis C virus HI Hexagonal normal phase HII Hexagonal inverted phase HIV Human immunodeficiency virus Hsp Heat shock protein HSV Herpes simplex virus HTS High throughput screening IFN Interferon KUNV Kunjin virus LD Lamellar liquid crystalline or disordered phase LE Lamellar organized (gel) phase LE’ Lamellar organized (gel) phase with tilted chains LEM Late endosome membranes LUV Large unilamellar vesicles MLV Multi lamellar vesicles NMR Nuclear magnetic resonance PA Phosphatidic acid PE’ Ripple organized (gel) phase with tilted chains 9 PC Phosphatidylcholine PE Phosphatidylethanolamine PG Phosphatidylglycerol PMO phosphorodiamidate morpholino oligomers PS Phosphatidylserine PSM N-stearoyl-D-erythro-sphingosylphosphorylcholine SARS-CoV Severe acute respiratory syndrome coronavirus SFV Simian foamy virus SIV Simian immunodeficiency virus SM Sphingomyelin SNARE Soluble N-ethylmaleimide sensitive Attachment protein Receptor SNR Signal-to-noise ratio SUV Small unilamellar vesicles TBEV Tick-borne encephalitis virus TFE 2,2,2-trifluoroethanol TGN Trans-Golgi network TLR Toll-like receptors Tm Temperature of the main transition TMA-DPH 1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene TPE Egg L-α-phosphatidylethanolamine, transphosphatidylated VSV Vesicular stomatitis virus WNV West Nile virus YFV Yellow Fever virus 10 INDEX Acknowledgments ................................................................................................................................................ 7 Abbreviations ......................................................................................................................................................... 9 Prefacio ................................................................................................................................................................... 13 Chapter 1. Introduction ............................................................................................................................. 17 1. Biological membranes and their lipid constituents ............................................................... 17 1.1. Relevance of the study of membranes ............................................................................... 17 1.2. Short historical introduction to the membrane model ............................................... 18 1.3. Lipid complexity and distribution in cells ........................................................................ 22 1.4. Bilayer structure ......................................................................................................................... 27 1.5. Lipid polymorphism .................................................................................................................