Universidad Autónoma de Madrid Departamento de Biología Molecular Facultad de Ciencias Analysis of the assembly and maturation processes of the Infectious Bursal Disease Virus capsid - TESIS DOCTORAL - Nerea Irigoyen Vergara Madrid, 2009 Universidad Autónoma de Madrid Departamento de Biología Molecular Facultad de Ciencias Memoria presentada para optar al grado de Doctor en Ciencias Biológicas por Nerea Irigoyen Vergara Universidad Autónoma de Madrid Mayo 2009 DIRECTORES DE TESIS: Dr. José Francisco Rodríguez Aguirre C.N.B.-C.S.I.C. Dr. José Ruiz Castón C.N.B.-C.S.I.C. El trabajo recogido en esta memoria ha sido realizado en el Centro Nacional de Biotecnología (C.N.B.-C.S.I.C.) bajo la dirección conjunta de los Drs., José Francisco Rodríguez Aguirre y José Ruiz Castón. Su financiación corrió a cargo de una beca de Formación Personal Universitario del Ministerio de Educación y Ciencia y una beca para estudiantes de tercer grado de la “Residencia de Estudiantes” a cargo del Gobierno de Aragón. Para la realización de estancias breves en el extranjero se contó con ayudas del Ministerio de Educación y Ciencia y de la Universidad Autónoma de Madrid. Abbreviations: MOI: Multiplicity Of Infection 3D: Three Dimensional mRNA: messenger RiboNucleic Acid 3DR: Three Dimensional Reconstrucción MW: Molecular Weight XX: antibody anti-XX α− MWM: Molecular Weight Marker aa: aminoacid NωV: Nudaurelia capensis ω virus AcPHV: Autographa californica polyhedrosis Virus N-terminal: Amino-terminal ATCC: American Type Cell Collection ORF: Open Reading Frame BBV: Black Beetle Virus PBS: Phosphate Buffered Saline BF: Bursa of Fabricius PCR: Polymerase Chain Reaction bp: base-pair PDB: Protein Data Bank BSA: Bovine Serum Albumin PepN: aminopeptidase N BTV: Bluetongue Virus PFU: Plaque Forming Unit C : -carbon α α PIPES: piperazine-N,N-bis(2-ethanesulfonic acid) CEF: Chicken Embryo Fibroblast PSA: Puromycin Sensitive Aminopeptidase CP: coat protein RdRp: RNA dependent RNA polymerase crioEM: electron cryomicroscopy rmsd: root square mean deviation C-terminal: carboxi-terminal RNA: Ribonucleic Acid Da:Dalton RNP: RiboNucleoProtein DNA: Desoxiribonucleic acid r.p.m.: revolutions per minute dsRNA: double stranded RNA rBV: recombinant BaculoVirus DMEM: Dulbecco Modified Eagle’s Medium rVV: recombinant Vaccinia Virus dNTP: desoxiNucleotide TriPhosphate qRT-PCR: Quantitative real time polymerase chain DTT : ditiotreitol reaction DXV: Drosophila X Virus T: triangulation number EDTA: Ethylene Diamine Tetraacetic Acid TBE: Tris-Borato-EDTA buffer EM: electron microscopy Tris: tri-(hidroximetil)-aminomethane FCS: Fetal Calf Serum SDS: Sodium Dodecil Sulfate FHV: Flock House Virus SDS-PAGE: SDS Polyacrilamide Gel hp.i.: postinfection hours Electrophoresis HT/His-tag: Histidine Tag sh: Short hairpin IBD: Infectious Bursal Disease ssRNA: single stranded RNA IBDV: Infectious Bursal Disease Virus (+) ssRNA: positive single stranded RNA IP: immunoprecipitation SVP: Subviral Particle IPNV: Infectious Pancreatic Necrosis Virus UTR: UnTranslated Region IPTG: isopropyl-β-D-thiogalactopyranoside VLP: Virus Like Particle kb: kilobase WB: Western Blot kDa: kilodalton Amino Acid Abbreviations Alanine Ala, A Arginine Arg, R Aspartic Acid Asp, D Asparagine Asn, N Cysteine Cys, C Glutamic Acid Glu, E Glycine Gly, G Glutamine Gln, Q Histidine His, H Isoleucine Ile, I Leucine Leu, L Lysine Lys, K Methionine Met, M Phenylalanine Phe, F Proline Pro, P Serine Ser, S Tyrosine Tyr, Y Threonine Thr, T Tryptophan Trp, W Valine Val, V INDEX Abstract p. 1 Chapter 1. Introduction p. 5 Capítulo 1. Introducción p. 5 1.1. Diferentes mecanismos de control para el ensamblaje de la cápsdida viral p. 8 1.1.1. Control del cambio conformacional p. 8 1.1.2. Proteínas formadoras de “cores” y de andamiaje p. 9 1.1.3. Cambios conformacionales mayores p. 9 1.1.4. Estructuras intermedias en el ensamblaje p. 10 1.1.5. Procesamiento proteolítico p. 10 1.2. Familia Birnaviridae: el virus de la bursitis infecciosa p. 11 1.2.1. Serotipos de IBDV p. 12 1.2.2. Organización genómica de IBDV p. 13 1.2.3. Estructura y morfogénesis de IBDV p. 14 1.2.3.1. Componentes estructurales del virión de IBDV p. 14 1.2.3.2. Sistemas de expresión basados en virus para el análisis del ensamblaje de IBDV p. 16 1.2.3.3. Polimorfismo estructural de la proteína de la cápsida de IBDV p. 17 1.2.3.4. Estructura atómica de VP2 y VP3 p. 19 1.3. Relaciones estructurales y funcionales de IBDV con virus ssRNA y dsRNA p. 20 Chapter 1. Introduction p. 25 1.1.Different control mechanisms for viral capsid assembly p. 27 1.1.1. Control of confomational switching p. 28 1.1.2. Cores and scaffolds p. 28 1.1.3. Major conformational changes p. 29 1.1.4. Assembly intermediates and pathways p. 29 1.1.5. Proteolytic processing p. 30 1.2. Birnaviridae family: Infectious bursal disease virus p. 30 1.2.1. IBDV serotypes p. 32 1.2.2. IBDV genomic organization p. 32 1.2.3. IBDV structure and morphogenesis p. 33 1.2.3.1. Structural components of IBDV virion p. 33 1.2.3.2. Virus based expression systems to analyze IBDV caspid asssemby p. 36 1.2.3.3. Structural polymorphism of IBDV coat protein p. 37 1.2.3.4. Atomic structures of VP2 and VP3 p. 38 1.3. IBDV structural and functional relationships with ssRNA and dsRNA viruses p. 40 Chapter 2.Aims p. 47 Chapter 3. Experimental procedures p. 51 3.1. Biological Material p. 51 3.1.1. Prokaryotic cells p. 51 3.1.2. Eukaryotic cells p. 51 3.1.3. Plasmids p. 51 3.1.4. Viruses p. 52 3.1.5. Antibodies p. 53 3.2. Manipulation and generation of recombinant vectors p. 53 3.2.1. PCR p. 53 3.2.2. DNA fragments ligation reactions p. 53 3.2.3. Enzymatic restriction reactions p. 53 3.2.4. Generation of recombinant plasmids p. 54 3.2.4.1. Plasticity domain of the C-terminal of pVP2 p. 54 3.2.4.2. Maturation of pVP2 p. 56 3.2.4.3. Implication of the catalytic residue in the viral assembly p. 57 3.2.4.4. The role of a cellular protease p. 57 3.2.5. Generation of recombinant baculoviruses p. 58 3.2.6. Generation of recombinant vaccinia viruses p. 58 3.2.7. Generation of DF1 silenced cells p. 59 3.2.8. Reverse genetic analysis p. 60 3.3. Infections and protein expression p. 61 3.3.1. rBV infection p. 61 3.3.2. rVV infection p. 61 3.3.3. IBDV infection p. 61 3.3.4. IBDV titration p. 61 3.3.5. rVV titration p. 62 3.4. Purification of IBDV polyprotein-derived stuctures p. 62 3.5. Biochemical analysis p. 63 3.5.1. Electrophoretic analysis in denaturing polyacrylamide gels p. 63 3.5.2. Electrotransference and immunodetection p. 63 3.5.3. Immunoprecipitation of VP2 p. 63 3.6. Microscopy p. 64 3.6.1. Electron microscopy p. 64 3.6.2. Immunofluorescence and confocal laser scanning micros. analysis p. 64 3.7. Crystallization and data collection p. 65 3.7.1. Structure refinement p. 65 3.7.2. Protein structure accession number p. 66 3.8. Quantitative real time polymerase chain reaction (qRT-PCR) p. 67 3.8.1. Total cellular RNA extraction p. 67 3.8.2. RNA purification and cDNA synthesis p. 67 3.8.3. Oligonucleotides design p. 67 3.8.4. Conditions and analysis of qRT-PCR p. 68 3.9. Computer programs p. 69 3.9.1. Sequence alignment p. 69 Chapter 4. Results p. 73 4.1. Platicity domain of the pVP2 C-terminal end p. 73 4.1.1. The role of the basic and acid segments of the His tag in T=13 VLP assembly p. 74 4.1.2. The emulation of VP3 C-terminal domain by the His tag p. 76 4.1.3. Interaction between VP3-based triggering factor and the pVP2 molecular switch p. 78 4.2. Maturation of pVP2 p. 82 4.2.1. VP2 candidates as catalytic residues for autoproteolysis p. 82 4.2.2. Effect of mutations on VP2 Asp-431 and Asp-391 p. 84 4.2.3. Decrease of IBDV infectivity by D431N substitution p. 86 4.2.4. pVP2 an intramolecular proteolytic processing p. 86 4.3. Implication of the catalytic D431 in viral assembly p. 88 4.3.1. Influence of pVP2→VP2 maturation blockade on assembly of IBDV-particles p. 88 4.3.2. The assembly of D431N VP2 trimers p. 89 4.3.3. D431N substitution in the context of HT-VP2-466 chimeric protein p. 92 4.4. The role of a cellular protease p. 93 4.4.1. The VP2-452 intermediate is required for IBDV correct assembly p. 93 4.4.2. Searching for the VP2-452 origin p. 97 4.4.3. Determination of the presence of the PSA in different IBDV- susceptible cell lines p. 98 4.4.4. Coexpression of PSA in H5 insect cells and its implication in VLP Assembly p. 99 4.4.5. The role of the cellular protease in viral infection context p. 104 4.4.5.1. Silencing of PSA in DF1 cells p. 104 4.4.5.2. Effect of silencing in IBDV infection p. 105 Chapter 5. Discussion: Insights in IBDV assembly p. 111 5. 1. Conformational polymorphism of the IBDV major coat protein p. 112 5.2. Molecular switch and triggering factors p. 114 5.3. Proteolytic processing of the capsid protein precursor p. 116 5.4. IBDV assembly model p. 119 5.4.1. Pentamer assembly p. 119 5.4.2.