RSC Biomolecular Sciences Edited by Mavis Agbandje-McKenna and Robert McKenna Structural Virology S t r u c t u r a l V i r o l o g y A g b a n d & je M -M c c K K e e n n n n a a Structural Virology RSC Biomolecular Sciences Editorial Board: Professor Stephen Neidle (Chairman), The School of Pharmacy, University of London, UK DrMariusClore,NationalInstitutesofHealth,USA ProfessorRoderickEHubbard,UniversityofYorkandVernalis,Cambridge,UK ProfessorDavidMJLilleyFRS,UniversityofDundee,UK Titles in the Series: 1:BiophysicalandStructuralAspectsofBioenergetics 2:ExploitingChemicalDiversityforDrugDiscovery 3:Structure-basedDrugDiscovery:AnOverview 4:StructuralBiologyofMembraneProteins 5:Protein–CarbohydrateInteractionsinInfectiousDisease 6:Sequence-specificDNABindingAgents 7:QuadruplexNucleicAcids 8: Computational and Structural Approaches to Drug Discovery: Ligand–Protein Interactions 9:Metabolomics,MetabonomicsandMetaboliteProfiling 10:RibozymesandRNACatalysis 11:Protein-NucleicAcidInteractions:StructuralBiology 12:TherapeuticOligonucleotides 13:ProteinFolding,MisfoldingandAggregation:ClassicalThemesandNovel Approaches 14:NucleicAcid-MetalIonInteractions 15:OxidativeFoldingofPeptidesandProteins 16:RNAPolymerasesasMolecularMotors 17:QuantumTunnellinginEnzyme-CatalysedReactions 18:NaturalProductChemistryforDrugDiscovery 19:RNAHelicases 20:MolecularSimulationsandBiomembranes:fromBiophysicstoFunction 21:StructuralVirology How to obtain future titles on publication: Astandingorderplanisavailableforthisseries.Astandingorderwillbringdeliveryof eachnewvolumeimmediatelyonpublication. Forfurtherinformationpleasecontact: BookSalesDepartment,RoyalSocietyofChemistry, ThomasGrahamHouse,SciencePark,MiltonRoad,Cambridge, CB40WF,UK Telephone:+44(0)1223420066,Fax:+44(0)1223420247,Email:[email protected] Visitourwebsiteathttp://www.rsc.org/Shop/Books/ Structural Virology Edited by Mavis Agbandje-McKenna and Robert McKenna Department of Biochemistry and Molecular Biology, University of Florida, FL, USA RSCBiomolecularSciencesNo.21 ISBN: 978-0-85404-171-8 ISSN: 1757-7152 AcataloguerecordforthisbookisavailablefromtheBritishLibrary rRoyalSocietyofChemistry2011 Allrightsreserved Apartfromfairdealingforthepurposesofresearchfornon-commercialpurposesorfor privatestudy,criticismorreview,aspermittedundertheCopyright,DesignsandPatents Act1988andtheCopyrightandRelatedRightsRegulations2003,thispublicationmaynot bereproduced,storedortransmitted,inanyformorbyanymeans,withouttheprior permissioninwritingofTheRoyalSocietyofChemistryorthecopyrightowner,orinthe caseofreproductioninaccordancewiththetermsoflicencesissuedbytheCopyright LicensingAgencyintheUK,orinaccordancewiththetermsofthelicencesissuedbythe appropriateReproductionRightsOrganizationoutsidetheUK. Enquiriesconcerning reproductionoutsidethetermsstatedhereshouldbesenttoTheRoyalSocietyof Chemistryattheaddressprintedonthispage. TheRSCisnotresponsibleforindividualopinionsexpressedinthiswork. PublishedbyTheRoyalSocietyofChemistry, ThomasGrahamHouse,SciencePark,MiltonRoad, CambridgeCB40WF,UK RegisteredCharityNumber207890 Forfurtherinformationseeourwebsiteatwww.rsc.org Preface Viruses can be grouped among the simplest biological systems that have the ability to evolve and adapt to exist in different environments. That is, they have the ability to ‘jump’ from one host to another, some carrying the neces- sary molecular machinery to transfer and modify their genetic information from one generation to the next, while others hijack the host machinery to effect the necessary modifications. Because of this innate ability, it would not be unreasonable to state that viruses have most likely infected every life form that has ever existed on our planet, from the simplest single-cell organisms to plants, animals, and humans. To achieve such biodiversity, viruses have evolved different and efficient strategies for host recognition, internalization, cellular trafficking, genome replication, capsid assembly, genome packaging, release of progeny (for re- infection) and host immune surveillance evasion, to optimize their life cycle in their unique niche. This has resulted in viruses of different shapes and sizes, fromsimplesingle-proteinsphericalorhelicalassemblages,tomultiplecomplex systems, assembled from hundreds of proteins without/with (enveloped) the incorporation of host lipids. Invariably the viral coat protein(s) (referred to throughout this monograph interchangeably as either CPs or VPs) form some sort of integral protective shell (a viral capsid) around the infectious genomic nucleicacid,whichcanbesingle-stranded(ss)DNA,ssRNA,double-stranded (ds) DNA or dsRNA, packaged as single or multiple, linear or circular mole- cule(s).ThepackagedviralgenomeencodesalltherequiredstructuralCPs/VPs and auxiliary non-structural proteins that are required in combination with hostproteinsforhostinfection.Theenvelopedvirusesincorporatetheirhost’s lipidsaseitheraninternaland/orexternalenvelopeduringtheirassembly.For a number of viruses, CP/VP recognition and encapsidation of the genomic nucleicacidisaprerequisiteforinfectiouscapsidformation,whereasforothers the genome is packaged into preformed capsids via interactions with viral or host encoded proteins. In addition to genome encapsidation and protection during cellular entry and trafficking, the CP/VP can also dictate many other RSCBiomolecularSciencesNo.21 StructuralVirology EditedbyMavisAgbandje-McKennaandRobertMcKenna rRoyalSocietyofChemistry2011 PublishedbytheRoyalSocietyofChemistry,www.rsc.org v vi Preface viral functions, including host receptor/vector recognition, transmission and the genomic transduction efficiency during infection. Forsphericalviruses,theCP/VPorganizationinthecapsidarchitecturetakes on the form of an icosahedron (a platonic solid with point group symmetry 5.3.2), a regular polyhedron which is assembled from 20 equilateral triangles. This symmetrical shell is a consequence of it consisting of identical (or almost identical) gene products, consistent with the argument that there is insufficient volume inside a virus to accommodate a more complicated protein coding strategy.Theexacttwofold,threefoldandfivefoldsymmetryoftheicosahedron permitsthe(quasi)equivalentsymmetryrequiredtoconstructstructureswith60 or multiples [denoted by a T (triangulation) number] of 60 subunits. This monograph will discuss viruses assembled from the simplest of icosahedral capsids,withT¼1triangulation(assembledfrom60CP/VPsubunits),tothose with more complicated VP shells assembles and lipid membrane envelopes. Viruses havebeenresponsibleformore humandeaths,eitherthroughdirect infection (such asinfluenzavirus)orinfection ofcrops,than anyother known human disease-causing agent. In addition, their ability to package efficiently and deliver genomic material to different living organisms and tissues also makesthemattractivevehiclesforthedeliveryoftherapeuticgeneticmaterialin situations where defective genes lead to disease phenotypes. Thus viruses are the subject of intense scientific study in many different disciplines, including structure biology, in efforts to (i) understand the basic biological processes governing viral infection and (ii) develop treatment strategies, including vac- cines, anti-virals and gene delivery vectors. The use of structure approaches in virology has given insight into the structural basis of assembly, nucleic acid packaging, particle dynamics and interactionswithcellularmoleculesandallowedtheelucidationofmechanistic pathways at the atomic and molecular level. Biological processes, such as the life cycle of a virus infection, are governed by numerous intricate macro- molecular interactions. The role of the structural virologist is thus to visualize these interactionsin three dimensions (3D), toprovidea full understandingof these interactions as ‘seeing is believing’. These structural characterizations of viruses then provide crucial platforms for the development of treatment and therapeutic strategies (Section 3 of this monograph). Therangeofbiophysicalmethodsusedinstructuralvirologyisvast,ranging fromhydrodynamictoscatteringtechniques(Section1ofthismonograph),and haveplayedafundamentalroleinourunderstandingofviralinfectioninrecent years.Themethod undertakenforaparticularstudyisoftendependentonthe resolution and type of information desired and also thesize and complexity of the macromolecule under investigation, the amount of material available, its solubility in aqueous environments (Chapter 1) and the type of interactions beingvisualized.Forexample,fortheimagingofwholevirusesduringinfection, confocal microscopy (Chapter 2) and cryo-electron tomography (cryo-ET) (Chapter4)areapplied,whichpermitstudiesatmolecularresolution.Andwhile both nuclear magnetic resonance (NMR) spectroscopy (Chapter 8) and X-ray crystallography(Chapters6and7)cangiveatomicresolutiondetailonprotein Preface vii backbone and side-chain placement, NMR also provides dynamic (ensemble) information and crystallography provides a ‘snapshot’ and is often considered static. Solution approaches, such as limited proteolysis combined with mass spectrometry and small-angle scattering approaches (Chapter 3), also provide dynamic information. In cryo-ET and cryo-electron microscopy (cryo-EM) (Chapter 5), macromolecules are frozen in their native state, allowing for dis- crete selection of dynamic states to be visualized, albeit at lower resolution. Generally, NMR spectroscopy is utilized for small protein molecules that are flexible, X-ray crystallography for medium-sized proteins and complexes that are compact, whereas very large macromolecular assemblages or membranous protein structures are determined by cryo-EM. The largest issue separating cryo-EM and cryo-ET from crystallography, in addition to size and the lim- itations of crystal formation, is resolution. Cryo-EM has generally been con- sideredalow-resolutiontechnique,givingreconstructionsaround15–30A˚ ,but withadvancesinsamplehandling,instrumentation,imageprocessingandmodel building, near-atomic resolution structures are now being achieved. For cryo- ET the resolution achieveable is still low. In reality, hybrid approaches, combining NMR, X-ray crystallography and cryo-EM, cryo-ET and solution data, are often adopted, which provides a powerfulmeansoffillinggapswhichcanariseinthestructuralcharacterization oflarge macromolecules.For example,instudieswherelargevirusescannotbe crystallized,subcomponents can be crystallized to obtain high-resolution infor- mation, which can then be used to interpret the structure at lower resolution obtained by cryo-EM or cryo-ET. Or atomic structures obtained from homo- logousviralproteins/viruscapsidscanbeusedfor3Dhomologymodelbuilding. These approaches permit the pseudo-atomic visualization of interaction inter- faces between protein–protein subunits, protein–nucleic acids and protein–lipid in virus capsids and also the visualization of virus capsid–host interactions. Combined with biochemical, biophysical and molecular biology analysis, structuralstudiesindicateahighdegreeoffidelityinthestepsthatresultinthe assemblyofmatureinfectious viruscapsids(Chapter 10).They also show that the fundmental principles governing successful viral capsid assembly, efficient polymerization of CP subunits utilizing specific interface interactions that spontaneously terminate, often employ structural polymorphisms to facilitate the required interactions. Structural virology approaches have also been plat- forms for the elegant description of the virus infection process, from initial receptor attachment to the interaction of the capsid with host antibodies (Section2),andprovidedthetargetsfortherapeuticinterventionandimproved viral capsid vectors for gene delivery (Section 3). This monograph is designed to provide a basic introduction to the use of structural virology and its applications in virus research towards functional annotation and is not intended to provide a detailed discussion of approaches utilized. Mavis Agbandje-McKenna Robert McKenna Contents SECTION 1 Chapter1 ProductionandPurificationofVirusesforStructuralStudies 3 Brittney L. Gurda and Mavis Agbandje-McKenna 1 Introduction 3 2 Expression Systems 4 Eukaryotic Systems 5 Plant Systems 9 Prokaryotic Systems 10 Cell-free Systems 12 Tissue Samples 12 3 Purification 12 Ultracentrifugation 13 Chromatography 14 4 Example Virus Capsid Production and Purification – Adeno-associated Virus Serotype 1 15 VLP Expression Using the BEVS 16 Production of VLPs in Sf9 Insect Cells 17 Purification of AAV1 VLPs from Infected Sf9 Cells 17 5 Summary 18 6 Acknowledgments 18 References 18 Chapter 2 Microscopic Analysis of Viral Cell Binding, Entry and Infection in Live Cells 22 Colin R. Parrish 1 Introduction 22 2 Endocytosis, Cytoplasmic Transport and Viral Entry 24 RSCBiomolecularSciencesNo.21 StructuralVirology EditedbyMavisAgbandje-McKennaandRobertMcKenna rRoyalSocietyofChemistry2011 PublishedbytheRoyalSocietyofChemistry,www.rsc.org ix