VIRUS-BASED ORGANELLES ENZYME AND DNA-BASED VIRUS NANOSTRUCTURES AND THEIR CELLULAR INTERACTIONS Mark V. de Ruiter VIRUS-BASED ORGANELLES ENZYME AND DNA-BASED VIRUS NANOSTRUCTURES AND THEIR CELLULAR INTERACTIONS Mark de Ruiter Graduation Committee Chairman: Prof. dr. J. L. Herek University of Twente Promoter: Prof. dr. J. J. L. M. Cornelissen University of Twente Members: Prof. dr. ir. P. Jonkheijm University of Twente Prof. dr. H. B. J. Karperien University of Twente dr. L. I. Segerink University of Twente Prof. dr. C. Wege University of Stuttgart Prof. dr. ir. J. C. M. van Hest Eindhoven University of Technology This work has been funded by the Consolidator Grant (Protcage) of the European Research Council (ERC), “Chemistry in the Confinement of Protein Cages” (project ID 616907). The research in this thesis was conducted within the department of Biomolecular Nanotechnology (BNT), the MESA+ Institute for Nanotechnology and at the faculty of Science and Technology (TNW) of the University of Twente. Copyright ©2019 Mark Vincent de Ruiter, Enschede, The Netherlands PhD Thesis, University of Twente, The Netherlands ISBN: 978-90-365-4703-1 DOI: 10.3990/1.9789036547031 Printed by: Ipskamp printing Cover design by: Mark de Ruiter and Laura de Ruiter - van de Weerd VIRUS-BASED ORGANELLES ENZYME AND DNA-BASED VIRUS NANOSTRUCTURES AND THEIR CELLULAR INTERACTIONS DISSERTATION to obtain the degree of doctor at the University of Twente, on the authority of the rector magnificus, prof. dr. T.T.M. Palstra, on account of the decision of the graduation committee, to be publicly defended on Friday the 1st of February 2019 at 14.45 hours by Mark Vincent de Ruiter born on the 16th of March 1990 in Rhenen, The Netherlands This dissertation has been approved by: Supervisor: Prof. dr. J.J.L.M. Cornelissen Table of Contents CHAPTER 1 PREFACE 1 General introduction 2 Aim and outline of this thesis 3 References 4 CHAPTER 2 PROTEIN-BASED NANOREACTORS 7 Introduction 8 Non-protein capsids applied as nanoreactors 9 Protein-based nanoreactors 9 Protein-based organelles derived from eukaryotes 11 Ferritins 11 Heat-shock proteins 13 Pyruvate Dehydrogenase multienzyme cages 14 Vault ribonucleoproteins 15 Protein-based organelles derived from prokaryotes 16 Bacterial microcompartments 17 Encapsulins 18 Lumazine synthase-based cages 22 Virus-like particles 23 Bacteriophage P22 24 Bacteriophage MS2 26 Bacteriophage Qβ 27 Simian virus 40 28 Cowpea chlorotic mottle virus 28 Other protein compartments 28 Cowpea chlorotic mottle virus (CCMV) 29 Structure, pH and salt response 30 Encapsulation of foreign materials in CCMV 31 CCMV-based nanoreactors by encapsulation of enzymes 33 Nanoreactor fabrication overview 34 Conclusions and outlook 36 References 37 i CHAPTER 3 VIRAL NANOREACTORS MADE FROM CCMV 47 Introduction 48 Results and discussion 51 Encapsulation of enzymes using DNA and PSS 51 Size of the particles 53 SDS-PAGE 56 Enzyme-linked immunosorbent assay 57 Catalysis 58 Cryo-EM reconstruction of GOx-ssDNA-CCMV 62 Cryo-EM reconstruction of ASNase-PSS-CCMV 66 Conclusions and outlook 68 Acknowledgments 69 Materials and methods 69 References 75 CHAPTER 4 VIRAL NANOREACTORS IN THE CELL 79 Introduction 80 Results and discussion 82 Intracellular kinetics of β-gal-CCMV 82 Cellular uptake of β-gal-CCMV 85 Stability of CCMV-based nanoreactors in the cell 86 Extracellular activity of encapsulated ASNase 88 Conclusions and outlook 90 Acknowledgments 90 Materials and methods 90 References 94 CHAPTER 5 VIRAL NANOSTRUCTURES CREATED WITH DNA 97 Introduction 98 Results and discussion 100 Minimal ssDNA length required for assembly 100 DNA to create different nanostructures with CCMV-CP 107 Cryo-EM analysis of the different viral structures 114 Conclusions and outlook 119 ii Acknowledgments 120 Materials and methods 121 Refereces 125 CHAPTER 6 UPTAKE OF VIRAL NANOSTRUCTURES IN THE CELL 127 Introduction 128 Results and discussion 130 Cellular uptake route of native CCMV in different cell lines 130 Shape depended cellular uptake in HeLa cells 134 Intracellular positioning of CCMV-based nanostructures 139 In vivo uptake of CCMV by immune cells 142 Plasmid DNA transfection using CCMV 143 Conclusions and outlook 146 Acknowledgments 147 Materials and methods 147 References 151 CHAPTER 7 DNA-BASED PROBES FOR VIRAL (DIS)ASSEMBLY 155 Introduction 156 Results and discussion 158 DNA assembly and purification 158 Formation of virus-like particles 159 Fluorescence analysis 161 Conclusions and outlook 165 Acknowledgments 165 Materials and methods 165 References 170 SUMMARY 173 SAMENVATTING 177 ACKNOWLEDGMENTS 181 ABOUT THE AUTHOR 185 LIST OF PUBLICATIONS 186 iii iv C h a p t e r 1 Preface 1 Chapter 1 | Preface General introduction One of the key features of life on earth is the organization of functional molecules in compartments. Such compartmentalization is even proposed to be one of the first essential steps that led to the formation of life as we know it.1, 2 Exterior compartments, composed of lipid membranes, are vital for the survival of cells,3, 4 because they separate their complex metabolic pathways and processes from the uncontrolled and harsh outside environment.5 However, some of these pathways within one cell could still interfere with each other or require different conditions for efficient functioning. Therefore, several cells evolved subcellular compartments, which are known as 1 organelles.6 Each organelle contains enzymes and is typically devoted to one or several metabolic pathways. This compartmentalization helps to reduce interference, creates controlled environments and enhances the reactions. Since the discovery of microscopes, we are able to visualize the organelles in cells, such as: the cell nucleus, the mitochondria and the Golgi apparatus, which are all made from lipid membranes.6 Cells that contain these lipid membrane- based organelles are classified as eukaryotes, while cells that do not show these lipid based organelles are classified as prokaryotes.7 As new techniques developed, many different smaller organelles were discovered.8 Also, protein- based structures, with sizes ranging from a few nm to one μm, were recognized in cells. For a long time, all of these structures were believed to be viruses,9 but further investigations, showed that some of these cages also contained enzymes that were encapsulated in a protein capsid.10 This revealed that some organelles are also made from proteins, which are found in many different species of life, including prokaryotes.11 This is a relatively new discovery, which gained the interest of an increasing number of researchers leading to the discovery of several different protein base organelles every year.12 However, the exact benefits and function of these protein-based organelles are still not completely understood.11 It is also not known why they are so different with respect to their lipid membrane-based counterparts. One way to gain more understanding on these protein organelles, is to mimic them with known components.13, 14 Since viruses have a similar structure and are well studied entities, they can be used to create artificial protein organelles.15 We use these 2 Chapter 1 | Preface capsids to encapsulate enzymes, creating so called nanoreactors. Several different virus capsids have been used for this purpose and have shown clear benefits over free enzymes.16 Therefore, both these artificial and non-artificial protein-based organelles can be applied in industrial catalysis, metabolic engineering and medicine. Aim and outline of this thesis The aim of this thesis is to create virus-based nanoreactors to gain more understanding on the natural protein-based organelles and to investigate their potential in medical applications. With special focus on their use in the treatment of cancer and enzyme deficiency diseases. In this approach we turn 1 the disease causing viruses in to useful assemblies that can make you better. To effectively create virus-based nanoreactors, it is advantages to know how the virus assembles. Furthermore, in working towards medical applications of the virus-based nanoreactors, more knowledge is required on the cellular interactions of these protein cages. Therefore, additional aims of this thesis are to understand the assembly of a virus capsid and to find the optimal virus-based nanostructure for cellular uptake. This increased understanding is valuable for the application of virus-based structures as drug carriers, transfection agents and new generations of vaccines. In this thesis the capsid proteins (CPs) of the cowpea chlorotic mottle virus (CCMV) are used to fabricate these nanoreactors and other nanostructures, since it is a well-studied virus that shows reversible disassembly behavior and because it is a plant virus that is safe for humans. Chapter 2 reviews natural and artificial protein-based organelles, discussing their structures, fabrication methods and applications. Furthermore, a more in- depth coverage on CCMV is given. Chapter 3 is focused on the fabrication of artificial organelles by encapsulation of several different enzymes inside the protein capsid of CCMV. These particles are then evaluated further, resolving their structure and showing a change in kinetics upon encapsulation of the enzyme. 3 Chapter 1 | Preface In Chapter 4, we show the potential of CCMV-based artificial organelles for medical applications. Towards this aim two of the nanoreactors described in Chapter 3 are used to study their extracellular and intracellular interaction with cancer cells. In Chapter 5 the assembly of the capsid proteins of CCMV around various lengths and sizes of DNA is investigated. In order to gain more understanding on the assembly process of CCMV and to create various virus-based nanostructures with different geometries. In Chapter 6 we investigate the cellular uptake mechanism of the native CCMV virus in different cell lines. Furthermore, we investigate how uptake is 1 influenced by shape of viral nanostructures by using the assemblies created in Chapter 5. In Chapter 7 we describe the development of a new FRET-based approach to study viral assembly and disassembly. The approach is based on the nucleic acid cargo and does not require exterior modification of the virus.