UNIVERSITY OF COPENHAGEN FACULTY OF SCIENCE DANISH ARCHAEA CENTRE PhD thesis Kristine Buch Uldahl Hyperthermophilic archaeal viruses as novel nanoplatforms A cademic supervisor: Xu Peng November 2015 UNIVERSITY OF COPENHAGEN FACULTY OF SCIENCE DANISH ARCHAEA CENTRE PhD thesis Kristine Buch Uldahl Hyperthermophilic archaeal viruses as novel nanoplatforms Academic supervisor: Xu Peng November 2015 Institutnavn: Biologisk Institut Name of department: Department of Biology Section: Functional Genomics Author: Kristine Buch Uldahl Titel: Hypertermofile arkæavirus som nye nanoplatforme Title / Subtitle: Hyperthermophilic archaeal viruses as novel nanoplatforms Subject description: This thesis aims at evaluating archaeal viruses as novel nanoplatforms. The focus will be on investigating the hyperthermophilic archaeal virus, SMV1, to gain insights into the viral life-cycle and to provide a strong knowledge base for developing SMV1 into a nanovector platform. Main supervisor: Associate Professor Xu Peng Co-supervisor: Professor Moein Moghimi Submitted: November 2015 Type: PhD thesis Cover: Top: Kristine Uldahl, sampling trip Yellowstone National Park, Left: TEM image of Sulfolobus monocaudavirus 1, Bottom: Morning Glory Hot spring, Yellowstone National Park Preface This thesis is the product of a three-year PhD project at the Faculty of Science, University of Copenhagen, based at the Danish Archaea Centre, Department of Biology. The thesis has been supervised by associate Professor Xu Peng with heavy involvement from co-supervisor Professor Moein Moghimi (Centre for Pharmaceutical Nanotechnology and Nanotoxiocology (CPNN), University of Copenhagen). Further guidance, collaboration, and advice were received in relation to specific chapters from Mark J. Young and Seth T. Walk. The thesis consists of two parts. The first part is a synopsis which gives an overview of the background and objectives of the thesis, summarizes and discusses the main findings, and outlines some perspectives for future research. The second part consists of 4 manuscripts, written as scientific papers, which comprise the core of the PhD project. During my PhD I had the pleasure of spending approximately half a year in the lab of Mark J. Young at Montana State University. Further, I was invited by Dr. Brent Peyton (Head of the Thermal Biology Institute, Montana State University) to participate in the annual sampling trip to the Hot Springs at Heart Lake, Yellowstone National Park. The stay at Montana State University was partially funded by Knud Højgaards Fond and Oticon Fonden. Besides the work contained in the chapters I have also assisted with teaching the course, Archaea Biology, for two consecutive years and supervised two bachelor students with laboratory experiments and troubleshooting. Kristine Buch Uldahl Copenhagen, Denmark November 2015 Table of contents 1. Preface v 2. Table of Contents vii 3. Summary 1 4. Sammenfatning 3 5. Acknowledgements 5 6. List of manuscripts 6 7. Synopsis 7.1 Introduction 7 7.2 Aims of the Thesis 8 7.3 Archaea: the new branch on the tree 9 7.4 The wondrous world of archaeal viruses 16 7.5 When viruses become useful 25 7.6 Design strategies of viral nanoplatforms 27 7.7 From engineering to applications in medicine 32 7.8 Summary of results 33 7.9 Outlook 38 7.10 References 40 8. Chapter I 45 9. Chapter II 69 10. Chapter III 97 11. Chapter IV 127 Summary Viruses are the most abundant biological entities on earth, and with an estimated 1031 virus-like particles in the biosphere, viruses are virtually everywhere. Traditionally, the study of viruses has focused on their roles as infectious agents. However, over the last decades with the development of a broad range of genetic and chemical engineering methods, viral research has expanded. Viruses are now emerging as nanoplatforms with applications in materials science and medicine. A great challenge in biomedicine is the targeting of therapeutics to specific locations in the body in order to increase therapeutic benefit and minimize adverse effects. Virus-based nanoplatforms take advantage of the natural circulatory and targeting properties of viruses, to design therapeutics that specifically target tissues of interest in vivo. Plant-based viruses and bacteriophages are typically considered safer nanoplatforms than mammalian viruses because they cannot proliferate in humans and hence are less likely to trigger adverse effects. Another group of viruses that fits this criterion is archaeal viruses yet their potential remains untapped. As a group, archaeal viruses offer distinct advantages such as unique morphotypes and inherent stability under extreme conditions. This thesis presents the first in depth investigation of any archaeal virus, SMV1, as a potential nanoplatform for applications in nanomedicine. In order to provide a strong foundation for downstream experiments and future applications, Chapter I presents an in depth investigation of the hyperthermophilic archaeal virus SMV. Decisive steps in the viral life-cycle are studied with focus on the early stages of infection. TEM observations suggest that SMV1 virions enter into host cells via a fusion entry mechanism, involving three distinct stages; attachment, alignment, and fusion. Upon infection, the intracellular replication cycle lasts 8 h at which point the virus particles are released as spindle-shaped tailless particles. Chapter II builds on the replication and purification methods in Chapter I to study the interaction between the two hyperthermophilic archaeal viruses, SMV1 and SSV2 and cells of human origin. This chapter provides the first results demonstrating that archaeal viruses can be taken up and internalized by human cells, thus indicating a potential as intracellular delivery agents. Chapter III investigates SMV1 particles as potential nanocarriers targeting the gut microbiome. Stability experiments proved SMV1 to be highly stable in both simulated conditions of the human gastrointesinal tract (in vitro) and when passaged orally in mice (in vivo). In general, high doses of SMV1 elicited a nearly undetectable murine inflammatory response and challenged mice showed no 1 observable signs of pain or distress. The stability of SMV1 was compared to that of the traditionally used Inovirus, M13KE. SMV1 outperformed this state-of-the-art vector as measured by in vitro and in vivo survival. Chapter III provides strong evidence that SMV1 in particular and archaeal viruses in general have intrinsically favorable in vivo characteristics for bioengineering applications, such as drug delivery in the gastrointestinal tract. Chapter IV presents an overview of all known archaeal viruses and discusses the application potential of archaeal viruses. 2 Sammenfatning Vira er de mest udbredte biologiske enheder på jorden. Vira er næsten overalt. Det er anslået at der er 1031 viruslignende partikler i biosfæren. Traditionelt set har studiet af virus fokuseret på deres rolle som sygdomsfremkaldende agenter. Men i de seneste årtier med udviklingen af en bred vifte af genetiske og kemiske teknikker har virusforskning udvidet sig. Vira finder nu anvendelse som organiske nanoplatforme indenfor materialevidenskab og medicin. En stor udfordring i biomedicin er målretningen af lægemidler til bestemte steder i kroppen for at øge terapeutisk fordel og minimere bivirkninger. Virus-baserede nanoplatforme drager fordel af naturlige virusegenskaber i form af nanostørrelse og symmetri, til at designe lægemidler, der er målrettet specifikt væv i kroppen. Vira der inficerer planter og bakterier, bliver typisk betragtet som ”mere sikre” i forhold til eukaryotiske vira, da de ikke kan reproducere sig i mennesker og dermed er mindre tilbøjelige til at forårsage bivirkninger. En anden gruppe af vira der passer til dette kriterium, er arkæavira. Deres potentiale er endnu ikke blevet udnyttet. Som gruppe tilbyder arkæavira flere fordele såsom unikke morfologiske former og naturlig stabilitet under ekstreme forhold. Denne PhD afhandling repræsenterer den første dybtgående evaluering af en arkæavirus, SMV1, som en potentiel nanoplatform til applikationer indenfor nanomedicin. For at give et stærkt fundament for efterfølgende eksperimenter og fremtidige applikationer, præsenterer Kapitel I en dybtgående undersøgelse af den hypertermofile arkæavirus SMV1. Afgørende faser i den virale livscyklus studeres med fokus på de tidligste faser af infektionen. TEM observationer tyder på at SMV1 partikler trænger ind i værtens celler via en fusionsmekanisme, der involverer tre forskellige stadier; vedhæftning, positionering, og fusion. Efter infektion varer den intracellulære replikationscyklus 8 timer, på hvilket tidspunkt viruspartiklerne frigives som haleløse partikler. Kapitel II bygger på oprensningsmetoder fra Kapitel I for at studere samspillet mellem de to hypertermofile arkæavira, SMV1 og SSV2 og celler af menneskelig oprindelse. Dette kapitel indeholder de første resultater der viser at arkæavira kan optages og internaliseres af humane celler, hvilket indikerer et potentiale som intracellulære leveringsagenter. Kapitel III undersøger SMV1 partikler som potentielle nanocarriers rettet mod tarmmikrobiomet. Stabilitetsforsøg viste at SMV1 partikler er særdeles stabile i både simuleret tarmmiljø (in vitro), og under tarmpassage hos en mus (in vivo). Generelt set gav høje doser af SMV1 et ikke målbart murin inflammatorisk respons og udfordrede mus viste ingen observerbare tegn på smerte eller stress. Stabiliteten af SMV1 blev 3 sammenlignet med den traditionelt anvendte