Cover Page The handle http://hdl.handle.net/1887/22114 holds various files of this Leiden University dissertation Author: Jiang, Xiaohong Title: Construction characterization and application of Flavivirus infectious clones Issue Date: 2013-11-06 Construction, Characterization and Application of Flavivirus Infectious Clones Xiaohong Jiang ISBN: 978-90-8891-733-2 Cover design: Xiaohong Jiang, Joe Zhou and Kerry Zhang (K&A Inc.) The image on the front cover is a schematic representation of the landmarks in Shanghai, China, which is sketched out by the use of predicted RNA structural elements in the 3’-UTR of flaviviruses as illustrated in the thesis. Layout and printing: Proefschriftmaken.nl || Uitgeverij BOXPress Published by: Uitgeverij BOXPress, ’s-Hertogenbosch Construction, Characterization and Application of Flavivirus Infectious Clones Proefschrift ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof. mr. C.J.J.M. Stolker, volgens besluit van het College voor Promoties te verdedigen op woensdag 6 november 2013 klokke 11:15 uur door Xiaohong Jiang geboren te Shanghai, China in 1983 PROMOTIECOMMISSIE Promotor: Prof. Dr. W.J.M. Spaan Copromotor: Dr. P.J. Bredenbeek Overige leden: Prof. Dr. E.J. Snijder Prof. Dr. R.C. Hoeben Dr. D. Franco (Katholieke Universiteit Leuven) Prof. Dr. J.H. Neyts (Katholieke Universiteit Leuven) 献给我挚爱的父母 5 6 CONTENTS Chapter 1 General Introduction 9 Chapter 2 Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs 31 Chapter 3 Molecular and immunological characterization of a DNA launched yellow fever virus 17D infectious clone 55 Chapter 4 An infectious Modoc virus cDNA as a tool to study conserved 3’-UTR RNA elements in flaviviruses with no known vector 79 Chapter 5 Mutagenesis and biochemical analysis of the XRN-1 stalling signal for subgenomic flavivirus (sf)RNA production 105 Chapter 6 Trans-complementation of ellowy fever virus cytopathogenicity by cells expressing subgenomic flavivirus (sf)RNA 127 Chapter 7 Epilogue 145 Summary 159 Samenvatting 163 Curriculum vitae 167 7 8 Chapter 1 Introduction 9 CHAPTER 1 Prologue: Virus Viruses, derived from the Greek word ‘poison’, are a group of obligatory parasites that hijack the hosts’ resources to produce their progeny. Indeed, viruses are notorious for the threats they pose to human beings, as the name suggests. From smallpox, one of human kind’s greatest scourges, to HIV, an enemy that tactfully disarms the human immune system; from the 1918 influenza pandemic to the 2003 SARS outbreak; viruses have often been associated with disease and death. This reputation is, in a sense, well deserved; however, the advent of genomics era has started to enrich our perception of these ‘killers’. The omnipresence and abundance of viruses turn out to go far beyond our wildest guess. In fact, viruses are so pervasive that they essentially infect any form of life, and even parasitizing other viruses [1]. Moreover, viruses have been proposed to play major roles in the global ecosystem [2], have influenced and are still shaping the evolution of cellular life forms, with elements of viral origin embedded in host genomes. The extent of their infiltration into the human genome is surprising yet undeniable [3;4]. We are, for better or worse, living in a virosphere with viruses lurking within us. Virus particles, or virions, consist of nucleic acids, the genetic material, as well as a protein coat, known as the capsid; in some cases, this nucleocapsid is further wrapped by an envelope of lipids. Different from ribosome-encoding organisms, i.e., bacteria, archaea, and eukarya, which all have DNA as their genetic material, viruses (defined as capsid-encoding organisms [5]) can also use RNA molecules to convey their genetic information. The genomic RNA for some viruses like reoviruses is double-stranded, while most of the RNA viruses possess single-stranded RNAs of either positive or negative polarity. Viruses with genomic RNA complementary to mRNA are denoted negative-strand RNA viruses, whereas those with genomic RNA of mRNA polarity are classified as positive-strand or plus-strand RNA (+ssRNA) viruses. The topics covered in this thesis are revolving around a group of +ssRNA viruses, the flaviviruses. Flavivirus: Phylogeny Flaviviruses form a genus within the Flaviviridae family that also includes the genera Pestivirus and Hepacivirus, as well as the newly proposed Pegivirus genus [6]. The Flavivirus genus comprises more than 70 viruses, which, based on phylogenetic analysis, can be grouped into three clusters of related viruses that are in general distinguished by their route of transmission: (i) mosquito-borne, (ii) tick-borne, and (iii) no known vector (NKV) flaviviruses [7]. The first two clusters of flaviviruses are transmitted by arthropods, 10 INTRODUCTION mosquitoes and ticks, respectively. They are exemplified by important human pathogens like dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV). The third cluster comprises a less-well studied group of viruses for which no arthropod vector has been implicated in transmission. These viruses have been exclusively isolated from bats (such as, Montana myotis leukoencephalitis virus [MMLV] [8] and Rio Bravo virus [RBV] [9]) and rodents (for example, Modoc virus [MODV] [10] and Apoi virus [APOIV]). In addition to the viruses assigned to any of the above-mentioned clusters within the Flavivirus genus, there are a few viruses that are currently considered tentative species of this genus. This group of viruses, such as cell fusing agent virus (CFAV), Kamiti River virus (KRV) and Culex flavivirus (CxFV), have all been exclusively isolated from mosquitoes or insect cell lines [11-13]. There is no evidence that these viruses are able to infect a vertebrate host and therefore they are referred to as insect-specific flaviviruses. Flavivirus: General Features Flaviviruses share similarities in virion morphology, genome organization and replication strategy. The enveloped viral particles are composed of a lipid bilayer surrounding a nucleocapsid that consists of a single-stranded, positive-sense genome RNA complexed with multiple copies of a basic capsid (C) protein [14]. 7 The genomic RNA is approximately 11 kb in length with a 5’ type I cap, m GpppAmpN2, while lacking a 3’ poly(A) tail. As other positive-strand RNA viruses, the flavivirus genome plays various roles in the viral life cycle: (i) directing the synthesis of viral proteins by ribosomes, (ii) serving as a template for the production of negative-strand RNA, and (iii) as the genetic material to be encapsidated into progeny virions. The genome is translated into a large polyprotein that is co- and post-translationally processed into functional viral proteins by cellular and viral proteases (Fig.1). The N-terminal third of the polyprotein yields the viral structural proteins: core (C), membrane (prM/M), and envelope (E) that participate in virus entry and assembly. The C-terminal two-thirds of the polyprotein is cleaved into at least seven nonstructural (NS) proteins, namely NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, which have been implicated directly or indirectly in viral RNA replication, evasion of the innate immune response, as well as virus assembly [14-21]. NS1 is a multifunctional glycoprotein that is localized to the replication complexes, expressed on the plasma membrane of infected cells and secreted into the extracellular milieu. The intracellular form of NS1 was proposed to interact with NS4A and NS4B and plays an important role in regulating negative-strand RNA synthesis, whereas its extracellular 11 CHAPTER 1 Figure 1. Flavivirus Genome Organization The flavivirus genome encompasses a single open reading frame that is flanked by ′5 and 3′ untranslated regions (UTRs), within which high-ordered RNA structures are formed. Stem loop (SL) structures are indicated with dots representing the loop and short lines for stems. Location of the conserved sequences (CS) and the upstream AUG region (UAR) that are involved in cyclization of the genomic RNA are indicated. The polyprotein is co- and post- translationally cleaved into three structural proteins (in green) and seven nonstructural proteins (in red). Putative functions of these proteins during infection are described. aa, amino acids; C, capsid protein; E, envelope; M, membrane; RdRp, RNA-dependent RNA-polymerase (Adapted from Cell Host & Microbe, 2009 Apr 23;5(4):318-28, Fernandez-Garcia MD, Mazzon M, Jacobs M, Amara A., reprinted with permission) form was implicated in immune evasion [22-27]. NS2A is a small hydrophobic protein that is essential for virus replication and assembly and may also modulate the host innate immune response [17;18;28;29]. NS2B, another small membrane-associated protein, is a cofactor for the NS2B-NS3 serine protease that mediates cleavage of NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/NS5 junction, and generates the C-termini of mature C and NS4A [14]. In addition to its protease activity, NS3 also contains RNA helicase, nucleotide triphosphatase and RNA triphosphatase activities [30-33]. Both NS4A and NS4B are small hydrophobic proteins that co-localize to ER-derived membrane structures presumed to be the site of replication [34]. NS5, a large and highly conserved flaviviral protein, functions as an RNA-dependent RNA polymerase and contains methyltransferase activity that is required for the formation of a 5’ type I cap as well as internal adenosine 2’-O methylation of the viral genome [14;35-37]. Flaviviruses enter the cell by receptor-mediated endocytosis via clathrin-coated pits [38;39]. The viral particles are subsequently trafficked to prelysosomal endocytic compartments where low pH induces a conformational change in the envelope protein, triggering the fusion between the viral and the endosomal membranes [38;40-43]. The nucleocapsid is thus released into the cytoplasm, where membrane-associated translation- 12 INTRODUCTION replication complexes are formed [34;44;45].