Molecular characterization of the Tick-borne Environments and replication

Wessam Melik ©Wessam Melik, Stockholm 2012

ISBN 978-91-7447-409-1

Södertörn Doctoral Dissertations 63 ISSN 1652-7399 ISBN 978-91-86069-42-1 (Södertörns högskola)

Printed in Sweden by Universitetsservice US-AB, Stockholm 2012 Distributor: Department of Genetics, Microbiology and Toxicology, Stockholm University

To my dearly beloved mother

Abstract

The genus is of major concern for world morbidity and mortality and includes causing both encephalitic as well as hemorrhagic diseases. The mammalian tick-borne flavivirus group (MTBFG) includes the Tick-borne encephalitis virus (TBEV), which can infect the human central nervous system (CNS) generating severe encephalitis. The incidence of Tick-borne encephalitis (TBE) is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus, at higher latitudes have putative connections to global warming. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains. In this study we have generated strategies for detection of broad types of tick-borne in pools of I. ricinus sampled in Sweden. The positive collection in the Stockholm archipelago on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self- replicating DNA based sub-genomic TBEV replicon capable of expressing egfp and luc reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/βPIX protein complex. In addition, the presence of TBEV NS5 impairs neurite outgrowth in neuronal growth factor (NGF) induced PC12 cells, and we postulate that this inhibition is due to improper activation of Rac1. We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins binding to the C-terminus of the TBEV and West-Nile virus NS5 proteins. These interactions might affect cellular pathways and suggests factors that might have a role in flavivirus replication. We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV comparing the Torö-2003 isolate with other TBEV strains. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in environmentally sampled ticks.

List of publication

This thesis is based on the following articles, which are referred to by their Roman numerals I to IV

I. Wessam Melik, Anders Nilsson, and Magnus Johansson, Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses, Archives of Virology, 2007; 152(5):1027-34

II. Annelie Elväng*, Wessam Melik*, Yann Bertrand, Mikael Lönn and Magnus Johansson, Sequencing of a Tick-borne encephalitis virus from Ixodes ricinus reveals a thermo sensitive RNA switch significant for virus propagation in ectothermic arthropods, Vector-Borne and Zoonotic Diseases, 2011; 11(6):649-58 (*) Authors contributed equally to this work

III. Michael Wigerius, Wessam Melik, Annelie Elväng and Magnus Johansson, Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5, Molecular and Cellular Neuroscience, 2010; 44(3):260-71

IV. Wessam Melik, Karin Ellencrona, Michael Wigerius, Annelie Elväng and Magnus Johansson, Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication, manuscript

Contents

List of publication ...... v

Introduction ...... 10 Flavivirus ...... 11 Mammalian Strains; Geographical Distributed Vectors and Clinical Manifestation ...... 13 Emergence of TBEV in Sweden ...... 16 The life cycle of the tick vector ...... 16 The flavivirus genome structure ...... 18 Tick-borne encephalitis virus NCR ...... 20 The cellular life cycle of the flavivirus ...... 21 Flavivirus replication ...... 24 Replicon ...... 25 Protein-Protein interaction domains ...... 26 PDZ domains ...... 27 The NS5 protein ...... 27 Scribble ...... 28 GIPC ...... 30 ZO-2...... 30

Aims of thesis ...... 32

Results and discussions ...... 33 Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses (Paper I) ...... 33 Sequencing of a Tick-borne encephalitis virus from Ixodes ricinus reveals a thermo sensitive RNA switch significant for virus propagation in ectothermic arthropods (Paper II) ...... 34 Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5 (Paper III) ...... 35 Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication (Paper IV) ...... 37

Conclusion and future perspective ...... 38

Material and methods ...... 41 Tick sampling ...... 41 Cell culture...... 41 Luciferase assay ...... 42 Real time PCR ...... 42 Nested PCR ...... 42

Confocal laser scanning microscopy...... 43 Co-immunoprecipitation ...... 43 Western blot ...... 43 Construction of the sub-genomic TBEV replicons ...... 44 Computer software ...... 51

Acknowledgements ...... 52

References ...... 55

Abbreviations

AP-1 Activator protein 1 BHK-21 Baby hamster kidney C Capsid CMV Human cytomegalovirus CNS Central nerve system CoIP Co-immunoprecipitation COS-7 Cercopithecus aethiops CS Cyclization sequence DIP Database of interacting proteins DNA Deoxyribonucleic acid E Envelope EGFP Enhanced green fluorescence protein ER FMDV Foot-and mouth-disease virus GIPC GAIP interacting protein C terminus HDV Hepatitis delta virus HPV Human papillomavirus IAV Influenza avian virus IL Interleukin LUC Luciferase M Membrane MDCK Madin-darby canine kidney MTBFG Mammalian tick-borne flavivirus group N2A Neuroblastoma NCR Non-coding region NLS Nuclear localization signals NS None structural ORF Open reading frame PBM peripheral blood mononuclear PC12 Pheochromocytoma PCR Polymerase chain reaction PDZ (PSD-95/Dlg/ZO-1) homology domain PH Pleckstrin homology PID Protein interaction domains prM Precursure membrane PU Homopurine PY Homopyrimidine RC Replication complex RdRp RNA dependent RNA polymerase RF Replicative form RLU Relative luciferase unit RNA Ribonucleic acid

SAM Sterile alpha motif SH3 SRC homology 3 SL Stem loop TBEV Tick borne encephalitis virus TGN Trans-golgi network TJ Tight junction UTR Un-translated region VLP Virus like particle WNV WW Tryptophan-tryptophan ZO-2 Zonula occludens protein

Introduction

Viruses are not considered as life-forms in the same sense as eukaryotic or prokaryotic organisms since they lack their own metabolism and the ability to replicate without a host cell. All viruses are simple infectious particles that have specialized to multiply in host cells e.g. mammals, plants, insects, ticks or bacteria where the virus highjack’s cellular systems such as proteases, endoplasmatic reticulum (ER), , polymerases and ribosomes to assemble new infectious virions. Different viruses vary in size from 20- 300 nm and can adopt different structures where the genome consists of either single- or double-stranded nucleic acid (DNA or RNA) surrounded by repetitive arrangement of the capsid protein. In addition, many viruses also have an outer envelope composed of lipids and proteins (Burke, 2001; Cann, 2001; Flint, 2003; Lindenbach, 2007), e.g. the flaviviruses (Fig. 1).

Figure 1) Drawing of the flavivirus structure. The nucleocapsid which surrounds the +ssRNA genome is enveloped by a lipid bilayer containing membrane (M) and envelope (E) glycoproteins surrounding. (Illustrated by; Irsa Melik).

The central dogma of life states that the coded genetic information (DNA) is transcribed into messenger RNA (mRNA), which is translated into proteins. This one-way dogma is often not manifested by viruses. Different modes of viral expression and replication are summarized in figure 2. The characterization of the viral genome is based on a number of parameters: 1) composition of nucleic acid (DNA or RNA); 2) size and number of strands; 3) terminal structure; 4) nucleotide sequence; 5) coding capacity; 6)

10 regulatory signal elements, transcriptional enhancer, promoters and terminators (Burke, 2001; Lindenbach, 2007).

Figure 2) Different mechanisms of viral expression and replication. a) dsDNA genomes e.g. , and Adenoviridae. b) ssDNA genomes e.g Parvoviridae. c) Gapped and circular dsDNA genome using positive ssRNA intermediate e.g. Hepadnaviridae. d) dsRNA e.g . e) Positive ssRNA e.g. , , Picornaviridae and Togaviridae. f) Negative ssRNA e.g , and . g) Positive ssRNA using ±DNA intermediate e.g Retroviridae.

Flavivirus At the beginning of the last century virus (YFV) was the first flavivirus isolated and cultivated in vivo. In rapid succession additional members of the genus were identified associated with specific diseases (Burke, 2001). The flavivirus genus includes about 70 viruses typically transmitted to vertebrate hosts by mosquito or tick vectors, but no known vector (NKV) flaviviruses have also been identified (Gould et al., 2001). The flavivirus genus is a member of the family flaviviridae which also includes hepaciviruses and pestiviruses (Heinz & Mandl, 1993; Lindenbach, 2001; Mandl et al., 1989). Human flaviviral diseases such as Dengue hemorrhagic fever (DHF), Yellow fever (YF), Tick-borne encephalitis (TBE), (JE), and (WN) cause extensive worldwide morbidity and mortality (Chambers et al., 1990). Even though the flaviviruses share the same genetic structure; the pathogenic patterns for different viruses within the genus vary extensively. An overview of disease symptoms for three flavivirus examples are illustrated in Table 1.

11

Table 1: Clinical manifestations of three common flaviviruses. Flavivirus complexes West Nile virus Western European tick (DENV) (WNV) borne encephalitis virus  Incubation period  20-40% of the  30 % of the infected

4-7 days infected show individuals show

 Occur with primary symptoms symptoms virus infection  Incubation period  Incubation period 2-  Fever, frontal 2-14 days 14 days headache, joint  Flue like  Flue like symptoms, pain, rash and symptoms, fever, fever, headache,

ral symptoms ral vomiting (Burke, headache, fatigue, fatigue, skin rash 2001) skin rash and and vomiting

Gene vomiting (Burke, (Burke, 2001; 2001) Gritsun et al., 2003b) Dengue hemorrhagic Neuroinvasive disease Secondary fever/dengue shock  1% of the infected neuroinvasive phase syndrome individuals  20-30% of the  Secondary dengue  Meningitis, infected individuals

infection encephalitis, acute  Neurological  2-3% infected flaccid entities symptoms patients (Ulbert, 2011)  Meningitis,  High fever, severe  Rhabdomyolysis meningoencephalitic headache, vomiting,  Myositis , poliomyelitic acute abdominal  Chorioretinis encephalitis pain  Fatality rate less  Capillary leakage than 2% (Burke, Specific symptoms Specific  Haemostasis 2001; Gritsun et al.,  Liver damage 2003b)  Fluid loss into tissue spaces led to shock

12 Mammalian Strains; Geographical Distributed Vectors and Clinical Manifestation The tick-borne flaviviruses currently comprise 12 species grouped into mammalian and seabird viruses placing TBEV into the mammalian tick- borne flavivirus group (MTBFG) (Thiel et al., 2005). The MTBFG includes several similar viruses distributed over the northern hemisphere e.g. the TBEV and virus (LIV) in Europe, virus (OHFV), Langat virus, Alkhurma hemorrhagic fever virus (AHFV) and virus (KFDV) in Asia and the and in North America (Gould et al., 2001). Of these, KFDV, AHFV and OHFV viruses cause hemorrhagic fever in humans, while the remaining species are encephalitogenic. There are three main lineages of TBEV; the Western European (W-TBEV), the far Eastern (FE-TBEV) and the Siberian subtypes (S-TBEV) (Burke, 2001; Charrel et al., 2004; Ecker et al., 1999; Gould et al., 2003). The foremost factors for determining the taxonomy of species and members within the genus are nucleotide and deduced amino acid sequence data, antigenic relationships, vector association and geographic incidence (Calisher & Gould, 2003; Thiel et al., 2005). The W-TBEV lineage is transmitted by Ixodes ricinus whereas the FE-TBEV and the S-TBEV subtypes are transmitted by I. persulcatus (Burke, 2001). A comparison between the severity of Siberian and Far- Eastern subtypes shows that FE-TBEV infections are usually severe with frequent encephalitic signs with a fatality rate of 5-35 %, whereas S-TBEV infections have a fatality rate of 1-3 %, although a chronic form of S-TBEV seems to be more frequent. W-TBEV infection develops into neurological disease in about 25 % of the patients and less than 2 % of the cases are fatal (Burke, 2001; Charrel et al., 2004; Gould et al., 2003). In nature TBEV is transmitted via co-feeding of infected and non-infected ticks mainly on rodents. Several reports note that differences in temperature and increased global warming increase the tick populations that can favor spread and TBEV in to new geographic regions (Estrada-Pena, 2009; Gould et al., 2003; Gritsun et al., 2003a; Gritsun et al., 2003b; Randolph, 2004; Randolph et al., 1999; Randolph et al., 2008). The LIV may cause fatal neurological disease in sheep and red-grouse but affects humans rarely (Gao et al., 1993; McGuire et al., 1998). Up to 60% of a sheep flock can die in endemic areas and the highest losses are among unvaccinated younger sheep that may have lost protection by maternal antibodies. The disease is extremely severe in red grouse populations where the mortality rate can be as high as 80% in experimentally infected birds. Reported human LIV infections occurs mainly among individuals with occupations that are in contact with the animals, e.g. sheep farmers, veterinarians and slaughterhouse workers (Burke, 2001; Charrel et al., 2004; Gould et al., 2003). Infected humans can develop flu-like- or neurological symptoms, but the illness is rarely fatal. There is no specific treatment, but

13 the TBEV vaccine confers immunity. LIV has so far only been detected in the British Isles, Ireland, Denmark and Norway and is transmitted to sheep by I. ricinus (Charrel et al., 2004; Jensen et al., 2004). OHFV is endemic in Siberia and Russia. The virus is transmitted mainly via Dermacentor ticks, I. persulcatus and other arthropods but can also be directly transmitted between muskrats (host) (Burke, 2001; Charrel et al., 2004; Gould et al., 2003). The virus can survive in water and therefore can be transmitted to human from contaminated water and through milk from goats or sheep (Holbrook et al., 2005). OHF is biphasic; the first symptoms of hemorrhagic fever are dehydration, hypotension, gastrointestinal symptoms, chills, headache, swollen glands in the neck, trace of blood in the eyes and often effects on the central nervous system. If the patient does not recover from the first phase then the second phase will develop with symptoms such as papulovesicular or petechial cutaneous rash, lymphadenopathy, ocular suffusion, and capillary perturbations, leading to oral and nasal mucosal bleeding as well as scattered visceral hemorrhage and also encephalitis (Charrel et al., 2004; Gritsun et al., 2003b; Holbrook et al., 2005; Ruzek et al., 2010). In the third phase the patient may experience neurological disorder with behavioral or psychological difficulties, hair loss and hearing loss if no recovery. Langat virus (LGTV) has been isolated in Malaysia in I. granulatus and later in Thailand in Haemaphysalis papuana which is associated with several species of forest rats (Rattus rattus, R. fulvescens, R. niviventor, R. surifer), suggesting that LGTV is spread through the forest in south east Asia. (Bancroft et al., 1976; Dobler, 2010). LGTV has also been isolated from I. persulcatus from the Krasnoyarsk region in central Siberia. Although there are no natural LGTV infections registered in humans, specific antibodies against LGTV have been detected in the sera of local people. Encephalitis occurred in some rare cases after vaccination with a live attenuated LGTV- based vaccine. The progression of encephalitis was similar to what is normally observed after infection with the typical W-TBEV (Gritsun et al., 2003b). Powassan virus (POWV) is a North American member of the TBE serological complex transmitted by several species of ticks from the genera Ixodes and Dermacentor (Burke, 2001; Ebel, 2010). The virus is the cause of rare but severe neuroinvasive disease in North America and Russia (Burke, 2001). Due to rare incidence of human infections, POWV is not as well studied as other human pathogenic flaviviruses (Ebel, 2010). Studies on POWV replication and pathogenesis are limited and it has been assumed that POWV shares the same features as other members of the TBEV sero- complex. However, increased number of human infections brought attention to the disease in North America and Russia (Burke, 2001; Charrel et al., 2004; Ebel, 2010; Ebel et al., 2001; Gould et al., 2003). POWV have life cycles in both ticks and mosquitoes. This might explain the wider geographical distribution of this virus compared to other Tick-borne flaviviruses. The mosquito life cycle still remains questionable and should

14 be further investigated as mosquitoes hosts does not support robust POW replication (Kuno, 2007). KFDV was identified in Karnataka State of southern India in 1957 and is the major tick-borne virus from southern Asia associated with Haemaphysalis spinigera (Mackenzie & Williams, 2009; Pattnaik, 2006). Kyasanur Forest disease (KFD) is a zoonotic disease and cause prostrating human febrile disease with hemorrhagic and encephalitic manifestations. The fatality rate associated with KFD is between 1% and 10% (Mackenzie & Williams, 2009; Pattnaik, 2006). Interestingly, a variant of KFDV was detected in 1992 in Saudi Arabia, (AHFV) where over 60 human developed Alkhurma hemorrhagic fever disease (Charrel et al., 2001; Pattnaik, 2006; Zaki, 1997). The AHFV share 89% sequence homology with KFDV suggesting a common ancestor and the Saudi Arabian outbreak demonstrates that that novel pathogenic flaviviruses may emerge at any time (Pattnaik, 2006).

15 Emergence of TBEV in Sweden To our knowledge W-TBEV is the only TBEV subtype that has been detected in Sweden, but the phylogenetic data on flaviviruses present in Sweden is limited. TBEV has been detected in Sweden mainly around Lake Mälaren, Vättern and in the coastal area from Roslagen to Kalmar region and Skåne (Haglund et al., 2003). A few cases of infected people have been reported from the south of Dalarna and TBEV isolation from ticks collected in the Göteborg region has been confirmed (Brinkley C. et al., 2008), suggesting that the disease now is established in the region (Brinkley C. et al., 2008; Jaenson & Lindgren, 2011). Even in the north of Sweden some cases of TBEV have been reported. Global warming could be one reason for the increased distribution of TBEV but there is ample evidence of indirectly linked changes in several different abiotic, biotic and non-biological factors which may have increased the risk of TBE (Randolph, 2004; Randolph et al., 2008). This makes it essential to develop rapid detection and sequencing strategies, but also to increase the understanding of other factors such as human urbanization and increased exposure to TBEV in endemic areas (Randolph, 2004; Randolph et al., 2008). It is also important to study the natural presence of flaviviruses e.g. in the Baltic Sea region where several MTBFG members have been found co-circulating (Golovljova et al., 2004; Han et al., 2001; Han et al., 2005; Lundkvist et al., 2001; Mavtchoutko et al., 2000). Other flaviviruses like the LIV, which has spread to Norway and Denmark presumably by imported sheep, might possibly also be found in Sweden (Charrel et al., 2004; Gao et al., 1993; Jensen et al., 2004; McGuire et al., 1998). S-TBEV was recently isolated from ticks collected in the Archipelago of Kokkola which might suggest a virus migration northwest of previously known locations in eastern Europe and Siberia (Jaaskelainen et al., 2006). This might suggest a putative future S-TBEV migration and establishment of new foci also in Sweden.

The life cycle of the tick vector The tick is related to mites and other arachnids (Jeyaprakash & Hoy, 2009). It has no eyes but has the ability to sense smells and movements in the vegetation. The ticks can also feel the heat radiation and secreted carbon dioxide by the victim (Waladde et al., 1993; Waladde et al., 1995). In Scandinavia, there are about 15 tick species but more than 300 different species are found around the world (Jaenson et al., 1994). The most common tick in Sweden is Ixodes ricinus (Jaenson et al., 1994; Jaenson et al., 2009). Mammals, birds and sometimes also reptiles and amphibians are the blood host for the parasitic tick (Balashov Iu, 1999; Kiszewski et al., 2001). The tick life cycle includes three blood meals to develop into the different life stages (Fig. 3). Humans can become infected with several

16 diseases from tick bites e.g. Lyme disease caused by the Borrelia bacteria and Ehrlichios caused by the Ehrlichia bacteria and TBE. A key factor in virus transmission between ticks is transfer from infected to uninfected ticks during co-feeding (Jones et al., 1987). Virus is also maintained by transovarial and transstadial transmission (Nuttall & Labuda, 2003). Once a tick is infected, it carries the virus its whole lifetime (Berrada & Telford, 2009).

Figure 3) The tick life cycle. The adult female releases about 2,000 eggs on the ground after feeding from a host. These eggs are under severe selection where drought may reduce the amount and the eggs are hatched as six-legged larvae. In the next phase the larvae attach to a small rodent to feed and molt into eight-legged nymphs. The nymphs seek and attach the second host, usually another rodent or lagomorph. After feeding on the second host the nymph will molt into an adult tick. The adult male and female ticks might now seek a larger herbivore, carnivore, or human. On the last host, the ticks will feed and mate. Females may reattach and feed multiple times until she gains 200 timers her own weight before it falls to the ground. (Illustrated by; Irsa Melik).

17 The flavivirus genome structure The flaviviruses contain an approximately 11 kb long, positive, single stranded RNA (+ssRNA) genome encoding one single polyprotein. The polyprotein is co- and post-translationally processed by viral (NS2B/NS3) and cellular proteases into 10 mature proteins; three structural proteins (the capsid [C], pre-membrane [prM] or membrane [M], and envelope [E]) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Fig. 4) (Burke, 2001; Chambers et al., 1990; Lindenbach, 2001).

Figure 4) Illustration representing the genome organisation of flavivirus. The viral genome consists of a positive sense 11 kb ssRNA. In light grey the CprME contain the structural genes. In dark grey the NS1-NS5 contains the non-structural genes. The flanking 5’ and 3’ ends are non-coding regions (NCR) containing both secondary structures and complementary regions cause cyclisation of the RNA genome.

The structural proteins are mainly involved in virus particle formation whereas the NS proteins are responsible for viral RNA replication and viral assembly (Chambers et al., 1990; Kummerer & Rice, 2002; Lindenbach, 2001; Liu et al., 2003). The NS proteins also play key roles in counteracting the host antiviral responses, to avoid the immune systems (Best et al., 2005; Guo et al., 2005; Liu et al., 2005; Munoz-Jordan et al., 2003; Werme et al., 2008). The regions flanking the polyprotein gene are the 5’ and 3’ non- coding regions (NCR or (un-translated region (UTR))), respectively which contains both secondary structures and complementary regions causing cyclisation of the RNA genome (Hahn et al., 1987; Khromykh et al., 2001a; Lindenbach, 2001; Mandl, 2005). The NCR structures contain both sequential and structural motifs that are important for flavivirus lifecycle processes such as translation, replication, and possibly assembly (Elvang et al., 2011; Gritsun & Gould, 2006, 2007a, b; Hoenninger et al., 2008; Mandl, 2005; Mandl et al., 1998). Many of the flavivirus proteins have multiple functional roles and activities (Hahn et al., 1987; Khromykh et al., 2001a; Lindenbach, 2001; Mandl, 2005), and some of the known functions are summarized in Table 2.

18 Table 2: Known functions of structural- and non-structural flavivirus protein. Protein Function (subunit weight, aa) Capsid (C, 114 Capsid protein forms the icosahedral nucleocapsid, surrounding the aa) RNA genome. This highly basic protein folds into a compact dimer. Both the C- and N terminal are important for association with the virus

RNA (Dokland et al., 2004; Khromykh & Westaway, 1996). Precursor The two forms manifest the maturation of the virus. In immature virions Membrane glycosylated precursor-M (prM) protein forms a hetrodimer with the E (prM, 165 aa/M, protein (Heinz et al., 1994). In the mature virion only the C-terminal 90 aa) region is retained in the viral membrane, as the precursor is cleaved by furin before the release of virions (Lindenbach, 2007). Envelope (E, 495 The envelope polypeptide forms homodimers, “head to tail”, that aa) completely covers the surface of the mature icosahedral virions. The E Structure Proteins Structure protein is responsible for receptor-binding and membrane fusion. The E protein contains 3 domains where domain 3 (DIII) is associated with receptor binding and domain 2 (DII) includes the membrane fusion peptide (Lindenbach, 2007; Rey et al., 1995). NS1 (352 aa) The NS1 protein has an early role in RNA replication where its association with NS4A is critical for the replicase complex (Lindenbach & Rice, 1999; Winkler et al., 1989; Winkler et al., 1988). NS1 is glycosylated and will induce an antibody response (Flamand et al., 1999; Huang et al., 2001). Each subunit forms a homodimer located in the ER lumen during replication (Lindenbach & Rice, 1999). NS2A (218 aa) The NS2A polypeptide binds strongly to the 3´NCR, NS3 and NS5

(Lanciotti et al., 1997; Lindenbach, 2001). NS2B (130 aa) NS2B is the co-factor of NS3, forming a serine protease complex essential for processing the virus polyprotein (Falgout et al., 1991). NS3 (618 aa) The NS3 protein is a multifunctional protein as it has protease activity, RNA stimulated NTPase activity and RNA triphosphate activity. The NS3 protein is an essential member of the replicase complex (Koonin, 1993; Lindenbach, 2001). NS4A (286 aa) The NS4A polypeptide has roles in rearrangement of the ER membrane

Structural Proteins Structural for viral replication. NS4A binds strongly to most of the other NS - proteins, and also participates in RNA replication by assembling

replicase component at the ER membrane (Lindenbach, 2007). Non NS4B (112 aa) NS4B is located at RNA replication sites and has roles in regulating virus replication. NS4B also has roles in the inhibition of the interferon (IFN) response by DENV (Lindenbach & Rice, 1999; Mackenzie et al., 1998; Miller et al., 2006; Miller et al., 2007; Munoz-Jordan et al., 2003). NS5 (900 aa) The multifunctional NS5 protein contains an N-terminal domain with S- adenosylmethioninie methyltransferase activity and a C-terminal domain with RNA dependent RNA polymerase (RdRp) activity (Lindenbach, 2007).

19 Tick-borne encephalitis virus NCR The terminal regions of the flavivirus genome, 5’- and 3’NCR contain secondary structures and RNA elements causing cyclization of the genome that is important for flavivirus lifecycle processes (Gritsun & Gould, 2007a, b; Li & Brinton, 2001; Markoff, 2003). The 3’ NCR of flaviviruses is relatively longer than the 5’ NCR and shows a significant heterogeneity in length and sequence even among closely related strains (Gritsun & Gould, 2006, 2007a, b). Both the 5’- and 3’ NCR can be divided into a highly conserved core element (C5’ and C3’, respectively) and a variable part (V5’ and V3’, respectively) in which the V3’NCR is characterized by low sequence conservation, sequence repeats (R) and internal poly (A) tracts that can range in size (Gritsun & Gould, 2007a, b; Wallner et al., 1995). The V3’NCR length variability has been suggested to be related to the number of laboratory passages the virus encounters, but the C3’NCR remains intact (Mandl et al., 1998; Wallner et al., 1995). Experimental passage of viruses with introduction of deletions into the C3’NCR has shown production of viruses with reduced neuroinvasiveness whereas viruses with removed V3’NCR retain similar levels of infectivity to wild type viruses under laboratory conditions (Mandl et al., 1998). Several computer predictions based on different methods have defined secondary structures that show a high degree of similarity for members of the MTBFG and mosquito borne flaviviruses, which reveal very little sequence identity with TBEV (Charlier et al., 2002; Gritsun et al., 1997; Leyssen et al., 2002; Proutski et al., 1997; Proutski et al., 1999; Rauscher et al., 1997; Thurner et al., 2004). By convention the secondary structures, stem loop (SL) of the genome have been numbered from the ends inwards as they appears. The C3’NCR of TBEV contains highly essential sequential and structural motifs defined as SL1-5, which are important for maintaining virus viability (Mandl et al., 1998; Pletnev, 2001). The 5’ region of the TBEV contains RNA stem loop structures (SL1–4), with the SL1–2 being present within the 5’-NCR, whereas the SL3–4 is situated in the N-terminal region of the ORF (Khromykh et al., 2001a; Kofler et al., 2006). Sequence motif within the core region of 3’NCR includes a pentanucleotide (GAGAG) motif exposed on the SL4 (Gritsun et al., 2001; Khromykh et al., 2003; Wengler & Castle, 1986). This motif is strictly conserved among the all flavivirus genomes. Other important elements present in the C3’ NCR of TBEV is the homopurine box, a homopyrimidine box and cyclization sequences (CS) motifs for circulazation of the genome (Khromykh et al., 2001a; Kofler et al., 2006; Mandl, 2005; Mandl et al., 1993; Thurner et al., 2004). Interactions between 5’-and 3’ NCRs were first identified in DENV (Hahn et al., 1987), and are believed to provide a signaling function either in single or double stranded form. Analogous sequences have been also identified for TBEV; CSA, CSb-2, and CSb-1(Khromykh et al., 2001a;

20 Kofler et al., 2006; Mandl et al., 1993; Thurner et al., 2004). The CSA is essential for functional replicase assembly (Khromykh et al., 2001a), whereas the CSB elements seem dispensable for functional TBEV replication (Kofler et al., 2006). The structure caused by the hybridization of inverted complementary sequences of viral RNA is also known as “panhandle-like structures” and is also observed in other viruses such as negative strand RNA bunya-, arena-, and orthomyxoviruses as well as in the double stranded RNA rotavirus (Flick & Hobom, 1999; Khromykh et al., 2001a; Kohl et al., 2004; Mir & Panganiban, 2005; Mir et al., 2006; Patton, 2001; Perez & de la Torre, 2003).

Figure 5) Schematic picture of the 3´-terminal part of the TBEV genome. The 3’NCR contain the variable region and the core element containing repetitive sequences (R1,R2 and R3), polyA tract, homopurine box (PU) and homopyrimidine box (PY). The core element contains also the pentanucleotide motif (CACAG) and cyclization motif CSA, CSb-1 and CSb-2). This figure is adapted from (Wallner et al., 1995).

The cellular life cycle of the flavivirus The flavivirus enters the host cell by receptor mediated endocytosis and endosomal traffic. Inside the , the acidic environment leads to conformational changes of the envelope protein that leads to membrane fusion. As the virion and the cell membranes fuse; the virus disassembles and the uncoated capsid releases the +ssRNA genome into the cytoplasm (Bressanelli et al., 2004; Modis et al., 2004). The +ssRNA genome is translated into the polyprotein precursor of approximately 3400 aa in length. The polyprotein traverses the ER membrane at several positions directed by hydrophobic signal sequences (Fig. 6). The polyprotein will be processed by the NS3/2B complex at the cytosolic side of the ER membrane, whereas lumenal regions are cleaved by host signal peptidases (Lindenbach, 2007). The topology of the polyprotein with respect to the ER membrane is illustrated in figure 6.

21

Figure 6) Illustration of the genome translation and processing of the polypotein. Signal sequences direct the polyprotein into the ER membrane were host signal peptidases and viral proteinases co-translationally and posttranslationally process the polyprotein into 10 viral proteins. Signal peptides and transmembrane peptides are indicated as white and blue cylinders, respectively.

The flavivirus replication occurs at the replication complex (RC) associated with the ER membrane. The replication is initiated as viral RNA associates with RC and a negative strand is synthesized, which generates dsRNA functioning as a template for the +ssRNA synthesis (Brinton, 2002). Most of the NS proteins are believed to be essential for the RC. Studies on have showed that NS1, NS2A, NS3, NS4A and NS5 interact with each other forming the RC (Fig. 7) (Lindenbach, 2007; Mackenzie & Westaway, 2001).

Figure 7) Proposed model explaining the assembly of the flaviviral RC. NS3 is assumed to bind N-terminal regions of NS5 perhaps also associated with NS2A. The complex NS3-NS5-NS2A attaches to the 3’NCR stem loops via binding of NS2A and subsequently also NS3 and NS5. A NS1 dimer located in the ER lumen binds to the NS4A dimer that is integrated into the ER membrane. Finally the RC assembles by interaction between NS2A and NS4A. RC is now complete and ready to start the synthesis of the negative strand (Westaway et al., 2003). Immature virion particles are assembled in the ER lumen. The C protein binds the new synthesized viral RNA, and the RNA-C complex is packaged within the ER-derived lipid bilayer containing the enveloped protein E and

22 preM by budding into the ER lumen. (Allison et al., 2003; Lorenz et al., 2003; Mackenzie & Westaway, 2001). The prM prevents premature fusion of the virus during its transport through the trans golgi network (TGN) (Li et al., 2008; Yu et al., 2009; Yu et al., 2008). In the last step of assembling and virus exocytosis; the prM proteins are cleaved by host protease furin, resulting in release of mature, infectious virions (Brinton, 2002; Mandl, 2005; Mavtchoutko et al., 2000). The virus lifecycle within the host cell is summarized in figure 8.

Figure 8) The flavivirus lifecycle. The virus enters the cell by receptor mediated endocytosis and membrane fusion. The genome is uncoated followed by translation and proteolytic processing of the polyprotein. Replication of the genome will take place at RC associated with the ER membrane. The cycle will end by formation of immature virions, which mature through the secretory pathway and finally after maturation by furin cleavage exit the cell by exocytosis. (Illustrated by; Irsa Melik).

23 Flavivirus replication The RC (Fig. 7) consists of several multifunctional NS proteins; NS1, NS2A, NS3, NS4A and NS5 (Lindenbach, 2007; Mackenzie & Westaway, 2001). Each protein is essential for replication and initially a negative complementary RNA strand to the viral genomic plus strand is synthesized, resulting in a double-stranded replicative form (dsRF) (Lindenbach, 2007). The synthesis of RNA is asymmetric; meaning that 10 to 100 fold more copies of plus sense RNA is synthesized compared to minus sense RNA during the flavivirus replication cycle (Cleaves et al., 1981; Muylaert et al., 1996). NS5 functions both as a RNA dependent RNA polymerase (RdRp) (Ackermann & Padmanabhan, 2001; Guyatt et al., 2001; Koonin & Dolja, 1993; Lindenbach, 2001) and as a methyltransferase (Egloff et al., 2002), where the methyltransferase activity is involved in the capping of the viral genomic RNA. NS3 is the second largest protein after NS5 and has 3 distinct activities essential for replication. The N-terminal domain has protease activity that depends on the cofactor NS2B for proper processing of the viral polyprotein (Chambers et al., 1993; Falgout et al., 1991; Jan et al., 1995). The C-terminal domain contains helicase activity required for the unwinding of dsRF (Matusan et al., 2001). The RNA triphosphatase activity is needed in the pre-step of viral RNA capping (Kuo et al., 1996; Lindenbach, 2001). NS1 is a soluble protein secreted from the host cell and its role in replication is relatively unknown. But it has been described that NS1 plays a role in the negative strand RNA synthesis (Lindenbach & Rice, 1997). The hydrophobic NS4A protein consists of four transmembrane helices and an N-terminal cytosolic region is integrated into the ER membrane, were it links NS1 in the ER lumen with the rest of the RC (Lindenbach & Rice, 1997; Mackenzie et al., 1998; Westaway et al., 1997). Furthermore, studies on DENV and Kunjin virus (KUNV) have shown that mature NS4A protein lacking the C-terminal region (2K fragment) promotes membrane rearrangements by inducing bends in the ER membrane, that harbor the viral RC (Mackenzie et al., 1998; Miller et al., 2007; Roosendaal et al., 2006). This membrane rearrangement is believed to protect the RNA from degradation. On the cytosolic side, NS4A associates with NS2A that attaches the secondary structures of 3'NCR and the NS3-NS5 complex which completes the RC formation (Mackenzie et al., 1998). The role of NS4B in the RC has been debated since it was found mainly in the nucleus, but colocalization has shown that it associates with NS3 and dsRNA in the ER- derived membrane structures, implying a role in RNA replication (Mackenzie et al., 1998; Miller et al., 2006; Westaway et al., 2002, 2003; Westaway et al., 1997).

24 Replicon In the early described flavivirus replicons, the viral genome was cloned downstream of the bacteriophage promoter SP6. The 11kb RNA was transcribed and purified in vitro and transfected into cells generating virus particles. Later approaches were developed for KUNV and Japanese encephalitis viruses (JEV) in which cDNA of the viral genome was cloned into plasmids under the control of eukaryotic promoters such as CMV allowing production of infectious virus particles using conventional plasmid transfection methodologies (Khromykh et al., 2001b; Varnavski et al., 2000; Yamshchikov et al., 2001b). One of the first designed flavivirus replicons was the KUNV replicon which provided a safe means of studying replication in the laboratory. This replicon is fully capable of replicating without the genes encoding the structural proteins. (Khromykh & Westaway, 1997; Khromykh et al., 2001a; Varnavski et al., 2000; Westaway et al., 2003). The vector genome was an ideal starting point for the development of a safe gene vector that can deliver suitable therapeutic proteins to cells, without the risk of producing infectious virus particles (Pierson et al., 2005; Westaway et al., 2003). The KUNV replicon contained the 5´- and 3´ NCR and seven non- structural proteins (NS1- NS5) that guided the expression of the gene of interest. The replicon was able to self-replicate in vivo, and the absence of structural genes means that the transfected cells are unable to produce viable virus particles (Aberle et al., 2005; Khromykh & Westaway, 1997; Khromykh et al., 2001a; Rossi et al., 2005; Varnavski et al., 2000; Westaway et al., 2003). The replicon plasmid also encoded a reporter gene such as green fluorescent protein (GFP) or luciferase (Luc), cloned directly upstream of the NS genes and the replicon was driven by the CMV promoter. In other replicons GFP or Luc were cloned into the 3´NCR under translational control by the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). (Hayasaka et al., 2004; Pierson et al., 2005; Yamshchikov et al., 2001a; Yamshchikov et al., 2001b). Transfection of cells with these DNA based replicon plasmids results in production of self-replicating replicons capable of mediating high-level expression of GFP in different cell types, (Fig. 9), including primary rat neurons and human monocyte derived dendritic cells (Hayasaka et al., 2004; Pierson et al., 2005; Yamshchikov et al., 2001a; Yamshchikov et al., 2001b). The GFP- expressing DNA replicon is an important powerful tool that generates a quantitative system for assaying virus replication and can be used to study many aspects of the viral lifecycle.

25

Figure 9) Confocal laser scanning microscopy (CLSM) image of N2A Cells transfected with Torö TBEV subgenomic replicon expressing the EGFP reporter gene at 72 h post transfection.

Protein-Protein interaction domains Many biological processes involve the formation of protein complexes essential for cellular functions, regulation and signaling. Protein-protein interactions may be dependent on protein interaction domains (PID) e.g. WW, SRC Homology 3 (SH3), Pleckstrin homology (PH), Sterile alpha motif (SAM) and Post-synaptic density-95/Disc large/ ZO-1 (PDZ) (Lee & Zheng, 2010; Stiffler et al., 2007). Finding interaction partners for a protein is relevant as it may clarify its function and determine the consequences of the interaction. These interactions might also enlighten the understanding of how cells react to agents or how viruses can develop strategies to escape or delay immune responses. Many methods have been developed for experimental approaches to evaluate the protein-protein interactions. With regards to sensitivity and specificity of the methods, each approach has its strengths and weaknesses. Over the years of protein-protein interaction studies, multiple databases have been assembled collecting such data. Some of these databases are BioGRID, STRING, and Database of Interacting Proteins (DIP). In this thesis we have focused on PDZ proteins interacting with TBEV as a previous study showed that a PDZ domain within Scribble binds specifically to the NS5 protein, which has a significant role in impairing cellular innate immunity. Here we reveal additional PDZ binding partners and study putative roles for these associations in cell polarity and virus replication.

26 PDZ domains The PDZ domains function as protein-protein interaction modules that play a central role in organizing diverse cell signaling assembly systems within eukaryotic organisms. It is believed that more than 300 proteins in mouse and over 400 proteins in human contain PDZ motifs (Ilinskaya et al., 2010; Ponting, 1997; Stiffler et al., 2007). Often several PDZ domains co-exist in a single polypeptide. PDZ associations are often involved in directing receptor trafficking and signaling molecules by the assembly of protein complexes (Lee & Zheng, 2010). PDZ domains are often important parts of scaffolding proteins that are involved in the regulation of cell polarity and cell plasticity of epithelial cells. These PDZ domains specifically recognize short C terminal peptide motifs, but can also recognize surface exposed internal sequences that structurally may mimic a C-terminus. (Ellencrona et al., 2009; Kim et al., 1995; Kornau et al., 1995; Niethammer et al., 1996; Werme et al., 2008). PDZ domain consists of 80-100 aa and folds into a conserved globular structure that comprises two α-helices and four to six β-strands (Doyle et al., 1996; Piserchio et al., 2002; Tochio et al., 2000). The active site of the PDZ domain is located in a pocket between the second β strand and the first α helix. Class I PDZ binds the C termini of proteins with the S/T-X-Φ-COOH consensus motif (where X stands for any amino acid and where Φ is a hydrophobic residue). Class II recognizes Φ-X- Φ-COOH motifs and Class III X-X-C-COOH (Appleton et al., 2006; Kang et al., 2003; Skelton et al., 2003; Tonikian et al., 2008; Zhang et al., 2007). It has also been shown that PDZ domains interact with internal protein sequences e.g. the cell polarity protein Par-6 binds to Pals-1 through an internal motif (Penkert et al., 2004). In addition, an internal motif of NS5 TBEV is bound by the PDZ4 of the scribble protein (Werme et al., 2008).

The NS5 protein NS5 is the largest and most conserved protein of all proteins within the flavivirus genus. Like other NS proteins, NS5 is multifunctional (Ackermann & Padmanabhan, 2001; Guyatt et al., 2001; Koonin & Dolja, 1993; Rice et al., 1985; Tan et al., 1996). Mutations in the active site of the polymerase disrupt the activity which demonstrated that NS5 is essential for virus replication. Activity could be restored by adding NS5 in trans using a replicon (Khromykh et al., 1998). NS5 binds to NS3 (Johansson et al., 2001; Kapoor et al., 1995) and to the 3'-SL of the genomic RNA (Chen et al., 1997). This (NS5-NS3-3'-SL) interaction might stimulate the NTPase and RTPase activity of NS3 (Cui et al., 1998; Yon et al., 2005). It has been shown that the dengue virus NS5 is located in the nucleus and contains a nuclear localization signal (NLS) recognized by both importin β1 and the

27 importin α / β complex (Brooks et al., 2002; Bruschke et al., 1997; Johansson et al., 2001; Jopling et al., 2005), whereas NS5 from other flaviviruses may not be located in the nucleus e.g. WNVNS5 (Mackenzie et al., 2007). In addition, it has been reported that the DENVNS5 contains a nuclear export sequence and is exported from the nucleus in a CRM1- dependent manner (Rawlinson et al., 2009). NS5 plays also a crucial role in the escape from or block of immune responses of the host cell (Best et al., 2005; Medin et al., 2005; Werme et al., 2008). A schematic representation of the NS5 protein is illustrated in figure 10.

Figure 10) Domains of TBEV NS5 with PDZ interacting motifs. The NS5 protein is approximately 900 aa and contains an MTase and an RdRp domain. PDZ dependent binding usually relies on a binding motif in the C-terminus, S/T-x-L/V/I- COO-, and this motif is also present in the C-terminus of TBEVNS5 Ser901, Ile902 and Ile903 (SII). The TBEV NS5 also containes an internal PDZ binding motif in the MTase domain Tyr222 and Ser223 (YS).

Scribble The Scribble protein was first identified in epithelial cells of Drosophila when studying cell adhesion, shape and polarity. Mutations in the scrib gene caused broad defects in epithelial organization and irregularly shaped cells was observed (Bilder & Perrimon, 2000; Bilder et al., 2000). Scribble is a large cytoplasmic scaffold protein that belongs to the LAP (Leucin rich repeats (LRR) and the PDZ)) protein family and contains 16 LRR at the N- terminus and four PDZ domains at the C-terminal end (Bilder & Perrimon, 2000; Fanning & Anderson, 1999; Legouis et al., 2003; Santoni et al., 2002). Other members of the LAP family are the Caenorhabditis elegans Let413, mammalian Erbin, Densin180 and Lano (Borg et al., 2000; Saito et al., 2001; Santoni et al., 2002; Strack et al., 2000). The fly scrib gene encodes a protein of approximately 1630 amino acids that are 39% identical and 49% similar to mammalian Scribble (Nakagawa & Huibregtse, 2000). Deficiency in Scribble impairs many aspects of cell polarity and cell movement. By forming a complex with the Lgl (lethal giant larvae) and Dlg (lethal discs large) proteins which are located in an apical belt in the lateral cell membrane at the septate junction; it controls and refines the segregation of apical and basolateral membrane domains maintaining cell polarity. Loss of scribble will cause misdistribution of apical proteins such as Crb (Crumbs), Dpatj (Drosophila PALS1 associated tight junction), E-cadherin and Armadillo, leading to loss of cell polarity (Bilder & Perrimon, 2000). Expression level of mammalian Scribble protein is relatively low in liver, lung, kidney and skeletal muscles and high in the skin, placenta, breast and intestine epithelial cells (Navarro et al., 2005). Along with Dlg1 protein

28 scribble is also present in mouse eyes and human colon epithelia (Gardiol et al., 2006; Navarro et al., 2005; Nguyen et al., 2005). The PDZ domain of scribble binds directly to the human T-lymphotropic virus, Tax1 protein and to human papillomavirus E6 (HPVE6) protein, the main agent responsible for cervical cancer (Kiyono et al., 1997; Nakagawa & Huibregtse, 2000; Okajima et al., 2008). Earlier the mammalian homolog of Drosophila Dlg1 had been shown to interact with tumor suppressor proteins, APC (adenomatous polyposis coli protein) and PTEN (protein tyrosine phosphatase and tensin homologue) as well as with viral oncoproteins such as the HPVE6 (Humbert et al., 2003). Overexpression of Dlg in fibroblasts inhibits cell proliferation by impairing the event in the cell cycle G0/G1 to S phase (Ishidate et al., 2000). However, Dlg1 like scribble is involved in the negative regulation of cell proliferation by interacting with the high risk HPVE6 oncoprotein, targeting Scribble and Dlg1 for degradation through an ubiquitin-mediated mechanism (Gardiol et al., 1999; Humbert et al., 2003; Nakagawa & Huibregtse, 2000). In addition, polarity complexes and cytoskeleton actin rearrangement are dynamically regulated by members of the Rho family of small GTPases and their effectors (Colicelli, 2004; Iden & Collard, 2008). It has been established that scribble is involved in the recycling of vesicle and signaling of transmembrane receptor TSHR (thyroid stimulation hormone receptor) via the βPIX /GIT1 (G-protein coupled receptor interacting protein 1)/ARF6 (ADP-ribosylation factor 6) pathway (Lahuna et al., 2005). This is a way to facilitate the βPIX for activation of Rac1, Cdc42 and other GTPases at the plasma membrane. Suppression of the Rac1/Cdc42 or Rhoa activity results in a loss of directional migration by neutrophils where polarity complexes are present at the leading edge of the plasma membrane (Xu et al., 2003). Several feedback loops may be involved in maintaining the Cdc42 or other members of GTPase activity, however, knockdown of scribble causes a decrease in Rac1 activation (Audebert et al., 2004; Osmani et al., 2006; Zhan et al., 2008). Additionally, my colleagues demonstrated that Scribble interacts with TBEVNS5 causing inhibition of interferon-stimulated JAK–STAT signaling by blocking the phosphorylation of STAT1, thus inhibiting the expression of antiviral genes (Werme et al., 2008).

29 GIPC GAIP interacting protein C terminus (GIPC) contains one class I PDZ domain, and is abundant in various cell types. GIPC has been reported to have several binding partners. One of the first proteins identified as a partner was RGS (regulator of G protein signaling), the GAIP protein (Bunn et al., 1999; De Vries et al., 1998). GAIP is located on clathrin-coated vesicles, and there are indications that it may be involved in modulating membrane trafficking. This interaction links the GIPC protein to the G coupled signaling pathway and localizes GIPC on the surface of intracellular vesicles (De Vries et al., 1998). GIPC has also been found to interact with several other proteins with a range of functional implications, including the neurotrophic tyrosine kinase A (TrkA), tyrosine kinase B (TrkB) receptors and APPL (adapter protein containing PH domain/PTB domain/leucine zipper motif), a GTPase Rab5 binding protein, which is a marker for signaling (Miaczynska et al., 2004). Expression of GIPC mutated in PDZ is unable to bind APPL and GIPC is no longer recruited to the incoming TrkA vesicles. PDZ mutated GIPC is also involved in the inhibition of neurite outgrowth and deactivation of activated Ras/mitogen activated protein (MAP) kinases (Lin et al., 2006; Lou et al., 2001; Varsano et al., 2006). The N-metyl-D-aspartat (NMDA) receptors are present in most glutamatergic synapses in the mammalian central nervous system (CNS) and GIPC has been found to interact with the cytoplasmic domains of the NR2 subunit, localizing primarily at the synapse. Changes in expression of GIPC also alter the number of NMDA receptors on the cell surface (Snyder et al., 2001; Yi et al., 2007). GIPC also interacts with the dopamine D2 and D3 receptors (Jeanneteau et al., 2004). GIPC knockdown showed that the protein affects transforming growth factor β (TGF-β) dependent gene expression and cell proliferation. (Favre-Bonvin et al., 2005). There are also numerous virus proteins interacting with GIPC including the E6 protein of high-risk human papillomavirus (HPLV) type 18, which triggers its degradation via the proteasome (Favre-Bonvin et al., 2005) and the Tax 1 protein of human T-cell leukemia Virus type 1 (HTLV-1) (Rousset et al., 1998).

ZO-2 Scaffolding protein Zonula occludens 2 (ZO-2) belongs to the membrane- associated guanylate kinase (MAGUK) protein family and is a multi-domain protein that contains a SH3 domain, a guanylate kinase-like (GK) domain and three PDZ domains (Beatch et al., 1996; Gumbiner et al., 1991; Haskins et al., 1998; Jesaitis & Goodenough, 1994). This domain organization is shared by the ZO proteins ZO-1, ZO-2 and ZO-3. The ZO-2 proteins also contain several NLS and NES sequences (Gonzalez-Mariscal & Nava, 2005;

30 Islas et al., 2002; Jaramillo et al., 2004). ZO-1 and ZO-2 can form both homo- and heterodimer via the PDZ domains (Im et al., 2003a; Im et al., 2003b; Itoh et al., 1999; Utepbergenov et al., 2006). Several functions have been demonstrated where ZO proteins in general act as a stage juncture for the recruitment or assembly of signaling complexes in response to epithelial and endothelial cell-cell adhesion (Angst et al., 2001; Zahraoui et al., 2000). It has also been shown that ZO proteins can act as a linker between integral junction proteins and the actin based cytoskeleton (Fanning et al., 1998; Itoh et al., 1997; Wittchen et al., 1999). Association with membrane proteins at tight junctions (TJs) (Anderson et al., 1988; Stevenson et al., 1986), cadherin-based adherens junctions (AJs) (Howarth et al., 1992) and the connexin 43 complex at gap junction (GJs) (Giepmans & Moolenaar, 1998; Singh & Lampe, 2003; Singh et al., 2005), reveals that ZO proteins are essential for cell structuring and communication. Moreover, ZO-2 proteins also take part in the regulation of cell- growth and proliferation by association in signal transduction pathways and transcriptional modulation (Huerta et al., 2007; Tapia et al., 2009). An interaction between the human scribble (hScribble) scaffolding protein and ZO-2 has been demonstrated, where hScribble acts as a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation (Metais et al., 2005; Nakagawa & Huibregtse, 2000). The hScribble is also an interaction partner to TBEVNS5 having a role in the viral blockage of the JAK/STAT pathway (Best et al., 2005; Werme et al., 2008). Interestingly in Paper IV we demonstrate a PDZ dependent interaction between TBEVNS5 and ZO-2. A subunit of the transcriptional AP-1 directly interacts with ZO-2 in the nucleus and at the plasma membrane site, which seems to down regulate the target gene expression enhanced by AP-1 (Betanzos et al., 2004).

31 Aims of thesis

Paper I The aim of this paper was to set up solid and successful strategies for detection of flavivirus members of the MTBFG present in Sweden. This was done by collecting ticks, extracting total RNA, and developing PCR strategies for TBEV detection and virus sequencing. We also studied the phylogeny of TBEV including sequences present in ticks collected at Torö.

Paper II The aim of this study was to present a complete TBEV sequence directly from the arthropod reservoir. We also investigated Torö- 2003 in silico which revealed novel secondary RNA structures, and a model that suggests a temperature dependent switch between a closed and opened genome structure which could have functions in the viral life cycle at different environmental temperatures.

Paper III The objective was to examine the putative effect of TBEV NS proteins on neuritogenesis in PC12 cells. These cells have the ability to differentiate into a neuronal-like phenotype upon NGF stimulation.

Paper IV The objective of this study was to find novel PDZ interactions partners for TBEVNS5 and examine whether the PDZ association with NS5 have effects on virus replication.

32 Results and discussions

Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses (Paper I) To increase the understanding of the natural geographical distribution of MTBFG, it is of great importance to establish novel rapid ways for detection and sequencing these viruses. In this study we developed several PCR strategies to test for TBEV presence in total RNA purified from ticks sampled in endemic TBEV regions north and south of Stockholm. Primers allowing broad virus detection within the MTBFG were designed for highly conserved regions, which would pick up putative emerging MTBFG members previously not detected within the region. Total Tick RNA was screened with nested RT-PCR covering 32% of the TBEV genome. PCR products at expected sizes were detected in RNA from a pooled Torö sample. The Torö pool contained 115 I. ricinus specimens including 9 adult ticks. Three genomic regions of the virus present at Torö were highlighted in the published paper (Melik et al., 2007). The 5´NCR clearly sequence classified Torö-2003 as a W-TBEV. By making an alignment of the 5´NCR we further identified two highly conserved regions. Within these conserved sequences we revealed two complementary stretches which suggests an interesting conserved RNA fold possibly essential for the viral life cycle i.e. replication and translation. The E protein is responsible for essential functions during virus entry as receptor binding and membrane fusion. The E gene is highly conserved and is also the part of tick-borne flavivirus genome that has the most sequences in the sequence database. E gene sequences from 49 strains from the MTBFG were aligned and analyzed using the maximum likelihood method. Torö clearly localizes within the cluster of W-TBEV but the absolute differentiation within the W-TBEV is small. The Torö E gene is most similar to the Croatian isolate Stara-Ves on the nucleotide level. However, on the protein level these strains differ at 6 amino acid residues whereas Torö is identical to the geographically closer isolate from the island of Åland, KumlingeA52. In Comparison with Neudoerfl, only one amino acid is different since Ile447 is a Val in Torö and in most other TBEV. Great TBEV divergence has previously been observed in the highly variable region of the 3’NCR. The 3’NCR of the Torö sequence was aligned with MTBFG sequences. Torö has a genotype most similar to the Slovenian W-TBEV strain Ljubljana and lacks the longer polyA stretch present e.g. in Neudoerfl. It has previously been shown that the core element present in the 3´end of

33 the 3´NCR is highly conserved and contains several interesting RNA structures. A tree based on the core elements illustrated that Torö clustered with W-TBEV. Notably, S-TBEV and W-TBEV co-cluster in the core tree rather differently from the tree generated with E.

Sequencing of a Tick-borne encephalitis virus from Ixodes ricinus reveals a thermo sensitive RNA switch significant for virus propagation in ectothermic arthropods (Paper II) In Paper II we describe the extension of the study from Paper I where we extend the partial sequencing of a TBEV pulled out of a pool of RNA extract from 115 ticks collected in Stockholm archipelago. The total RNA was sufficient for all sequencing of a TBEV genome (Torö- 2003), without conventional enrichment procedures such as cell culturing or suckling mice amplification. To our knowledge this is the first time that the genome of TBEV has been sequenced directly from an arthropod source. We also characterized and compared the ORF of Torö-2003 genome with other TBE viruses. We define five unique aa characteristics for Torö sequence compared to other MTBFG members. Out of these five amino acids three changes are within the highly conserved NS5 protein, where the other two are within the helicase domain of NS3 and NS4B, respectively. Interestingly, all three NS5 substitutions are in rather close proximity within the finger region of the RdRp domain. Several research groups have described RNA secondary structures within the flaviviral 5’ and 3’NCRs. These RNA structures are fundamental and carry both sequential and structural motifs that are important for flavivirus lifecycle processes such as translation, replication and possible assembly. The 3’NCR of TBEV can be divided into a variable region part (V3´-NCR) and an extremely conserved core element (C3´-NCR), which is important for maintaining virus viability. The V3´- NCR region is highly heterogenic, both in nucleotide sequence and in length between strains. The variability in length has been postulated to be linked to the number of laboratory passages the virus encounters. Experimental passage of the virus with truncated V3´-NCR has demonstrated that deletions in the region could be dispensable during laboratory conditions. An alternative interpretation rests on the well-established observation that arthropod-borne viruses are genetically constrained as they require a functional life cycle in two highly different cellular environments. Because the variable region is present in strains freshly isolated from ticks, and multiple passages in vertebrate cells trigger spontaneous losses of significant fragments within it, some have suggested that the V3´-NCR region probably plays a role during the viral lifecycle in tick hosts. In this paper, we further

34 interpret the biological significance of the V3´-NCR for differential replication/translation dynamics in mammalian and tick cells. Our In silico analysis brings new evidence that the V3´-NCR region also carries Y-shaped SL11-12 RNA motifs that might have a role in the adaptation of the viral cycle to different environmental conditions. These motifs have a high level of sequence identity with the Y-shaped SL7-8 within both the stem and loops. The strength of this folding is highlighted even more as exposed sequences such as GCAGC, UGGUCG and GAGAG are identical between the Y-shaped structures. We propose a temperature-sensitive trans-acting riboswitch, similar to a temperature-regulated translation mechanism that was recently found in a bacterial model system (Neupert et al., 2008). Cyclization of the 5´- and 3´-NCRs might prevent TBEV translation by masking the ribosomal access to AUG. We suggest that mechanism is temperature dependent and the melting of CSA/CS-b1 or CSb-2 acts as a thermosensitive riboswitch in conjunction with the unfolding of individual low temperature secondary structural enhancers such as Y-shaped SL7–8 and SL11–12. This open and closed conformation acts as a switch for on/off setting of TBEV translation and explains low virus levels often found in the questing ticks sampled at environmental conditions.

Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5 (Paper III) In a recent investigation, colleagues have demonstrated the role of viral proteins in immune responses during TBEV infection. It was shown that NS5 protein of TBEV act as an interferon antagonist and is able to inhibit interferon-stimulated JAK–STAT signaling by blocking the phosphorylation of STAT1, thus inhibiting the expression of antiviral genes (Werme et al., 2008). TBEV is potentially a fatal neurotropic flavivirus and causes severe central nerve system diseases with thousands of cases reported annually throughout Europe and across a belt in the northern hemisphere all the way to Japan. In this study we investigate the effect of TBEVNS5 in the development of PC12 cells stimulated with NGF. One advantage which PC12 cells have is its ability to develop a neuronal like phenotype as it differentiates and develops neurites in the presence of NGF. To investigate the role of TBEV in PC12 cell differentiation we used the TBEV replicon (TBEVrep) expressing the NS genes. We observe a strong inhibition of neurite outgrowth during expression of TBEVrep in PC12 cells stimulated with NGF. This effect was absent when expressing a TBEV replicon lacking NS5 (TBEVrep∆NS5). We also showed that TBEV replicon repress GAP43luc activity, a marker of neurite outgrowth, where TBEVrep∆NS5 did not have any effect. Taken together, these findings indicate that

35 TBEVNS5 might have a crucial role in impairing neurite outgrowth. To determine whether the effect on neurite outgrowth was a direct link to NS5, the PC12 cells were transfected with YFP-tagged TBEVNS5 and WNVNS5. TBEVNS5 generates few and short neurites compared to YFP expressing controls. WNVNS5 also caused an abnormal phenotype compared to control cells. The plasmid reporter confirmed that TBEVNS5 was responsible for the neurite outgrowth suppression as the GAP43luc activity was repressed in a dose dependent manner. WNVNS5 had a minor influence on the reporter activity, which correlates with the observed abnormality of neurite outgrowth. To map the responsible domain for effect we tested MTase and RdRp constructs and observed that the suppression was mainly associated with the MTase domain. We could also show that the effect is dependent on binding between NS5 and Scribble. We have previously defined an internal motif (YS) in the MTase domain which gets targeted by Scribble. When (YS> AA) mutants of full-length NS5 and the MTase domain was expressed in PC12 cells, the effect on neuritogenesis was reduced. Limited effect on neuritogenesis was also observed when we expressed NS5 or MTase domain in Scribble depleted PC12 cells. The Rho GTPase, Rac1 forms a complex with Scribble and is constitutively active during elevated neurite outgrowth (Osmani et al., 2006; Zhang et al., 2005). We examined the association between Rac1 and Scribble by co-immunoprecipitation in presence of NS5. We found that the Rac1/Scribble association was hindered by NS5 in a concentration dependent manner. We also studied the involvement of βPIX, the guanine exchange factor of Rac1 and also the βPIX/Scribble association was inhibited by NS5. Our results define Rac1 and βPIX, as indirect targets of NS5 via the Scribble association. Thus, we suggest that NS5 interference with Scribble disturbs the association with Rac1 and βPIX, which inhibits neurite outgrowth, possibly due to inactivation of Rac1.

36 Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication (Paper IV) PDZ domains are common protein interaction modules often found in scaffolding proteins involved in regulating cell polarization and epithelial cell plasticity. In a previous investigation, colleagues extensively characterized a protein-protein interaction between host protein Scribble and TBEVNS5 (Werme et al., 2008), and found that this interaction depends on an internal PDZ binding motif in the MTase domain Tyr222 and Ser223 (YS). The internal PDZ motif was further studied using the TranSignal PDZ domain arrays (Panomics), which revealed additional PDZ protein targets (Ellencrona et al., 2009). PDZ association usually relies on a binding motif in the C-terminus, S/T-x-L/V/I-COO-, and this motif is also present in the C- terminus of TBEVNS5 Ser901, Ile902 and Ile903 (SII). In this study we show that the typical class 1 PDZ binding motifs (-SII) in TBEVNS5 and a similar motif in WNVNS5 (-TVL) are targeting human PDZ domains. TBEVNS5 has affinity to Zonula occludens-2 (ZO-2), Gaip-C-terminus interacting protein (GIPC), Calcium/calmodulin-dependent serine protein kinase (CASK) and interleukin 16 (Il-16), whereas NS5 of West-Nile virus showed a different pattern with several putative interaction partners. We also demonstrate that the PDZ binding motifs found in TBEVNS5 and WNVS5 are important for sufficient virus replication in mammalian cells. This was done in the light of a recent study that described species specific differences within C-terminus motif of influenza A virus (IAV) NS1 connected to host adaptation, replication and virulence (Soubies et al.).

37 Conclusion and future perspective

Flaviviruses have shown a significant propensity to emerge and establish in new geographic areas. This was first observed some 400 years ago when yellow fever virus spread from West Africa to the New World through the slave trade, but more recently has been seen with a number of other members of the family. Thus WNV jumped from the Middle East to North America in 1999 (Jia et al., 1999; Lanciotti et al., 1999); spread from Africa to emerge in Austria as a disease of blackbirds (Weissenbock et al., 2002); and JE virus has spread from south-east Asia into Australasia, first into Papua New Guinea (Johansen et al., 2000) and then into the Torres Strait of Northern Australia (Hanna et al., 1996). Like other arthropod vectors; ticks are ectothermic and thus especially sensitive to climatic factors. Weather influences the survival and reproduction rates of the vectors, in turn influencing habitat suitability, distribution, and abundance. This together with temporal pattern of vector activity throughout the year will indirectly also influence the survival and reproduction of pathogens within vectors. It is likely that climate change has already led to changes in the distribution of I.ricinus populations in Europe. Several reports have emphasized the expansion of I.ricinus into higher latitudes, which has been related to increases in average temperatures. The pattern of vector distribution is also noted in Sweden in where TBE cases have increased and to higher latitudes related to milder and shorter winters, resulting in longer tick-activity seasons. These changes in climate also explain the distribution of migrating birds and increased number of deer population which is also influencing tick abundance. In this thesis I describe our studies regarding a journey of biology and molecular biology in where it started by collecting ticks direct from nature extracting total RNA. Also, I established novel rapid ways for detection and sequencing these viruses collected in Swedish natural foci. In order to obtain a more detailed picture of viral distribution and able to detect possible new members of the MTBFG in the Swedish landscape, it is important to gather more viruses and broaden collection. But to understand the distribution it is also important to ensure the viral distribution in the Baltic Sea region, by extending the collection in surrounding country. A pool of 115 ticks from the Torö archipelago of Stockholm was found to contain TBEV, which later proved to be the W-TBEV in a phylogenetic analysis. This complete virus sequence straight from the tick reservoir at natural foci is to our knowledge the first sequenced genome of a Swedish TBEV. As Torö-2003 was sequenced without enrichment and compared with other TBEV; in silico analysis of secondary RNA structures formed by two NCR revealed a temperature sensitive structural shift between a closed replicative form and an open AUG accessible form. We also discuss additional novel phylogenetic conserved structures in the variable region of

38 3’NCR suggesting a function as an enhancer element in virus replication and translation. SL1-5 structures are proposed to act as virus promoter, while the SL6-8 has been suggested to function as enhancers for virus replication and translation (Gritsun & Gould, 2007b; Gritsun et al., 1997; Proutski et al., 1999). We have shown additional RNA structures (SL9-SL14), which may explain the relevance of V3’ NCR and its role as an enhancer element for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells (Elvang et al., 2011). We further argue that this adaptation is performed through a temperature-sensitive trans-acting riboswitch; altering a closed form of the secondary structures at lower temperatures and an open form at higher temperatures allowing translation. Because the variable region is present in strains freshly isolated from ticks, and multiple passages in vertebrate cells trigger spontaneous losses of significant fragments within it; more genome sequences obtained without cell propagation should in the future give insight into the role of V’NCR (Elvang et al., 2011; Hayasaka et al., 2001; Mandl et al., 1998; Melik et al., 2007; Wallner et al., 1995). These mechanisms may explain the low TBEV level observed in environmentally sampled ticks. The dynamics and variability of viral RNA folding should be studied carefully as virus adaptation to environmental temperatures might play a key role in viral lifecycle in different organisms. In research, the design of virus replicons has provided an important tool for the understanding of fundamental viral processes and for the development of antiviral drugs. The benefit of using replicons as a tool is that fundamental processes can be studied without the need for growing infectious virus. Taken this together we had excellent material to extend the sequenced Torö- 2003 and further used it in construction of a DNA sub-genomic replicon encoding the NS genes and GFP or luciferase as reporter. The developed TBEV replicon is a suitable tool in studies on TBEV genome cyclization dynamics. Passaging the replicon in different arthropod and mammalian cell lines and at different temperatures will shed light into the viral adaptation issue. Introducing the C-terminal NS1 sequence RSKV (typically found in human IAV) in an infectious IAV clone increased the replication rate of avian IAV, H7N1 (low pathogenic) in human cells and ducks. Introducing, the C-terminal ESEV sequence (typical for avian IAV) increased the replication rate and IAV pathogenicity in mice (Soubies et al., 2010). We also found that replication is affected when the PDZ motif in NS5 is mutated. This may be related to pathway mechanisms and its PDZ binding proteins affecting the viral replication within the cell. TransSignal PDZ domain arrays revealed that the C-terminus of NS5 binds several novel human PDZ domains, such as IL-16, ZO-2 and GIPC, associates with TJ, vesicle movements and different pathways related with cell proliferation and regulation of transcriptional factors. Several flavivirus species induce or reduce transcriptional factors and it is of interest to test

39 whether the replicon and its PDZ mutants have this effect on transcriptional regulation in association to PDZ binding proteins. IL-8 secretion is an essential mediator of the inflammatory response in the innate immune system. Reporter assays have shown that IL-8 induced by DENVNS5 through CAAT/enhancer binding protein (c/EBP) activity protein and to a lesser extent NF-κB activity and AP-1; indicating the role of NS5 in DEN2V infection. The recruitment and activation of potential target cells may contribute to viral replication as well as to the inflammatory components of dengue virus disease. We have indications that TBEVNS5 induces IL-8 and AP-1 like DENVNS5 and JEVNS3. These tests should be considered and a relation to the detected proteins should be studied. Even though all these proteins contain PDZ domains their connection might not involve PDZ binding directly with TBEVNS5. There is evidence that IL-16 stimulated peripheral blood mononuclear (PBM) cells induces other cytokines and tumor necrosis factor-α (TNF- α). This stimulation might be important in the inflammatory response. It is also believed that IL-16 plays a role as an inhibitor of HIV replication. A homologue to IL-16 in the brain; NIL-16 associates with NR2A, a subunit of the NMDA receptor. As TBEV is a neurotropic virus, it is of great interest to look into NS5/IL-16 interaction and there may be factors that could affect the disease outcome. In paper III, we observed that the TBEV replicon had a strong inhibition effect on neurite outgrowth in PC12 cells stimulated with NGF. We also showed that the TBEV replicon represses GAP43luc activity, a marker of neurite outgrowth. These findings were linked to TBEVNS5 MTase domain targeting Rac1 and βPIX via Scribble association, suggesting that NS5 interference with Scribble disturbs the association with Rac1 and βPIX, which inhibits neurite outgrowth, possibly due to inactivation of Rac1 (Wigerius et al., 2010).

40 Material and methods

Tick sampling From 2003 to 2007 at different time periods, ticks were sampled from different areas in the Stockholm archipelago and southern Sweden. Nymph and adult I. ricinus specimens were sampled by flagging, i.e. dragging a white flannel blanket (0.75 x 1.5 m) slowly (approximately 1 m/s) over meadow forest landscape. The blankets were harvested on both sides every 50 meter and ticks from each sampling site were pooled and stored in RNAlater (Ambion) at -20°C until use. I. ricinus ticks are relatively common in the Swedish open fields, but also in forest vegetation. During hot sunny days or rainy days the ticks are harder to find as they avoid dehydration or drowning, respectively. During the hot summer days the ticks were collected in shady areas. I. ricinus thrive in vegetation nearby small ponds or streams and nymphs were the most common life stage found during the sampling.

Cell culture Cell culture technique involves the cultivating processes of eukaryotic cells under controlled conditions typically in a flask or in wells on a plate. Cells grown directly from a tissue are known as primary cell culture, but they have a limited life span. Established cell lines have the ability to grow and divide indefinitely as long as provided with suitable nutrients. The advantage with established cell lines is the homologous source and no need to sacrifice animals, whereas a major advantage of primary culture is mimicking of the in vivo state more closely. Both cell lines have advantages and disadvantages, but together both techniques are significant and can complement each other. Several different cell lines have been used in our studies, which were chosen to resemble specific virus environments as possible. The African green monkey derived kidney cell line COS-7 is well known for expressing heterologous proteins at high levels. For cell differentiation studies the rat adrenal medulla pheochromocytoma cells (PC12) were used. PC12 cells are useful as a model system for neuronal differentiation because it stops dividing and terminally differentiate when treated with NGF. The mouse neuroblastoma (N2A) and the epithelial dog madin-darby canine kidney (MDCK) cell lines were also used for cell imaging of the replicons and NS5, respectively. The baby hamster kidney cells (BHK-21) were used for real time studies of the virus replication.

41 Luciferase assay To study transcriptional regulation a reporter gene is often used. The promoter under study is linked to a detectable reporter gene and expression of the reporter can be assayed under different conditions. The luciferase gene is a common reporter gene. Different assay systems have been developed, such as the Dual-Luciferase® Reporter (DLR™) Assay System which provides an accurate, convenient and sensitive technique to analyze and measure the firefly (Photinus pyralis) and Renilla (Renilla reniformis or sea pansy) luciferase. The luciferase enzyme has luciferin as substrate and catalyzes a process emitting light that can be quantified in a luminometer. In transient expression assays, the constitutive expression of a second co- transfected reporter gene provides an internal standard (Renilla).

Real time PCR Real-time PCR; also known as quantitative PCR (qPCR) is a valuable tool in life science research. It is excessively accurate, sensitive and quantitative method to measure and monitor nucleic acid sequences. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of PCR product during each cycle. To bypass the problem with RNA instability an additional cycle of reverse transcription that leads to transcript of a cDNA is required. Two well defined techniques can be used to detect the fluorescent reporter molecules. The SYBR Green dye technique is a non-specific detection system where the dye intercalates into the dsDNA molecules, while Taqman technique use a fluorescent probe to be more sequence specific. The probe in Taqman consists of a fluorescent molecule attached to the 5’ end and a quencher molecule at the 3’ end. When the fluorescent molecule and the quencher are in proximity the probe will not fluoresce. During the elongation step, the exonuclease activity of taq polymerase will digest the probe and release the fluorescent molecule from the quencher. The free fluorescent molecule will be registered at emission at the end of each cycle. The SYBR Green dye has higher affinity to dsDNA than ssDNA or RNA and in solution, the unbound dye exhibits very little fluorescence and increases its emission 1000 fold when intercalated to dsDNA. An advantage of using SYBR Green is that no additional probes are required.

Nested PCR The nested PCR method is a form of the classical PCR technique. The specificity of PCR fragment is dependent on primers binding to the template. Unspecific binding of primer may give more than one amplified DNA segment. To control the specificity of primer binding and their potential to

42 enhance accurate gene of interest the nested PCR method is an outstanding method to employ. In this method, two pairs of primers are used for the amplification of a single gene of interest. The first pair of primers will amplify the gene as in any classical PCR method. The second pair of primers binds within the first PCR product and generates a second PCR product. The strength of this approach is both increased specificity and sensitivity.

Confocal laser scanning microscopy CLSM is an important tool for obtaining high resolution cell images at various depths of the specimen, a procedure known as optical sectioning. CLSM is capable of examining varied conjugated fluorochromes exhibiting fluorescence at different wavelengths. Precise regulation of wavelength and excitation intensity allows studying dynamic living or fixed biological specimens. Due to the risk of overlapping emission it is preferred to use fluorophores with nonoverlapping spectra. The choice of fluorochromes should be according to the LASER lines of the system. Overlapping can be overcome by detecting the channels sequentially instead of in parallel. A commonly used fluorophore that exhibits green fluorescence is green fluorescent protein (GFP) often used in protein localization and protein- protein interaction studies. GFP has its excitation-emission peaks at 495 nm and 509 nm, respectively.

Co-immunoprecipitation Co-immunoprecipitation (Co-IP) is a method for the analysis of protein- protein interactions. The method can be divided into different stages where the first step is that a specific antibody is added to the cell lysate which will bind to the protein of interest. This results in an antibody-protein complex which is precipitated by using protein-G or protein-A sepharose which binds most antibodies. The sepharose binds to antibodies bound to the protein that might associate with another protein which is co-precipitated. Co- precipitated proteins of interest can be identified e.g. by Western blot analysis.

Western blot Western blot was introduced by Harry Towbin in 1979, since then the method has become a routine technique e.g for protein analysis. Western blot is also called immunoblotting for the reason that an antibody is used to detect its antigen. Shortly, the method includes separation of proteins using

43 gel electrophoresis and subsequently transferring the biological samples from the gel to a membrane for detection on the surface of the membrane. Both qualitative and semi-quantitative data can be analyzed by this method. The specificity of the antibody-antigen interaction allows a target protein to be identified in the complex protein mixture. The membranes commonly used in Western blotting are nitrocellulose or polyvinylidene difluoride (PVDF) membrane, which must first be blocked to prevent nonspecific binding of antibodies. There are several techniques to develop a Western blot membrane which could either be “direct” or “indirect” methods. The direct method implies that the detection depend on a primary antibody labeled with an enzyme or fluorescent dye, whereas in the indirect method using a primary and a secondary antibody. Primary antibody is added first to bind to the antigen. The secondary antibody binds the primary antibody and is labeled by biotin, fluorescent probes such as fluorescein or rhodamine, or enzyme conjugates such as horseradish peroxidase or alkaline phosphatase. Finally, this antigen-enzyme-probe complex is detected by adding an appropriate substrate to the enzyme which produces a detectable product e.g. chemiluminescent. The light output produced by the chemical reaction between the probe and substrate can be captured using a light sensitive film, a CCD camera or a phosphorimager designed for chemiluminescent detection.

Construction of the sub-genomic TBEV replicons In Paper I and Paper II, we present the primers used to amplify and sequence the Torö TBEV genome from a total RNA extract; generating cDNA fragments of the complete genome. To construct a TBEV sub-genomic replicon, we used the overlapping regions and suitable restriction enzymes to clone and merge suitable fragments. Each amplified fragment was first cloned into pcDNA™3.1/V5-His-TOPO® 5523 bp (Invitrogen). The construction of the sub-genomic replicon is described stepwise below with important restriction enzymes presented in the figures.

44 Strategy 1, Fragment 2256/5034 was joined with the vector containing the 4828/6163 fragment by using the HindIII site. This generated continuous fragment that includes the NS1, the NS2A/B and a large part of the NS3 gene.

Strategy 2, Fragment 9650/11122 was joined with the 7611/9834 fragments. Initially the 9650/11122 plasmid was digested with PmeI and the extracted fragment of 1594 bp was again digested with EagI to obtain a desired fragment of 1339 bp with PmeI/EagI overhang. To generate a suitable vector, the 9650/11122 was subsequently digested with BstXI/PmeI resulting in a fragment of 5367 bp with a BstXI/PmeI overhang. The 7611/9834 was digested with BstXI/EagI to obtain the desired fragment of 2180 bp with BstXI/EagI overhang. By joining these three fragments we merged the NS5-3’NCR part of the TBEV genome.

45 Strategy 3, Due to difficulty to find suitable restriction enzyme sites at the overlapping regions we decided to bypass this problem by cloning the fragment into the pET-15b vector, 5708 bp (Novagen). The pET-15b was digested with MluI/BssHII. The 4809/6182 plasmid was digested with BssHII/BalI and the 6046/7888 plasmid was digested with BalI/MluI. The extracted fragments of 325 bp and 808 bp from respective plasmid was joined and cloned into the digested pET-15b vector.

46 Strategy 4, Next, the fragments assembled in Strategy 1 and 3 were merged. Both plasmids were digested with NruI/BssHII and the fragment of Strategy 1 was cloned into the vector generated in Strategy 3.

Strategy 5 The 6046/7888 vector was digested with MluI/BsmBI to obtain a 1325 bp fragment. The vector from Strategy 2 was first digested with MluI, followed by partial digestion with BsmBI to generate a 7998 bp vector fragment. The 1325 bp fragment was cloned into the vector that merged the genomic region of NS3-NS5.

47 Strategy 6, To replace the 5’NCR+C20 of a WNV replicon (pCMVWRepGFP) with equivalent regions of TBEV, a WNV replicon (Pierson et al., 2006) was digested with SacI/MunI and a fragment of 4554 bp was extracted. WNV replicon was also partially digested with MunI/MluI and a fragment of 800 bp was extracted. A plasmid containing a fragment of the Torö capsid gene and 5´NCR extended with consensus sequence at 5´ terminus was amplified by PCR using a modified forward primer containing a suitable part of the vector + SacI (5’- GAGCTCGTTTAGTGAACCGAGATTTTCTTGCACGTGCATGCGTT- 3’). The reverse primer used introduced a MluI site (5’- ACGCGTTTTCGACACTCGTCGAGGGGGACCG-3’) at a suitable position. The PCR fragment was purified and was introduced with the 800 bp and 4554 bp fragment of the WNV replicon.

Strategy 7, WNV replicon was partial digested with MluI/KasI. A fragment of 4928 bp was extracted. The plasmid from the Strategy 5 was used as a template for PCR with a forward primer MluI site (5’- ATATACGCGTCTGGGCGTCGCAGCT-3’) and a reverse primer that covers the very end of Torö and modified with the ribozyme part of the WNV replicon, followed with a KasI site (5’- GGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCAGCGGGTGTTT TTCCGAGTCAC-3’). The thermal PCR profile was 25 cycles of 94ºC for 15s, 55ºC for 30s, and 72ºC for 6 min. Amplified fragment was ligated with the extracted 4928 bp fragment.

48 Strategy 8, The vector from Strategy 6 was used as a PCR template with forward primer (5’-TATTGAAGCATTTATCAGGG-3’), and reverse primer introducing a KpnI site (5’-GGTACCAGGACCAGGGTTACTTTCAAC-3’). The amplified fragment was cloned into a Topo vector followed by digestion with ClaI/KpnI generating fragment of 1936 bp.

Strategy 9, The vector from Strategy 4 was used as a PCR template where the forward primer was (5’-AGAACGAAGCGGCGTCGAAG-3’), and a reverse primer modified with a KpnI site (5’-GGTACCCTGAACATGAGAAACCCTAC- 3’). The amplified fragment was cloned into a Topo vector and digested with MluI/KpnI generating a fragment of 4129 bp.

49 Strategy 10, The vector from Strategy 7 was digested with ClaI/MluI which generated a fragment of 8706 bp. This fragment together with fragments from Strategy 8 and 9 were ligated and completed the construction ToröTBEVrepGFP replicon. The complete TBEV replicon was further used to construct the TBEV replicon expressing luciferase as reporter. To do this, the EGFP was removed by MluI cleavage and was substituted with a PCR amplified luc gene modified with suitable MluI restriction sites.

CMV promoter Human cytomegalovirus immediate early promoter TBEV 5'NCR+60C Torö TBEV 5´NCR extended with (60bp) Capsid pEGFP Enhanced green fluorescent protein FMDV 2a protease 2a protease of foot-and mouth-disease virus (FMDV) mediates cleavage on the polyprotein releasing the reporter protein E-3'NCR Envelope protein (84bp) extended with the rest of the genome; NS1-3´NCR HDV Ribozyme Hepatitis delta virus (HDV) ribozyme for post transcriptional self-cleavage generating “wild type” TBEV 3’ termini Sv40 polyA Simian virus 40 polyA; regulate the final outcome from a transcriptional unit

50 Computer software The impressive development of computers and software has made it easier to process and analyze genetic data. Software used in this thesis is summarized below. The molecular biology programs Vector NTI and BioEdit were used for various analyses of overviewed DNA/aa aligning, restriction enzyme analysis, sequence analysis and the creation of a plasmid data base. Vector NTI along with FastPCR and PerlPrimer was operated for primer design and testing. PAUP 4.0 and MEGA 5 were used for phylogenetic analysis. For statistical analysis and illustration, the software Statistica 8.0, Microsoft Excel and the (rmcdr) package in the software application R was utilized. For graphical images, Adobe Photoshop, Macromedia FreeHand, Image J and Multi Gauge, were used. Microsoft Word, Adobe illustrator and PowerPoint were used for word processing and presentations.

51 Acknowledgements

First and foremost I would like to thank my supervisor, Magnus Johansson for accepting me as doctoral student and scientific support I have received over years. Magnus, you have taught me allot in molecular biology, biochemistry and collecting ticks. I became the best tick collector. I am especially grateful to you Magnus for your friendship and all memories. For kindly reading and helping me with articles and reading this thesis several times making it better and better. I would like to thank Professor Elisabeth Haggård for carefully reading through the thesis and for your helpful comments during my time as a PhD student. I would also like to express my gratitude and humble thanks to the SH administration for all help during these years and in particular to Andres and Tord for your time and assistance and friendship. Thanks to Mike for your friendship and many discussions we have had about everything under the sun. You gave science another dimension and in you I found a friend. Barca seems to win “El Clásico” all the time. Thank you Karin for you are such a wonderful person and for all scientific and non-scientific talk we have had and all lunches. I miss you very much. Annelie, I want to thank you for always being there. You are an ocean of knowledge and life experience. Thanks for all the discussions and experience that you have shared. Naveed, Thank you for being a good friend and a “bangra sidekick dancer”. I have never seen anyone more positive than you. You have some years ahead of you and many exciting results in front of you to analyze. Keep up the good work high and Torö-2003 will never die. Justina, Thanks for your friendship and you made our group brighter. I am sure you will see the world before I finish this acknowledgement. Ingela, Thank you for your friendship and I hope it goes well for you in Switzerland. We went to same classes from the first day at SU. I miss you very much and all our coffee break and lunches together with Karin. Who shall I tell all my stories for when you and Karin is gone? Robert, my friend, you are google and encyclopedia together. I appreciate our time on SH and the time before at SU, where we also went to the same education. From the first day I saw a friend in you and I hope to see you more often. I would like to acknowledge many people who have passed my way and contributed directly or indirectly to this thesis. I would like to thank all my colleagues in different groups: Anthony W., Patrick D., Stefan, Helmi, Chiounan, Petter, Sara, Nasim, Christina, Julian (miss you), Ida, Fergal, Fredrik B, Yann, Agata, Juan

52 Carlos, John B, Ivailo (Jag uppskattar din vänskap och vi kommer bli etoro guru:)), Julian, Anna J, Carlos, Petter, kristina, Tiina, Inger, Jan-Eric, Paulina, Josefin, Per, Kari, Patrick (thanks for your time and help with statistics), Anders N(GMT). Extra thanks to Einar Hallberg's group for their close friendship and all the help I have received with various questions over the years; Einar Hallberg, Marie, Santhosh, Veronica, Ricardo, Ellinor, Lotta, Robert. Also want to thank my friends for all the nice time we had together. Very are little to write here and I appreciating your friendship Henri, Ihab (mattias), Jamal, Fared, Fuad (abu Hasan). Henri, you are truly a good friend and I have found something most people do not find in their life; your friendship. I miss our fishing days and movie nights. Henri, I told you Volvo is a great car! Vill tacka Securitas Sverige AB, Fredrik V, Personalplanerarna, Jan, Fredrik H, Göte , Tomas och mfl, Arbetledare och alla väktare som jag har fått lära känna genom alla mina år där.

Lastly, I would like to thank my family for all their love and encouragement. To my brother Hossam and his family, adorable Ibtisam, my sweet lovely sweetheart Mina (Håll mitt hjärta, håll min själ, lägg mitt huvud i ditt knä..., Ta mig till havet och gör mig till kung...och galeb galeb wen wen ghayeb 3laya yomen) and Mr Emin the serious one with the beautiful smile and sharp eyes. To my dear sweet Dalia and Marc; I loved your wedding, so beautiful and elegant in every single way, and the Barca museum trip:) I understood that it is more than just a game! To Hassanpour family, Dr Gholamreza and Parvaneh, father and mother in law, for your support and believing in me. I know you are with me on my dissertation day. To the beautiful and artistic Sayna. I believe in you and your art and thank you very much for the great typography design illustrated on dedication page. The world is ready for your arts. To my mother, you made this mom! At a time when humanity was humiliation; when the sound of bombs echoed like a constant wind breeze in our life and the tears poured as Tigris and Euphrates over the cheeks of children; mother you saved us from that humiliation. This great evil! Where does it come from? How did it steal into the world? What seed, what root did it grow from? Who is doing this? Love, where does it come from? Who lit this flame in us? No war can put it out or conquer it. I was a prisoner, you set me free. Like a mother lion saving her cubs, you saved us over borders, country to country, language to language, water to water and from sun to snow. We were born in the cradle of civilization, but you took us to the supremacy of paradise. In the new, we built our hearts and set seed in the ground. The ground welcomed us and made us human again. Mother, I can breathe again! You taught us how to

53 build; how to build with hands and taste the sweetness of sweat. I am so thankful till may last breath. Lastly, and most of all for my sweet loving, supportive, encouraging, and patient wife Irsa whose faithful support during the final stages of this Ph.D. is so appreciated. Thank you and I love you deeply. We did this Irsajoooooooooooooooooooooooooooooooooooooooooooooooooooon

Thank you all!

54 References

Aberle, J. H., Aberle, S. W., Kofler, R. M. & Mandl, C. W. (2005). Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus. J Virol 79, 15107-15113. Ackermann, M. & Padmanabhan, R. (2001). De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. J Biol Chem 276, 39926-39937. Allison, S. L., Tao, Y. J., O'Riordain, G., Mandl, C. W., Harrison, S. C. & Heinz, F. X. (2003). Two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus. J Virol 77, 11357- 11366. Anderson, J. M., Stevenson, B. R., Jesaitis, L. A., Goodenough, D. A. & Mooseker, M. S. (1988). Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J Cell Biol 106, 1141-1149. Angst, B. D., Marcozzi, C. & Magee, A. I. (2001). The cadherin superfamily. J Cell Sci 114, 625-626. Appleton, B. A., Zhang, Y., Wu, P., Yin, J. P., Hunziker, W., Skelton, N. J., Sidhu, S. S. & Wiesmann, C. (2006). Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity. J Biol Chem 281, 22312-22320. Audebert, S., Navarro, C., Nourry, C., Chasserot-Golaz, S., Lecine, P., Bellaiche, Y., Dupont, J. L., Premont, R. T., Sempere, C. & other authors (2004). Mammalian Scribble forms a tight complex with the betaPIX exchange factor. Curr Biol 14, 987-995. Balashov Iu, S. (1999). [The role of blood-sucking ticks and insects in natural foci of infections]. Parazitologiia 33, 210-222. Bancroft, W. H., Scott, R. M., Snitbhan, R., Weaver, R. E., Jr. & Gould, D. J. (1976). Isolation of Langat virus from Haemaphysalis papuana Thorell in Thailand. Am J Trop Med Hyg 25, 500-504. Beatch, M., Jesaitis, L. A., Gallin, W. J., Goodenough, D. A. & Stevenson, B. R. (1996). The tight junction protein ZO-2 contains three PDZ (PSD-95/Discs- Large/ZO-1) domains and an alternatively spliced region. J Biol Chem 271, 25723-25726. Berrada, Z. L. & Telford, S. R., 3rd (2009). Burden of tick-borne infections on American companion animals. Top Companion Anim Med 24, 175-181. Best, S. M., Morris, K. L., Shannon, J. G., Robertson, S. J., Mitzel, D. N., Park, G. S., Boer, E., Wolfinbarger, J. B. & Bloom, M. E. (2005). Inhibition of interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist. J Virol 79, 12828-12839. Betanzos, A., Huerta, M., Lopez-Bayghen, E., Azuara, E., Amerena, J. & Gonzalez-Mariscal, L. (2004). The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells. Exp Cell Res 292, 51-66. Bilder, D. & Perrimon, N. (2000). Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676-680.

55 Bilder, D., Li, M. & Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113-116. Borg, J. P., Marchetto, S., Le Bivic, A., Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Adelaide, J., Margolis, B. & other authors (2000). ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor. Nat Cell Biol 2, 407-414. Bressanelli, S., Stiasny, K., Allison, S. L., Stura, E. A., Duquerroy, S., Lescar, J., Heinz, F. X. & Rey, F. A. (2004). Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J 23, 728-738. Brinkley C. , Nolskog P. , Golovljova I. , Lundkvist Å. & T., B. (2008). Tick- borne encephalitis virus natural foci emerge in western Sweden. Int J Med Microbiol, 73–80. Brinton, M. A. (2002). The molecular biology of West Nile Virus: a new invader of the western hemisphere. Annu Rev Microbiol 56, 371-402. Brooks, A. J., Johansson, M., John, A. V., Xu, Y., Jans, D. A. & Vasudevan, S. G. (2002). The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals. J Biol Chem 277, 36399-36407. Bruschke, C. J., Hulst, M. M., Moormann, R. J., van Rijn, P. A. & van Oirschot, J. T. (1997). Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species. J Virol 71, 6692-6696. Bunn, R. C., Jensen, M. A. & Reed, B. C. (1999). Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton. Mol Biol Cell 10, 819-832. Burke, D. S., Monath, T.P. (2001). Fields virology, 4. ed. edn, vol. 2 vol.(3087 s.). Philadelphia: Lippincott-Raven. Calisher, C. H. & Gould, E. A. (2003). Taxonomy of the virus family Flaviviridae. Adv Virus Res 59, 1-19. Cann, A. J. (2001). Principles of molecular virology, 3.ed. edn. San Diego Academic Press, cop. 2001. Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44, 649-688. Chambers, T. J., Nestorowicz, A., Amberg, S. M. & Rice, C. M. (1993). Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J Virol 67, 6797-6807. Charlier, N., Leyssen, P., Pleij, C. W., Lemey, P., Billoir, F., Van Laethem, K., Vandamme, A. M., De Clercq, E., de Lamballerie, X. & other authors (2002). Complete genome sequence of Montana Myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the 3' untranslated region of flaviviruses with no known vector. J Gen Virol 83, 1875-1885. Charrel, R. N., Zaki, A. M., Attoui, H., Fakeeh, M., Billoir, F., Yousef, A. I., de Chesse, R., De Micco, P., Gould, E. A. & other authors (2001). Complete coding sequence of the Alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in Saudi Arabia. Biochem Biophys Res Commun 287, 455-461.

56 Charrel, R. N., Attoui, H., Butenko, A. M., Clegg, J. C., Deubel, V., Frolova, T. V., Gould, E. A., Gritsun, T. S., Heinz, F. X. & other authors (2004). Tick-borne virus diseases of human interest in Europe. Clin Microbiol Infect 10, 1040-1055. Chen, C. J., Kuo, M. D., Chien, L. J., Hsu, S. L., Wang, Y. M. & Lin, J. H. (1997). RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA. J Virol 71, 3466-3473. Cleaves, G. R., Ryan, T. E. & Schlesinger, R. W. (1981). Identification and characterization of type 2 dengue virus replicative intermediate and replicative form RNAs. Virology 111, 73-83. Colicelli, J. (2004). Human RAS superfamily proteins and related GTPases. Sci STKE 2004, RE13. Cui, T., Sugrue, R. J., Xu, Q., Lee, A. K., Chan, Y. C. & Fu, J. (1998). Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology 246, 409-417. De Vries, L., Lou, X., Zhao, G., Zheng, B. & Farquhar, M. G. (1998). GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP. Proc Natl Acad Sci U S A 95, 12340-12345. Dobler, G. (2010). Zoonotic tick-borne flaviviruses. Vet Microbiol 140, 221-228. Dokland, T., Walsh, M., Mackenzie, J. M., Khromykh, A. A., Ee, K. H. & Wang, S. (2004). West Nile virus core protein; tetramer structure and ribbon formation. Structure 12, 1157-1163. Doyle, D. A., Lee, A., Lewis, J., Kim, E., Sheng, M. & MacKinnon, R. (1996). Crystal structures of a complexed and peptide-free membrane protein- binding domain: molecular basis of peptide recognition by PDZ. Cell 85, 1067-1076. Ebel, G. D. (2010). Update on Powassan virus: emergence of a North American tick-borne flavivirus. Annu Rev Entomol 55, 95-110. Ebel, G. D., Spielman, A. & Telford, S. R., 3rd (2001). Phylogeny of North American Powassan virus. J Gen Virol 82, 1657-1665. Ecker, M., Allison, S. L., Meixner, T. & Heinz, F. X. (1999). Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J Gen Virol 80 ( Pt 1), 179-185. Egloff, M. P., Benarroch, D., Selisko, B., Romette, J. L. & Canard, B. (2002). An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J 21, 2757-2768. Ellencrona, K., Syed, A. & Johansson, M. (2009). Flavivirus NS5 associates with host-cell proteins zonula occludens-1 (ZO-1) and regulating synaptic membrane exocytosis-2 (RIMS2) via an internal PDZ binding mechanism. Biol Chem 390, 319-323. Elvang, A., Melik, W., Bertrand, Y., Lonn, M. & Johansson, M. (2011). Sequencing of a Tick-Borne Encephalitis Virus from Ixodes ricinus Reveals a Thermosensitive RNA Switch Significant for Virus Propagation in Ectothermic Arthropods. Vector Borne Zoonotic Dis 11, 649-658. Estrada-Pena, A. (2009). Tick-borne pathogens, transmission rates and climate change. Front Biosci 14, 2674-2687.

57 Falgout, B., Pethel, M., Zhang, Y. M. & Lai, C. J. (1991). Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65, 2467-2475. Fanning, A. S. & Anderson, J. M. (1999). Protein modules as organizers of membrane structure. Curr Opin Cell Biol 11, 432-439. Fanning, A. S., Jameson, B. J., Jesaitis, L. A. & Anderson, J. M. (1998). The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273, 29745- 29753. Favre-Bonvin, A., Reynaud, C., Kretz-Remy, C. & Jalinot, P. (2005). Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP- 2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome. J Virol 79, 4229-4237. Flamand, M., Megret, F., Mathieu, M., Lepault, J., Rey, F. A. & Deubel, V. (1999). Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol 73, 6104-6110. Flick, R. & Hobom, G. (1999). Interaction of influenza virus polymerase with viral RNA in the 'corkscrew' conformation. J Gen Virol 80 ( Pt 10), 2565-2572. Flint, S. J., Enquist, L.W., Ranciello V.R.,Skalka, A.M. (2003). Principles of Virology - Molecular Biology, Pathogenesis, and Control of Animal Viruses. AMERICAN SOCIETY FOR MICROBIOLOGY. Gao, G. F., Jiang, W. R., Hussain, M. H., Venugopal, K., Gritsun, T. S., Reid, H. W. & Gould, E. A. (1993). Sequencing and antigenic studies of a Norwegian virus isolated from encephalomyelitic sheep confirm the existence of louping ill virus outside Great Britain and Ireland. J Gen Virol 74 ( Pt 1), 109-114. Gardiol, D., Zacchi, A., Petrera, F., Stanta, G. & Banks, L. (2006). Human discs large and scrib are localized at the same regions in colon mucosa and changes in their expression patterns are correlated with loss of tissue architecture during malignant progression. Int J Cancer 119, 1285-1290. Gardiol, D., Kuhne, C., Glaunsinger, B., Lee, S. S., Javier, R. & Banks, L. (1999). Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation. Oncogene 18, 5487-5496. Giepmans, B. N. & Moolenaar, W. H. (1998). The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr Biol 8, 931-934. Golovljova, I., Vene, S., Sjolander, K. B., Vasilenko, V., Plyusnin, A. & Lundkvist, A. (2004). Characterization of tick-borne encephalitis virus from Estonia. J Med Virol 74, 580-588. Gonzalez-Mariscal, L. & Nava, P. (2005). Tight junctions, from tight intercellular seals to sophisticated protein complexes involved in drug delivery, pathogens interaction and cell proliferation. Adv Drug Deliv Rev 57, 811- 814. Gould, E. A., de Lamballerie, X., Zanotto, P. M. & Holmes, E. C. (2001). Evolution, epidemiology, and dispersal of flaviviruses revealed by molecular phylogenies. Adv Virus Res 57, 71-103.

58 Gould, E. A., de Lamballerie, X., Zanotto, P. M. & Holmes, E. C. (2003). Origins, evolution, and vector/host coadaptations within the genus Flavivirus. Adv Virus Res 59, 277-314. Gritsun, T. S. & Gould, E. A. (2006). The 3' untranslated region of tick-borne flaviviruses originated by the duplication of long repeat sequences within the open reading frame. Virology 350, 269-275. Gritsun, T. S. & Gould, E. A. (2007a). Origin and evolution of flavivirus 5'UTRs and panhandles: trans-terminal duplications? Virology 366, 8-15. Gritsun, T. S. & Gould, E. A. (2007b). Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission. Adv Virus Res 69, 203-248. Gritsun, T. S., Desai, A. & Gould, E. A. (2001). The degree of attenuation of tick- borne encephalitis virus depends on the cumulative effects of point mutations. J Gen Virol 82, 1667-1675. Gritsun, T. S., Nuttall, P. A. & Gould, E. A. (2003a). Tick-borne flaviviruses. Adv Virus Res 61, 317-371. Gritsun, T. S., Lashkevich, V. A. & Gould, E. A. (2003b). Tick-borne encephalitis. Antiviral Res 57, 129-146. Gritsun, T. S., Venugopal, K., Zanotto, P. M., Mikhailov, M. V., Sall, A. A., Holmes, E. C., Polkinghorne, I., Frolova, T. V., Pogodina, V. V. & other authors (1997). Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'- UTRs. Virus Res 49, 27-39. Gumbiner, B., Lowenkopf, T. & Apatira, D. (1991). Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1. Proc Natl Acad Sci U S A 88, 3460-3464. Guo, J. T., Hayashi, J. & Seeger, C. (2005). West Nile virus inhibits the signal transduction pathway of alpha interferon. J Virol 79, 1343-1350. Guyatt, K. J., Westaway, E. G. & Khromykh, A. A. (2001). Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin. J Virol Methods 92, 37-44. Haglund, M., Vene, S., Forsgren, M., Gunther, G., Johansson, B., Niedrig, M., Plyusnin, A., Lindquist, L. & Lundkvist, A. (2003). Characterisation of human tick-borne encephalitis virus from Sweden. J Med Virol 71, 610- 621. Hahn, C. S., Hahn, Y. S., Rice, C. M., Lee, E., Dalgarno, L., Strauss, E. G. & Strauss, J. H. (1987). Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences. J Mol Biol 198, 33- 41. Han, X., Aho, M., Vene, S., Peltomaa, M., Vaheri, A. & Vapalahti, O. (2001). Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in Finland. J Med Virol 64, 21-28. Han, X., Juceviciene, A., Uzcategui, N. Y., Brummer-Korvenkontio, H., Zygutiene, M., Jaaskelainen, A., Leinikki, P. & Vapalahti, O. (2005). Molecular epidemiology of tick-borne encephalitis virus in Ixodes ricinus ticks in Lithuania. J Med Virol 77, 249-256. Hanna, J. N., Ritchie, S. A., Phillips, D. A., Shield, J., Bailey, M. C., Mackenzie, J. S., Poidinger, M., McCall, B. J. & Mills, P. J. (1996). An outbreak of

59 Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 165, 256-260. Haskins, J., Gu, L., Wittchen, E. S., Hibbard, J. & Stevenson, B. R. (1998). ZO- 3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol 141, 199-208. Hayasaka, D., Yoshii, K., Ueki, T., Iwasaki, T. & Takashima, I. (2004). Sub- genomic replicons of Tick-borne encephalitis virus. Arch Virol 149, 1245- 1256. Hayasaka, D., Ivanov, L., Leonova, G. N., Goto, A., Yoshii, K., Mizutani, T., Kariwa, H. & Takashima, I. (2001). Distribution and characterization of tick-borne encephalitis viruses from Siberia and far-eastern Asia. J Gen Virol 82, 1319-1328. Heinz, F. X. & Mandl, C. W. (1993). The molecular biology of tick-borne encephalitis virus. Review article. Apmis 101, 735-745. Heinz, F. X., Stiasny, K., Puschner-Auer, G., Holzmann, H., Allison, S. L., Mandl, C. W. & Kunz, C. (1994). Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198, 109-117. Hoenninger, V. M., Rouha, H., Orlinger, K. K., Miorin, L., Marcello, A., Kofler, R. M. & Mandl, C. W. (2008). Analysis of the effects of alterations in the tick-borne encephalitis virus 3'-noncoding region on translation and RNA replication using reporter replicons. Virology 377, 419-430. Holbrook, M. R., Aronson, J. F., Campbell, G. A., Jones, S., Feldmann, H. & Barrett, A. D. (2005). An animal model for the tickborne flavivirus--Omsk hemorrhagic fever virus. J Infect Dis 191, 100-108. Howarth, A. G., Hughes, M. R. & Stevenson, B. R. (1992). Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am J Physiol 262, C461-469. Huang, J. L., Huang, J. H., Shyu, R. H., Teng, C. W., Lin, Y. L., Kuo, M. D., Yao, C. W. & Shaio, M. F. (2001). High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen. J Med Virol 65, 553-560. Huerta, M., Munoz, R., Tapia, R., Soto-Reyes, E., Ramirez, L., Recillas-Targa, F., Gonzalez-Mariscal, L. & Lopez-Bayghen, E. (2007). Cyclin D1 is transcriptionally down-regulated by ZO-2 via an E box and the transcription factor c-Myc. Mol Biol Cell 18, 4826-4836. Humbert, P., Russell, S. & Richardson, H. (2003). Dlg, Scribble and Lgl in cell polarity, cell proliferation and cancer. Bioessays 25, 542-553. Iden, S. & Collard, J. G. (2008). Crosstalk between small GTPases and polarity proteins in cell polarization. Nat Rev Mol Cell Biol 9, 846-859. Ilinskaya, A., Heidecker, G. & Derse, D. (2010). Opposing effects of a tyrosine- based sorting motif and a PDZ-binding motif regulate human T- lymphotropic virus type 1 envelope trafficking. J Virol 84, 6995-7004. Im, Y. J., Park, S. H., Rho, S. H., Lee, J. H., Kang, G. B., Sheng, M., Kim, E. & Eom, S. H. (2003a). Crystal structure of GRIP1 PDZ6-peptide complex reveals the structural basis for class II PDZ target recognition and PDZ domain-mediated multimerization. J Biol Chem 278, 8501-8507.

60 Im, Y. J., Lee, J. H., Park, S. H., Park, S. J., Rho, S. H., Kang, G. B., Kim, E. & Eom, S. H. (2003b). Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization. J Biol Chem 278, 48099-48104. Ishidate, T., Matsumine, A., Toyoshima, K. & Akiyama, T. (2000). The APC- hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase. Oncogene 19, 365-372. Islas, S., Vega, J., Ponce, L. & Gonzalez-Mariscal, L. (2002). Nuclear localization of the tight junction protein ZO-2 in epithelial cells. Exp Cell Res 274, 138- 148. Itoh, M., Morita, K. & Tsukita, S. (1999). Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem 274, 5981- 5986. Itoh, M., Nagafuchi, A., Moroi, S. & Tsukita, S. (1997). Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments. J Cell Biol 138, 181-192. Jaaskelainen, A. E., Tikkakoski, T., Uzcategui, N. Y., Alekseev, A. N., Vaheri, A. & Vapalahti, O. (2006). Siberian subtype tickborne encephalitis virus, Finland. Emerg Infect Dis 12, 1568-1571. Jaenson, T. G. & Lindgren, E. (2011). The range of Ixodes ricinus and the risk of contracting Lyme borreliosis will increase northwards when the vegetation period becomes longer. Ticks Tick Borne Dis 2, 44-49. Jaenson, T. G., Talleklint, L., Lundqvist, L., Olsen, B., Chirico, J. & Mejlon, H. (1994). Geographical distribution, host associations, and vector roles of ticks (Acari: Ixodidae, Argasidae) in Sweden. J Med Entomol 31, 240-256. Jaenson, T. G., Eisen, L., Comstedt, P., Mejlon, H. A., Lindgren, E., Bergstrom, S. & Olsen, B. (2009). Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden. Med Vet Entomol 23, 226-237. Jan, L. R., Yang, C. S., Trent, D. W., Falgout, B. & Lai, C. J. (1995). Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases. J Gen Virol 76 ( Pt 3), 573-580. Jaramillo, B. E., Ponce, A., Moreno, J., Betanzos, A., Huerta, M., Lopez- Bayghen, E. & Gonzalez-Mariscal, L. (2004). Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells. Exp Cell Res 297, 247-258. Jeanneteau, F., Diaz, J., Sokoloff, P. & Griffon, N. (2004). Interactions of GIPC with dopamine D2, D3 but not D4 receptors define a novel mode of regulation of G protein-coupled receptors. Mol Biol Cell 15, 696-705. Jensen, P. M., Skarphedinsson, S. & Semenov, A. (2004). [Densities of the tick (Ixodes ricinus) and coexistence of the Louping ill virus and tick borne encephalitis on the island of Bornholm]. Ugeskr Laeger 166, 2563-2565. Jesaitis, L. A. & Goodenough, D. A. (1994). Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J Cell Biol 124, 949-961. Jeyaprakash, A. & Hoy, M. A. (2009). First divergence time estimate of spiders, scorpions, mites and ticks (subphylum: Chelicerata) inferred from mitochondrial phylogeny. Exp Appl Acarol 47, 1-18.

61 Jia, X. Y., Briese, T., Jordan, I., Rambaut, A., Chi, H. C., Mackenzie, J. S., Hall, R. A., Scherret, J. & Lipkin, W. I. (1999). Genetic analysis of West Nile New York 1999 encephalitis virus. Lancet 354, 1971-1972. Johansen, C. A., van den Hurk, A. F., Ritchie, S. A., Zborowski, P., Nisbet, D. J., Paru, R., Bockarie, M. J., Macdonald, J., Drew, A. C. & other authors (2000). Isolation of Japanese encephalitis virus from mosquitoes (Diptera: Culicidae) collected in the Western Province of Papua New Guinea, 1997-1998. Am J Trop Med Hyg 62, 631-638. Johansson, M., Brooks, A. J., Jans, D. A. & Vasudevan, S. G. (2001). A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin- beta and the viral helicase, NS3. J Gen Virol 82, 735-745. Jones, L. D., Davies, C. R., Green, B. M. & Nuttall, P. A. (1987). Reassortment of Thogoto virus (a tick-borne influenza-like virus) in a vertebrate host. J Gen Virol 68 ( Pt 5), 1299-1306. Jopling, C. L., Yi, M., Lancaster, A. M., Lemon, S. M. & Sarnow, P. (2005). Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309, 1577-1581. Kang, B. S., Cooper, D. R., Devedjiev, Y., Derewenda, U. & Derewenda, Z. S. (2003). Molecular roots of degenerate specificity in syntenin's PDZ2 domain: reassessment of the PDZ recognition paradigm. Structure 11, 845- 853. Kapoor, M., Zhang, L., Ramachandra, M., Kusukawa, J., Ebner, K. E. & Padmanabhan, R. (1995). Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5. J Biol Chem 270, 19100-19106. Khromykh, A. A. & Westaway, E. G. (1996). RNA binding properties of core protein of the flavivirus Kunjin. Arch Virol 141, 685-699. Khromykh, A. A. & Westaway, E. G. (1997). Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol 71, 1497-1505. Khromykh, A. A., Kenney, M. T. & Westaway, E. G. (1998). trans- Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells. J Virol 72, 7270-7279. Khromykh, A. A., Meka, H., Guyatt, K. J. & Westaway, E. G. (2001a). Essential role of cyclization sequences in flavivirus RNA replication. J Virol 75, 6719-6728. Khromykh, A. A., Varnavski, A. N., Sedlak, P. L. & Westaway, E. G. (2001b). Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus. J Virol 75, 4633-4640. Khromykh, A. A., Kondratieva, N., Sgro, J. Y., Palmenberg, A. & Westaway, E. G. (2003). Significance in replication of the terminal nucleotides of the flavivirus genome. J Virol 77, 10623-10629. Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N. & Sheng, M. (1995). Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378, 85-88. Kiszewski, A. E., Matuschka, F. R. & Spielman, A. (2001). Mating strategies and spermiogenesis in ixodid ticks. Annu Rev Entomol 46, 167-182.

62 Kiyono, T., Hiraiwa, A., Fujita, M., Hayashi, Y., Akiyama, T. & Ishibashi, M. (1997). Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proc Natl Acad Sci U S A 94, 11612-11616. Kofler, R. M., Hoenninger, V. M., Thurner, C. & Mandl, C. W. (2006). Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses. J Virol 80, 4099-4113. Kohl, A., Dunn, E. F., Lowen, A. C. & Elliott, R. M. (2004). Complementarity, sequence and structural elements within the 3' and 5' non-coding regions of the Bunyamwera orthobunyavirus S segment determine promoter strength. J Gen Virol 85, 3269-3278. Koonin, E. V. (1993). Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. J Gen Virol 74 ( Pt 4), 733-740. Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28, 375-430. Kornau, H. C., Schenker, L. T., Kennedy, M. B. & Seeburg, P. H. (1995). Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 1737-1740. Kummerer, B. M. & Rice, C. M. (2002). Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol 76, 4773-4784. Kuno, G. (2007). Host range specificity of flaviviruses: correlation with in vitro replication. J Med Entomol 44, 93-101. Kuo, M. D., Chin, C., Hsu, S. L., Shiao, J. Y., Wang, T. M. & Lin, J. H. (1996). Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein. J Gen Virol 77 ( Pt 9), 2077-2084. Lahuna, O., Quellari, M., Achard, C., Nola, S., Meduri, G., Navarro, C., Vitale, N., Borg, J. P. & Misrahi, M. (2005). Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway. EMBO J 24, 1364- 1374. Lanciotti, R. S., Gubler, D. J. & Trent, D. W. (1997). Molecular evolution and phylogeny of dengue-4 viruses. J Gen Virol 78 ( Pt 9), 2279-2284. Lanciotti, R. S., Roehrig, J. T., Deubel, V., Smith, J., Parker, M., Steele, K., Crise, B., Volpe, K. E., Crabtree, M. B. & other authors (1999). Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286, 2333-2337. Lee, H. J. & Zheng, J. J. (2010). PDZ domains and their binding partners: structure, specificity, and modification. Cell Commun Signal 8, 8. Legouis, R., Jaulin-Bastard, F., Schott, S., Navarro, C., Borg, J. P. & Labouesse, M. (2003). Basolateral targeting by leucine-rich repeat domains in epithelial cells. EMBO Rep 4, 1096-1102. Leyssen, P., Charlier, N., Lemey, P., Billoir, F., Vandamme, A. M., De Clercq, E., de Lamballerie, X. & Neyts, J. (2002). Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector. Virology 293, 125-140.

63 Li, L., Lok, S. M., Yu, I. M., Zhang, Y., Kuhn, R. J., Chen, J. & Rossmann, M. G. (2008). The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319, 1830-1834. Li, W. & Brinton, M. A. (2001). The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs. Virology 287, 49-61. Lin, D. C., Quevedo, C., Brewer, N. E., Bell, A., Testa, J. R., Grimes, M. L., Miller, F. D. & Kaplan, D. R. (2006). APPL1 associates with TrkA and GIPC1 and is required for nerve growth factor-mediated signal transduction. Mol Cell Biol 26, 8928-8941. Lindenbach, B. D. & Rice, C. M. (1997). trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 71, 9608-9617. Lindenbach, B. D. & Rice, C. M. (1999). Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J Virol 73, 4611-4621. Lindenbach, B. D., Rice, C.M. (2001). Fields Virology, 4. ed. edn, vol. 2 vol.(3087 s.). Philadelphia: Lippincott-Raven. Lindenbach, B. D., Thiel H-J, Rice C.M. (2007). Fields virology, 5th edn. Philadelphia: Lippincott-Raven. Liu, W. J., Chen, H. B. & Khromykh, A. A. (2003). Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication. J Virol 77, 7804-7813. Liu, W. J., Wang, X. J., Mokhonov, V. V., Shi, P. Y., Randall, R. & Khromykh, A. A. (2005). Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins. J Virol 79, 1934-1942. Lorenz, I. C., Kartenbeck, J., Mezzacasa, A., Allison, S. L., Heinz, F. X. & Helenius, A. (2003). Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus. J Virol 77, 4370-4382. Lou, X., Yano, H., Lee, F., Chao, M. V. & Farquhar, M. G. (2001). GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways. Mol Biol Cell 12, 615-627. Lundkvist, k., Vene, S., Golovljova, I., Mavtchoutko, V., Forsgren, M., Kalnina, V. & Plyusnin, A. (2001). Characterization of tick-borne encephalitis virus from Latvia: evidence for co-circulation of three distinct subtypes. J Med Virol 65, 730-735. Mackenzie, J. M. & Westaway, E. G. (2001). Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 75, 10787-10799. Mackenzie, J. M., Kenney, M. T. & Westaway, E. G. (2007). West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis. J Gen Virol 88, 1163-1168. Mackenzie, J. M., Khromykh, A. A., Jones, M. K. & Westaway, E. G. (1998). Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology 245, 203-215. Mackenzie, J. S. & Williams, D. T. (2009). The Zoonotic Flaviviruses of Southern, South-Eastern and Eastern Asia, and Australasia: The Potential for Emergent Viruses. Zoonoses and public health.

64 Mandl, C. W. (2005). Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis. Virus Res 111, 161-174. Mandl, C. W., Heinz, F. X., Stockl, E. & Kunz, C. (1989). Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses. Virology 173, 291-301. Mandl, C. W., Holzmann, H., Kunz, C. & Heinz, F. X. (1993). Complete genomic sequence of Powassan virus: evaluation of genetic elements in tick-borne versus mosquito-borne flaviviruses. Virology 194, 173-184. Mandl, C. W., Holzmann, H., Meixner, T., Rauscher, S., Stadler, P. F., Allison, S. L. & Heinz, F. X. (1998). Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus. J Virol 72, 2132-2140. Markoff, L. (2003). 5'- and 3'-noncoding regions in flavivirus RNA. Adv Virus Res 59, 177-228. Matusan, A. E., Pryor, M. J., Davidson, A. D. & Wright, P. J. (2001). Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J Virol 75, 9633-9643. Mavtchoutko, V., Vene, S., Haglund, M., Forsgren, M., Duks, A., Kalnina, V., Horling, J. & Lundkvist, A. (2000). Characterization of tick-borne encephalitis virus from Latvia. J Med Virol 60, 216-222. McGuire, K., Holmes, E. C., Gao, G. F., Reid, H. W. & Gould, E. A. (1998). Tracing the origins of louping ill virus by molecular phylogenetic analysis. J Gen Virol 79 ( Pt 5), 981-988. Medin, C. L., Fitzgerald, K. A. & Rothman, A. L. (2005). Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion. J Virol 79, 11053-11061. Melik, W., Nilsson, A. S. & Johansson, M. (2007). Detection strategies of tick- borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses. Arch Virol 152, 1027- 1034. Metais, J. Y., Navarro, C., Santoni, M. J., Audebert, S. & Borg, J. P. (2005). hScrib interacts with ZO-2 at the cell-cell junctions of epithelial cells. FEBS Lett 579, 3725-3730. Miaczynska, M., Christoforidis, S., Giner, A., Shevchenko, A., Uttenweiler- Joseph, S., Habermann, B., Wilm, M., Parton, R. G. & Zerial, M. (2004). APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment. Cell 116, 445-456. Miller, S., Sparacio, S. & Bartenschlager, R. (2006). Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B. J Biol Chem 281, 8854-8863. Miller, S., Kastner, S., Krijnse-Locker, J., Buhler, S. & Bartenschlager, R. (2007). The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner. J Biol Chem 282, 8873-8882. Mir, M. A. & Panganiban, A. T. (2005). The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding. J Virol 79, 1824-1835.

65 Mir, M. A., Brown, B., Hjelle, B., Duran, W. A. & Panganiban, A. T. (2006). Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle. J Virol 80, 11283-11292. Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313- 319. Munoz-Jordan, J. L., Sanchez-Burgos, G. G., Laurent-Rolle, M. & Garcia- Sastre, A. (2003). Inhibition of interferon signaling by dengue virus. Proc Natl Acad Sci U S A 100, 14333-14338. Muylaert, I. R., Chambers, T. J., Galler, R. & Rice, C. M. (1996). Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse neurovirulence. Virology 222, 159- 168. Nakagawa, S. & Huibregtse, J. M. (2000). Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Mol Cell Biol 20, 8244-8253. Navarro, C., Nola, S., Audebert, S., Santoni, M. J., Arsanto, J. P., Ginestier, C., Marchetto, S., Jacquemier, J., Isnardon, D. & other authors (2005). Junctional recruitment of mammalian Scribble relies on E-cadherin engagement. Oncogene 24, 4330-4339. Neupert, J., Karcher, D. & Bock, R. (2008). Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic Acids Res 36, e124. Nguyen, M. M., Rivera, C. & Griep, A. E. (2005). Localization of PDZ domain containing proteins Discs Large-1 and Scribble in the mouse eye. Mol Vis 11, 1183-1199. Niethammer, M., Kim, E. & Sheng, M. (1996). Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci 16, 2157-2163. Nuttall, P. A. & Labuda, M. (2003). Dynamics of infection in tick vectors and at the tick-host interface. Adv Virus Res 60, 233-272. Okajima, M., Takahashi, M., Higuchi, M., Ohsawa, T., Yoshida, S., Yoshida, Y., Oie, M., Tanaka, Y., Gejyo, F. & other authors (2008). Human T- cell leukemia virus type 1 Tax induces an aberrant clustering of the tumor suppressor Scribble through the PDZ domain-binding motif dependent and independent interaction. Virus Genes 37, 231-240. Osmani, N., Vitale, N., Borg, J. P. & Etienne-Manneville, S. (2006). Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration. Curr Biol 16, 2395-2405. Pattnaik, P. (2006). Kyasanur forest disease: an epidemiological view in India. Rev Med Virol 16, 151-165. Patton, J. T. (2001). Rotavirus RNA replication and gene expression. Novartis Found Symp 238, 64-77; discussion 77-81. Penkert, R. R., DiVittorio, H. M. & Prehoda, K. E. (2004). Internal recognition through PDZ domain plasticity in the Par-6-Pals1 complex. Nat Struct Mol Biol 11, 1122-1127. Perez, M. & de la Torre, J. C. (2003). Characterization of the genomic promoter of the prototypic lymphocytic choriomeningitis virus. J Virol 77, 1184-1194.

66 Pierson, T. C., Diamond, M. S., Ahmed, A. A., Valentine, L. E., Davis, C. W., Samuel, M. A., Hanna, S. L., Puffer, B. A. & Doms, R. W. (2005). An infectious West Nile virus that expresses a GFP reporter gene. Virology 334, 28-40. Pierson, T. C., Sanchez, M. D., Puffer, B. A., Ahmed, A. A., Geiss, B. J., Valentine, L. E., Altamura, L. A., Diamond, M. S. & Doms, R. W. (2006). A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection. Virology 346, 53-65. Piserchio, A., Pellegrini, M., Mehta, S., Blackman, S. M., Garcia, E. P., Marshall, J. & Mierke, D. F. (2002). The PDZ1 domain of SAP90. Characterization of structure and binding. J Biol Chem 277, 6967-6973. Pletnev, A. G. (2001). Infectious cDNA clone of attenuated Langat tick-borne flavivirus (strain E5) and a 3' deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice. Virology 282, 288- 300. Ponting, C. P. (1997). Evidence for PDZ domains in bacteria, yeast, and plants. Protein Sci 6, 464-468. Proutski, V., Gould, E. A. & Holmes, E. C. (1997). Secondary structure of the 3' untranslated region of flaviviruses: similarities and differences. Nucleic Acids Res 25, 1194-1202. Proutski, V., Gritsun, T. S., Gould, E. A. & Holmes, E. C. (1999). Biological consequences of deletions within the 3'-untranslated region of flaviviruses may be due to rearrangements of RNA secondary structure. Virus research 64, 107-123. Randolph, S. E. (2004). Evidence that climate change has caused 'emergence' of tick-borne diseases in Europe? Int J Med Microbiol 293 Suppl 37, 5-15. Randolph, S. E., Miklisova, D., Lysy, J., Rogers, D. J. & Labuda, M. (1999). Incidence from coincidence: patterns of tick infestations on rodents facilitate transmission of tick-borne encephalitis virus. Parasitology 118 ( Pt 2), 177-186. Randolph, S. E., Asokliene, L., Avsic-Zupanc, T., Bormane, A., Burri, C., Gern, L., Golovljova, I., Hubalek, Z., Knap, N. & other authors (2008). Variable spikes in tick-borne encephalitis incidence in 2006 independent of variable tick abundance but related to weather. Parasit Vectors 1, 44. Rauscher, S., Flamm, C., Mandl, C. W., Heinz, F. X. & Stadler, P. F. (1997). Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities. RNA (New York, NY 3, 779-791. Rawlinson, S. M., Pryor, M. J., Wright, P. J. & Jans, D. A. (2009). CRM1- mediated nuclear export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and virus production. J Biol Chem 284, 15589- 15597. Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C. & Harrison, S. C. (1995). The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375, 291-298. Rice, C. M., Lenches, E. M., Eddy, S. R., Shin, S. J., Sheets, R. L. & Strauss, J. H. (1985). Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 229, 726-733.

67 Roosendaal, J., Westaway, E. G., Khromykh, A. & Mackenzie, J. M. (2006). Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein. J Virol 80, 4623-4632. Rossi, S. L., Zhao, Q., O'Donnell, V. K. & Mason, P. W. (2005). Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Virology 331, 457-470. Rousset, R., Fabre, S., Desbois, C., Bantignies, F. & Jalinot, P. (1998). The C- terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular proteins. Oncogene 16, 643-654. Ruzek, D., Dobler, G. & Donoso Mantke, O. (2010). Tick-borne encephalitis: pathogenesis and clinical implications. Travel Med Infect Dis 8, 223-232. Saito, H., Santoni, M. J., Arsanto, J. P., Jaulin-Bastard, F., Le Bivic, A., Marchetto, S., Audebert, S., Isnardon, D., Adelaide, J. & other authors (2001). Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells. J Biol Chem 276, 32051-32055. Santoni, M. J., Pontarotti, P., Birnbaum, D. & Borg, J. P. (2002). The LAP family: a phylogenetic point of view. Trends Genet 18, 494-497. Singh, D. & Lampe, P. D. (2003). Identification of connexin-43 interacting proteins. Cell Commun Adhes 10, 215-220. Singh, D., Solan, J. L., Taffet, S. M., Javier, R. & Lampe, P. D. (2005). Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage- specific manner. J Biol Chem 280, 30416-30421. Skelton, N. J., Koehler, M. F., Zobel, K., Wong, W. L., Yeh, S., Pisabarro, M. T., Yin, J. P., Lasky, L. A. & Sidhu, S. S. (2003). Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain. J Biol Chem 278, 7645-7654. Snyder, E. M., Philpot, B. D., Huber, K. M., Dong, X., Fallon, J. R. & Bear, M. F. (2001). Internalization of ionotropic glutamate receptors in response to mGluR activation. Nat Neurosci 4, 1079-1085. Soubies, S. M., Volmer, C., Guerin, J. L. & Volmer, R. (2010). Truncation of the NS1 protein converts a low pathogenic avian influenza virus into a strong interferon inducer in duck cells. Avian Dis 54, 527-531. Soubies, S. M., Volmer, C., Croville, G., Loupias, J., Peralta, B., Costes, P., Lacroux, C., Guerin, J. L. & Volmer, R. Species-specific Contribution of the Four C-terminal Amino Acids of Influenza A NS1 Protein to Virulence. Journal of virology. Stevenson, B. R., Siliciano, J. D., Mooseker, M. S. & Goodenough, D. A. (1986). Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103, 755-766. Stiffler, M. A., Chen, J. R., Grantcharova, V. P., Lei, Y., Fuchs, D., Allen, J. E., Zaslavskaia, L. A. & MacBeath, G. (2007). PDZ domain binding selectivity is optimized across the mouse proteome. Science 317, 364-369. Strack, S., Robison, A. J., Bass, M. A. & Colbran, R. J. (2000). Association of calcium/calmodulin-dependent kinase II with developmentally regulated splice variants of the postsynaptic density protein densin-180. J Biol Chem 275, 25061-25064.

68 Tan, B. H., Fu, J., Sugrue, R. J., Yap, E. H., Chan, Y. C. & Tan, Y. H. (1996). Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. Virology 216, 317- 325. Tapia, R., Huerta, M., Islas, S., Avila-Flores, A., Lopez-Bayghen, E., Weiske, J., Huber, O. & Gonzalez-Mariscal, L. (2009). Zona occludens-2 inhibits cyclin D1 expression and cell proliferation and exhibits changes in localization along the cell cycle. Mol Biol Cell 20, 1102-1117. Thiel, H.-J., Collett, M. S., Gould, E. A. H., F.X.; , Houghton, M., Meyers, G., Purcell, R. H. & Rice, C. M. (2005). In Virus Taxonomy. VIIIth Report of the International Committee on Taxonomy of Viruses; Fauquet, C.M.; Mayo, M.A.; Maniloff, J.; Desselberger, U.; Ball, L.A.;. 979-998. Thurner, C., Witwer, C., Hofacker, I. L. & Stadler, P. F. (2004). Conserved RNA secondary structures in Flaviviridae genomes. J Gen Virol 85, 1113- 1124. Tochio, H., Hung, F., Li, M., Bredt, D. S. & Zhang, M. (2000). Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density- 95. J Mol Biol 295, 225-237. Tonikian, R., Zhang, Y., Sazinsky, S. L., Currell, B., Yeh, J. H., Reva, B., Held, H. A., Appleton, B. A., Evangelista, M. & other authors (2008). A specificity map for the PDZ domain family. PLoS Biol 6, e239. Ulbert, S. (2011). West Nile Virus: The Complex Biology of an Emerging Pathogen. Intervirology. Utepbergenov, D. I., Fanning, A. S. & Anderson, J. M. (2006). Dimerization of the scaffolding protein ZO-1 through the second PDZ domain. J Biol Chem 281, 24671-24677. Waladde, S. M., Young, A. S., Ochieng, S. A., Mwaura, S. N. & Mwakima, F. N. (1993). Transmission of Theileria parva to cattle by Rhipicephalus appendiculatus adults fed as nymphae in vitro on infected blood through an artificial membrane. Parasitology 107 ( Pt 3), 249-256. Waladde, S. M., Young, A. S., Mwaura, S. N., Njihia, G. N. & Mwakima, F. N. (1995). Optimization of the in vitro feeding of Rhipicephalus appendiculatus nymphae for the transmission of Theileria parva. Parasitology 111 ( Pt 4), 463-468. Wallner, G., Mandl, C. W., Kunz, C. & Heinz, F. X. (1995). The flavivirus 3'- noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus. Virology 213, 169-178. Varnavski, A. N., Young, P. R. & Khromykh, A. A. (2000). Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors. J Virol 74, 4394-4403. Varsano, T., Dong, M. Q., Niesman, I., Gacula, H., Lou, X., Ma, T., Testa, J. R., Yates, J. R., 3rd & Farquhar, M. G. (2006). GIPC is recruited by APPL to peripheral TrkA endosomes and regulates TrkA trafficking and signaling. Mol Cell Biol 26, 8942-8952. Weissenbock, H., Kolodziejek, J., Url, A., Lussy, H., Rebel-Bauder, B. & Nowotny, N. (2002). Emergence of Usutu virus, an African mosquito- borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg Infect Dis 8, 652-656.

69 Wengler, G. & Castle, E. (1986). Analysis of structural properties which possibly are characteristic for the 3'-terminal sequence of the genome RNA of flaviviruses. J Gen Virol 67 ( Pt 6), 1183-1188. Werme, K., Wigerius, M. & Johansson, M. (2008). Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon- stimulated JAK-STAT signalling. Cell Microbiol 10, 696-712. Westaway, E. G., Mackenzie, J. M. & Khromykh, A. A. (2002). Replication and gene function in Kunjin virus. Curr Top Microbiol Immunol 267, 323-351. Westaway, E. G., Mackenzie, J. M. & Khromykh, A. A. (2003). Kunjin RNA replication and applications of Kunjin replicons. Adv Virus Res 59, 99-140. Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K. & Khromykh, A. A. (1997). Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus- induced membrane structures. J Virol 71, 6650-6661. Wigerius, M., Melik, W., Elvang, A. & Johansson, M. (2010). Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5. Mol Cell Neurosci 44, 260-271. Winkler, G., Maxwell, S. E., Ruemmler, C. & Stollar, V. (1989). Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 171, 302-305. Winkler, G., Randolph, V. B., Cleaves, G. R., Ryan, T. E. & Stollar, V. (1988). Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162, 187-196. Wittchen, E. S., Haskins, J. & Stevenson, B. R. (1999). Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem 274, 35179- 35185. Xu, J., Wang, F., Van Keymeulen, A., Herzmark, P., Straight, A., Kelly, K., Takuwa, Y., Sugimoto, N., Mitchison, T. & other authors (2003). Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114, 201-214. Yamshchikov, V., Mishin, V. & Cominelli, F. (2001a). A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli. Virology 281, 272-280. Yamshchikov, V. F., Wengler, G., Perelygin, A. A., Brinton, M. A. & Compans, R. W. (2001b). An infectious clone of the West Nile flavivirus. Virology 281, 294-304. Yi, Z., Petralia, R. S., Fu, Z., Swanwick, C. C., Wang, Y. X., Prybylowski, K., Sans, N., Vicini, S. & Wenthold, R. J. (2007). The role of the PDZ protein GIPC in regulating NMDA receptor trafficking. J Neurosci 27, 11663- 11675. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V. K., Murthy, K. H. & Padmanabhan, R. (2005). Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase. J Biol Chem 280, 27412-27419.

70 Yu, I. M., Holdaway, H. A., Chipman, P. R., Kuhn, R. J., Rossmann, M. G. & Chen, J. (2009). Association of the pr peptides with dengue virus at acidic pH blocks membrane fusion. J Virol 83, 12101-12107. Yu, I. M., Zhang, W., Holdaway, H. A., Li, L., Kostyuchenko, V. A., Chipman, P. R., Kuhn, R. J., Rossmann, M. G. & Chen, J. (2008). Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834-1837. Zahraoui, A., Louvard, D. & Galli, T. (2000). Tight junction, a platform for trafficking and signaling protein complexes. J Cell Biol 151, F31-36. Zaki, A. M. (1997). Isolation of a flavivirus related to the tick-borne encephalitis complex from human cases in Saudi Arabia. Trans R Soc Trop Med Hyg 91, 179-181. Zhan, L., Rosenberg, A., Bergami, K. C., Yu, M., Xuan, Z., Jaffe, A. B., Allred, C. & Muthuswamy, S. K. (2008). Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865-878. Zhang, H., Webb, D. J., Asmussen, H., Niu, S. & Horwitz, A. F. (2005). A GIT1/PIX/Rac/PAK signaling module regulates spine morphogenesis and synapse formation through MLC. J Neurosci 25, 3379-3388. Zhang, Y., Appleton, B. A., Wu, P., Wiesmann, C. & Sidhu, S. S. (2007). Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain. Protein Sci 16, 1738-1750.

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