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Molecular aspects of Dengue virus replication

for correspondence 3-V Biosciences, c/o Institute of Biochemistry, Schafmattstrasse 18, ETH Hoenggerberg HPME 17, CH-8093 Zurich, Switzerland Tel.: +41 446 336 019; Fax: +41 446 331 091; [email protected]

Dengue virus (DENV) a mosquito transmitted pathogen is the causative agent of Dengue fever, the most important arboviral disease of humans, which affects an estimated 50100 million people annually. Despite the high morbidity and mortality associated with DENV infections, an effective DENV vaccine and antiviral therapies are still missing. An improved understanding of the molecular mechanisms underlying the different steps of the DENV replication cycle, for example, genome replication and virus maturation, could help to develop such substances. Over the past several years, many important findings have been published with respect to a better understanding of DENV replication. In this review we will highlight recent insights into the molecular mechanisms of the viral replication cycle.

Keywords: cyclization sequence, Dengue virus, double-membrane vesicles, membrane rearrangements, nonstructural proteins, nuclear localization, polyprotein, replication, replication complex, untranslated region
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The mosquito-transmitted Dengue virus (DENV) is the causative agent of Dengue fever (DF), the most prevalent arthropod-borne viral disease in humans worldwide [1]. DF is characterized by high fever, chills, body aches and skin rash and ranges from mild, influenza-like symptoms to severe forms, which are Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS). During the last 50 years, the prevalence of DF, DHF and DSS has increased exponentially, with approximately two-fifths of the worlds population at risk, and 50100 million cases reported annually, particularly in Southeast Asia, the Western Pacific and the Americas [101]. Decreases in mosquito control efforts during the late 20th century, as well as societal factors (e.g., increased transportation and dense urbanization) and global warming, have contributed to the tremendous spread of the virus and its vector. However, despite the high morbidity and mortality associated with DENV infections, neither a protective vaccine nor specific antiviral therapies are available. The four serotypes of DENV identified so far (DENV 14) belong to the genus flavivirus within the Flaviviridae family. The flavivirus genus consists of more than 70 viruses, many of which are arthropod-borne human pathogens causing a variety of diseases, including fevers, encephalitis and hemorrhagic fevers. Among them are the widespread yellow fever virus, the West Nile virus and the Japanese encephalitis virus [2]. Flaviviruses are small, enveloped viruses (a50 nm in diameter) containing a singlestranded genomic RNA of positive polarity ([+]RNA) approximately 11 kb in length [3]. Since, in the following, we will focus exclusively on recent advances in our understanding of

DENV molecular virology, the interested reader is referred to further references for more detailed and up-to-date reviews of the pathogenesis, transmission and evolution of this medically important pathogenic agent [48].
The viral replication cycle

The DENV nucleocapsid, which consists of the genomic (+)RNA and the basic capsid (C) protein, is surrounded by a lipid bilayer into which the viral membrane protein (M) and the envelope glycoprotein (E) are embedded (Figure 1). The E protein is the viral determinant mediating the binding of the virus to specific receptors found on the surface of DENV-permissive host cells, including dendritic cells (DCs), B cells, T cells, monocytes/hepatocytes and neuronal cells (for further details, see [9]). Several receptors for DENV have been identified so far, among them heparan sulfate, CD14, 37/67-kDa highaffinity laminin receptor, GRP78/BiP, and the well-characterized DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) [1016]. Upon binding, the virus is internalized via receptor-mediated endocytosis (Figure 1A). The low pH of the endosomal pathway induces an irreversible trimerization of the E protein, which thereupon mediates the fusion of the virion envelope with cellular membranes. Following uncoating of the nucleocapsid, the viral genome is released into the cytoplasm of the infected cells, where it is translated at the rough endoplasmic reticulum (ER), giving rise to a polyprotein of approximately 3400 amino acids. This polyprotein is co- and post-translationally cleaved by a combination of cellular proteases and the viral nonstructural (NS)2B-3 protease into three structural and seven NS proteins.
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Figure 1. The Dengue virus replication cycle and structure of the viral particle.

1. Binding

2. Receptor-mediated endocytosis

Endosome 3. RNA release

Virus-induced membranes

4. Translation and replication Nucleus Golgi Rough ER

5. Maturation 6. Virus release

(+) Genomic RNA Translation and processing

Viral proteins

Intracellular EprM heterodimer

Extracellular

() RF (+) Formation of replication complex (membrane bound) () (+) RI (+) E-trimer (+) (+) (+) Encapsidation E protein M protein Endosomal (low pH) C protein pr part of prM protein (+) RNA E-dimer

Progeny RNA

(A) Overview of the different steps of the DENV replication cycle, from binding to the host cell up to virus release. (B) Close-up of the RNA replication strategy of DENV. (C) Schematic of the DENV particle. Depicted are the different states of the envelope proteins as they mature during intracellular assembly, egress (extracellular) and infection (endosomal). C: Capsid protein; DENV: Dengue virus; E: Envelope protein; ER: Endoplasmic reticulum; M: Membrane protein; RF: Replicative form; RI: Replicative intermediate.

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Following translation and processing, a protein complex is assembled that is composed of the NS5 RNA-dependent RNA-polymerase (RdRp), accessory NS proteins, viral (+)RNA and, probably, host cell factors. Within this so-called replication complex (RC), which is associated with virusinduced intracellular membrane structures, viral RNA replication takes place. Replication starts with the synthesis of genome-length (-)RNA, which then serves as a template for the production of (+)RNA progeny (Figure 1B). During completion of transcription, the newly synthesized (-)RNA undergoes base pairing with the (+)RNA template, leading to the formation of an extended RNA duplex called replicative form (RF). It functions as a template for the generation of new (+)RNA via a replicative intermediate (RI) in an asymmetric and semiconservative manner. The newly produced (+)RNA strand is released from the RI and either attaches to ribosomes to initiate a new translation cycle (dashed line in Figure 1B) or assembles into virions, which most likely form at the ER. Virion maturation appears to require a passage through the Golgi compartment where glycosylation of the viral membrane proteins, as well as proteolytic cleavage of the precursor of mature M (prM) protein into the components pr and M by the Golgi-resident protease Furin or a related enzyme, takes place. The uncleaved prM prevents E from undergoing an acid-catalyzed transition into the fusogenic form during the transit through the secretory pathway by forming a prME heterodimeric complex. Following cleavage, the pr-fragment is released, E forms homodimeric complexes and the mature virions exit the infected cell.
Genome organization & protein functions

The genome of DENV consists of a singlestranded (+)RNA of approximately 11 kb in size, which contains a 5 untranslated region (UTR; a100 nucleotides), a single open reading frame encoding for the viral polyprotein and a 3 UTR approximately 400 nucleotides in length (Figure 2A). Attached to the 5 end of the viral genome is a type I 7-methyl guanosine cap structure, but unlike cellular mRNAs the DENVgenome is not 3 polyadenylated. The 5 and 3 UTRs are not well conserved between different flaviviruses, but in all cases secondary structures have been identified within these regions. An important function of the 5 UTR probably resides in the complementary region of the negative strand, which serves as the initiation site for the synthesis of (+)RNA during replication. A
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stem-loop (SL) structure near the 5 terminus of the genome plays an important role for RNA replication: deletions within this structure of DENV4 were lethal for replication [17]. Furthermore, the 5 UTR influences the translation of the RNA genome [18,19]. The 3 UTR of DENV encompasses three regions: a variable region (VR) immediately 3 of the stop codon of the long open reading frame, a core region downstream of the VR and a 3-terminal region. The latter contains a very conserved 3 terminal SL, which is absolutely required for replication [20], and a cyclization sequence (CS) just upstream of the 3 SL. The CS is complementary to a sequence at the 5-end and mediates together with further sequences (see below) 5-3 long-range RNARNA interactions, which have been proposed to be necessary for RNA replication of flaviviruses [21,22]. Since DENV genomes with mutations in the VR and the core region of the 3 UTR were shown to be viable in cell culture, their functional importance remains to be established [23,24]. However, even though it has not been possible so far to establish a clear correlation between a particular DENV-serotype or genotype and the severity of disease outcome [4], full-length sequencing experiments of different DENV genotypes let us assume that sequence differences in the 5 and 3 UTRs as well as nucleotide differences in the coding region of the genome might play a role in modulating the severity of DENV infection [25]. The DENV open reading frame encodes for a polyprotein, which is co- and post-translationally processed into three structural proteins (C, prM and E) and seven NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5; Figure 2B). Processing of the polyprotein into the individual components is carried out by a combination of different host cell enzymes and the cytoplasmic viral NS2B-3 protease complex (Figure 2B) [2629]. The highly basic C protein forms the viral nucleocapsid together with the genomic RNA. The immature prM prevents the activation of the fusion activity of the E protein during virus maturation, and E is important for virus binding and fusion of viral and cellular membranes during receptor-mediated endocytosis (see above). Most, if not all, NS proteins are involved in viral RNA replication, which occurs in close association with cellular membranes within the viral RCs (see below). NS5 is the RdRp and carries in addition an N-terminal methyltransferase domain important for the formation of the RNA
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Figure 2. Genome structure and putative topology and functions of the Dengue virus proteins.

5 UTR (~ 100 bases) Structural region 5 SL

3 UTR Open reading frame Nonstructural region 3 SL Single-stranded (+) RNA C prM E 1 2A 2B 3 4A 4B 5 VR Binding, fusion Replication, pathogenesis Protease cofactor 3 CS
3 UAR

(~ 400 bases)

CAP

5 CS Capsid

Prevention of premature fusion

Methyltransferase, guanylytransferase, RdRp IFN resistance Protease, IFN Viral protease (NS2B-3) helicase, resistance ? Unknown host cell protease RNA triphosphatase IFN resistance, membrane induction X Host cell signalase Furin Cytoplasm

C 2A M pr X E 1 ?

2K

2B

4A

4B

ER lumen

(A) Organization of the DENV genome and structures of RNA elements. The regions encoding for structural proteins (purple) and nonstructural proteins (green), as well as the 5 and 3 UTRs containing SL structures, are depicted. Complementary sequences in the 5 and 3 UTRs are highlighted; positions of the VR in the 3 UTR, as well as 5 and 3 UARs are marked. Functions (putative) of the individual proteins are given. (B) Putative topology of the DENV structural and nonstructural proteins in the membrane of the ER. Transmembrane regions, as well as the cellular and viral proteases required for polyprotein processing, are indicated. C: Capsid protein; CS: Cyclization sequence; DENV: Dengue virus; E: Envelope protein; ER: Endoplasmic reticulum; IFN: Interferon; M: Membrane protein; RdRp: RNA-dependent RNA-polymerase; SL: Stem loop; UAR: Upstream AUG region; UTR: Untranslated region; VR: Variable region.

cap structure [3033]. NS3 acts as the viral serine protease, which requires the cofactor NS2B for full activity. Furthermore, NS3 contains a RNA helicase and nucleotide triphosphatase activity important for replication of the viral RNA [3439]. The role of the glycoprotein NS1 in viral replication is unknown, but it is assumed that NS1 acts at an early stage in viral RNA replication [4042]. Furthermore, it may also be important for the pathogenesis of DHF [43]. Only little is known about the functions of the small hydrophobic proteins NS2A, NS4A and NS4B. It has been suggested that they may serve to anchor the viral replicase to cellular membranes [44]. In addition, it has been shown that they contribute to the inhibition of the IFN-D/E response of the infected host cell [45,46]. Recent results indicate that NS4A plays a role in the induction of
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membrane alterations in infected cells, which may serve as a scaffold to anchor the viral RC (see below) [47,48]. NS4B seems to be involved in viral replication, for which an interaction with NS3 appears to be required [49,50].
Viral replication
Intracellular sites of viral replication

All (+)RNA viruses investigated so far replicate their RNA in close association with virusinduced intracellular membranous structures, which may provide a scaffold for anchoring the viral RC. The biological function of such structures is not completely understood, but it is assumed that they help to increase the local concentration of components required for replication, confine RNA replication to a distinct subcellular site, and tether viral RNA during
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Molecular aspects of Dengue virus replication REVIEW

unwinding. In addition, the induced membranes may aid in preventing the activation of dsRNAinduced host defense mechanisms, such as interferon-induced pathways in humans. Furthermore, the encryption of viral dsRNA in virusinduced membrane alterations could explain why DENV is able to efficiently replicate in its mosquito vector, despite the existence of a dsRNA-triggered antiviral RNA interference pathway in this organism [51]. The best-characterized flavivirus in terms of induction of intracellular membrane rearrangements is Kunjin virus (KUNV), the Australian variant of West Nile virus (for a review, see [52]). In KUNV-infected cells, clusters of doublemembrane vesicles each vesicle approximately 50150 nm in diameter have been described. These structures are called vesicle packets (VP) or smooth membrane structures (SMSs). Furthermore, so-called convoluted membranes (CMs) and paracrystalline structures occur, which were found adjacent to the VP/SMS. Since the components of the KUNV-RC were found by immunolabeling studies to localize to VP/SMS, it is assumed that viral replication occurs in this compartment. However, the viral serine-protease colocalizes with the CM, indicating that they are the sites of viral polyprotein processing. The membrane reorganization induced by KUNV might therefore give rise to adjacent, but distinct, subcellular structures where different steps of the viral replication cycle are carried out [53].

Clusters of double membrane vesicles comparable to the VP/SMS of KUNV have also been observed for DENV (Figure 3) and other flaviviruses. The detection of viral RNA and several DENV NS proteins within these structures by electron and immunofluorescence microscopy suggests that they represent the sites of DENV RNA replication [S Miller, R Bartenschlager, Unpublished Data] [47,49,50,54]. Whether there are different compartments for polyprotein processing and replication in DENV-infected cells, as was described for KUNV, remains to be clarified.
Cyclization of the viral genome during replication

A 25-nucleotide-long region (CS1) just upstream of the 3 SL within the 3 UTR was found to base pair with a complementary sequence (5 CS) in the 5 coding region of the capsid gene (Figure 2A) [55,56]. Such 5-3 long-range RNARNA interactions across a region of approximately 10 kb have been proposed to be necessary, but not sufficient, for RNA replication of DENV. In addition, further sequences, named 5 and 3 UARs (upstream AUG region), were found to be required for successful RNARNA complex formation [21]. Recent results indicate that RNARNA interactions between 5- and 3-end sequences of the viral genome enhance DENV RNA synthesis only in the presence of an intact 5 SL [57]. In these excellent studies it was found that the 5 SL functions as a promoter for the viral RdRp. Using atomic force microscopy, Filomatori et al.

Figure 3. Ultrastructural analysis of Dengue virus-infected cells.

Virus-induced structures

Virus particles 200 nm 200 nm

(A) Resin-embedded sections of Dengue virus-infected cells reveal double membrane vesicles and membranous structures that contain accumulations of viral particles. It is unclear whether these are naked or (more likely) enveloped particles. (B) Immunogold labeling of a thawed cryosection prepared from Dengue virus-infected cells, with antibodies raised against the viral nonstructural protein NS3 and 10 nm protein-A gold.

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demonstrated that DENV NS5 the RdRp of the virus was bound to the viral RNA only when the 5 SL was present. A model was developed in which interactions between the 5- and the 3ends of the viral RNA position the promoter adjacent to the 3-end of the genome (Figure 4). The RdRp binds to the 5 SL and is transferred to the transcription initiation site at the 3-end of the genome, which is made possible by long-range RNARNA interactions. These findings provide
Figure 4. Proposed model for Dengue virus minus-strand RNA-synthesis.
3 SL 5 SL

an explanation for the strict requirement of genome cyclization during DENV replication. Genome cyclization may be an important control mechanism to ensure that only full-length templates are amplified, to increase RNA stability and to control the levels of minus-strand RNA synthesis. Furthermore, cyclization may be important for the coordination of translation and RNA synthesis by overlapping signals at the 5- and 3ends of the genome and it could localize the viral RdRp or accessory proteins of the RC at the appropriate start site.
Host cell proteins involved in viral replication

Cyclization of genome 53 UAR 5 3

53 CS

Binding of NS5 ( = RdRp)

5 3

Up to now, only very few host cell factors contributing to the flavivirus replication cycle have been identified. One example is the eukaryotic translation elongation factor eF1-D, which interacts with the 3 UTR of West Nile virus [58]. Further host cell proteins possibly involved in the viral replication cycle of flaviviruses are MOV34, which interacts with the 3 UTR of Japanese encephalitis virus [33], tubulin and the tumor susceptibility protein 101, which interact with Japanese encephalitis virus NS3 [59], and the mosquito La protein, which binds to the 3 UTR of (+) and (-)RNA of DENV [60]. Although these proteins were shown to interact with viral proteins or RNA, their role in the viral replication cycle is not understood thus far. Taking advantage of large-scale high-throughput screens, Chu and Yang recently identified the c-Src protein kinase as an important host cell factor required for DENV assembly [61]. This example illustrates the power of such high-content screening approaches and we can assume that more cellular factors and pathways required for flaviviral replication will be discovered with these methods.
Replication & the cellular nucleus

Transcription

5 5

3 (+) RNA 5 () RNA

53 UAR and 53 CS hybridization mediates Dengue virus genome circularization. The RdRp of Dengue virus binds to the 5-end of the genome and is transferred to the site of RNA synthesis initiation at the 3-end by long range RNARNA interactions. CS: Cyclization sequence; RdRp: RNA-dependent RNA-polymerase; SL: Stem loop; UAR: Upstream AUG region. Adapted from [57].

It is textbook knowledge that the replication of flaviviruses takes place in the cytoplasm of virusinfected cells. However, in several studies some flavivirus structural and NS proteins have been found to localize to the nucleus. One prominent example is DENV NS5, which has been detected in the nucleus of cells already at very early time points after infection (Figures 5AC) [62]. This result is perplexing because RdRp activity is required in the cytoplasmic RC. Nuclear translocation appears to depend on the phosphorylation status of NS5 and is modulated by interaction with NS3. Nuclear hyperphosphorylated NS5 was found to be unable to bind to NS3, whereas hypophosphorylated NS5 is cytoplasmic and
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Figure 5. Nuclear localization of Dengue virus proteins.

NS5

DAPI

Merge

Capsid

DAPI

Merge

(AC) In Dengue virus-infected cells, the great majority of NS5 is detectable in the nucleus by immunoflorescence microscopy after labeling with a NS5-specific antibody. (DF) Nuclear dot-like structures were detectable after the transfection of cells with a full-length Dengue virus genomic RNA, which encodes for a capsid protein, fused to the green fluorescent protein. In each case (NS5 and capsid), staining of the nucleus with DAPI is shown in the middle; merged pictures are given on the right. DAPI: 4,6-diamidino-2-phenylindole dihydrochloride.

associated with NS3. Likewise, NS5 of the related flavivirus yellow fever virus was reported to localize mainly to the nucleus, where it may exert some function not directly required for RNA replication [63]. The nuclear localization of proteins with a molecular mass higher than 45 kDa is an active process that requires the recognition of a nuclear localization signal (NLS) within the corresponding protein by the nuclear transport machinery [64]. Previous studies have shown that DENV NS5 contains two potential NLS in its central region that are recognized by the importin D/E and importin E1 nuclear transport proteins, respectively [65]. Both NLS are highly conserved among various members of the flavivirus genus. Intriguingly, DENV NS5 also possesses the ability to be exported from the nucleus, for which the nuclear export receptor CRM1 (exportin 1) is required [66]. The role of nuclear NS5 for DENV replication is unknown, but recent experiments allow us to assume that nuclear NS5 suppresses IL-8 production, and thus protects against the antiviral activity of this cytokine [67]. As well as NS5, flavivirus C protein also localizes to the nucleus, where it is concentrated in small dot-like structures, which may represent
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nucleoli (Figures 5DF) [6871]. Similar to NS5, C also contains a bipartite NLS that is responsible for nuclear transport. The function of C in the nucle(ol)us remains to be clarified. However, it is possible that the C protein is not only the building block of nucleocapsids, but is also involved in regulating the DENV replication cycle, for example, by modulating host cell transcription profiles [72]. Apart from NS5 and core, a recent study suggests that NS3, and presumably up to 20% of active replication complexes, resides in the nucleus of DENV-infected cells [73]. Although this is an interesting observation, nuclear localization of NS3 could not be confirmed by other groups [49,53] [Miller S, Bartenschlager R, Unpublished Data]. The reason for this discrepancy is not clear, but it may be due to different experimental conditions. Further studies will be required to clarify the possibility of nuclear DENV RNA replication. Further studies are required to investigate the role of nuclear localized flavivirus proteins. On the one hand, a better understanding could lead to the development of new recombinant vaccines based on viral proteins that are deficient in nuclear trafficking and thus lead to an attenuation of the virus. On the other hand, nuclear localized viral proteins could be an ideal tool to
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unravel the cell biological details of nuclear processes and functions. Finally, nuclear localized DENV proteins may be important determinants of pathogenesis, for example, by altering host cell transcriptome profiles, as suggested for the C protein, or by subverting innate antiviral defense mechanisms, as discussed for NS5.
Conclusions & future perspective

In recent years, many new important findings have contributed to a better understanding of several steps of the DENV replication cycle. However, much work remains to be done, in particular with respect to DENVhost cell interaction and the pathogenesis of DF, DHF and DSS. Advanced methods, such as genomewide RNA interference studies, as well as modern microscopy techniques (e.g., tomography or live cell imaging), will help to unravel the biogenesis of the viral RC, to identify host cell factors involved in the various steps of the DENV replication cycle and to understand the dynamics of DENVhost cell interaction. In addition, 3D reconstruction of virus-induced membrane compartments should help to
Executive summary
Postgenome: understanding Dengue virus replication

decipher their interconnection with the subcellular environment, the topology of the membranous vesicles and eventually define compartments where distinct steps of the DENV replication cycle occurs (RNA translation, replication and assembly). Finally, more studies are required to characterize the role of nuclear localized DENV proteins for viral replication and their possible contribution to pathogenesis. An improved understanding of the molecular mechanisms of the DENV replication cycle may help to develop an effective vaccine, as well as therapeutic strategies to treat infections with this insidious pathogen.
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

The mosquito-transmitted Dengue virus (DENV) is the causative agent of Dengue fever, the most prevalent arthropod-borne viral disease in humans worldwide. Neither a protective vaccine nor specific antiviral therapies are currently available. DENV belongs to the flavivirus genus within the Flaviviridae family and contains an RNA genome of positive polarity, which is infectious per se. Even though much is known about the DENV replication cycle, further studies are required to completely unravel this complex process and to better understand DENVhost cell interaction. Genome organization & protein functions The (+)RNA genome of DENV contains a 5 untranslated region (UTR), a single open reading frame encoding a viral polyprotein, a 3 UTR, and a type I 7-methyl guanosine cap structure at the 5-end. The viral polyprotein is cleaved by a combination of cellular proteases and a viral protease into three structural and seven nonstructural proteins. Viral replication takes place in cytoplasmic, virus-induced intracellular membrane compartments that serve as a scaffold for anchoring the viral replication complex (RC). The viral RC consists of viral RNA, viral proteins and yet undefined host cell factors. Cyclization of the DENV genome is important for RNA replication, since it localizes the NS5 RNA-dependent RNA polymerase (RdRp) which binds to the 5 UTR to the appropriate transcription start site at the 3 end of the genome. Even if the replication of DENV takes place in the cytoplasm of infected cells, several viral proteins localize in the nucleus. The role of these nuclear proteins for the viral replication cycle remains to be clarified. Conclusion & future perspective In recent years, many new important findings have contributed to a better understanding of the DENV replication cycle. Nevertheless, much work remains to be done to completely unravel the complex replication strategy of this virus. Advanced methods such as inhibitor and genome-wide RNA interference studies, as well as modern microscopy techniques will help to achieve this goal and will contribute to the development of an effective vaccine and selective antiviral therapies.

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Website
101. World Health Organization. Dengue and

dengue haemorrhagic fever. www.who.int/mediacentre/factsheets/fs117/ en/

Affiliations
Ralf Bartenschlager Department of Molecular Virology, Im Neuenheimer Feld 345, University of Heidelberg, D-69120 Heidelberg, Germany Tel.: +49 622 156 4569; Fax: +49 622 156 4570; [email protected] Sven Miller 3-V Biosciences, c/o Institute of Biochemistry, Schafmattstrasse 18, ETH Hoenggerberg HPME 17, CH-8093 Zurich, Switzerland Tel.: +41 446 336 019; Fax: +41 446 331 091; [email protected]

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