Future Microbiol 2008 155-65

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10.2217/17460913.3.2.155 2008 Future Medicine Ltd ISSN 1746-0913

REVIEW

Future Microbiol. (2008) 3(2), 155165 155

Molecular aspects of Dengue virus replication

Ralf Bartenschlager &

Sven Miller

Author for correspondence3-V Biosciences, c/o Instituteof Biochemistry,Schafmattstrasse18, ETHHoenggerbergHPME 17,CH-8093 Zurich,SwitzerlandTel.: +41 446 336 019;Fax: +41 446 331 091;[email protected]

Keywords: cyclizationsequence, Dengue virus,double-membrane vesicles,membrane rearrangements,nonstructural proteins,nuclear localization,polyprotein, replication,replication complex,untranslated region

Dengue virus (DENV) a mosquito transmitted pa thogen is the causative agent of Dengue

fever, the most important arboviral disease of humans, which a ffects an estimated

50100 million people annually. Despite the high morbidity and mortality assoc iated 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 replica tion 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 o f DENV replication. In this review we

will highlight recent insights into the molecular mechanisms of the viral replication cycle.

The mosquito-transmitted Dengue virus

(DENV) is the causative agent of Dengue fever(DF), the most prevalent arthropod-borne viraldisease in humans worldwide[1]. DF is charac-terized by high fever, chills, body aches and skinrash and ranges from mild, influenza-likesymptoms to severe forms, which are Denguehemorrhagic fever (DHF) and Dengue shocksyndrome (DSS). During the last 50years, theprevalence of DF, DHF and DSS has increasedexponentially, with approximately two-fifths ofthe worlds population at risk, and50100million cases reported annually, particu-

larly in Southeast Asia, the Western Pacific andthe Americas[101]. Decreases in mosquito controlefforts during the late 20th century, as well associetal factors (e.g., increased transportationand dense urbanization) and global warming,have contributed to the tremendous spread ofthe virus and its vector. However, despite thehigh morbidity and mortality associated withDENV infections, neither a protective vaccinenor 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 flavivirusgenus consists of more than 70viruses, many ofwhich are arthropod-borne human pathogenscausing a variety of diseases, including fevers,encephalitis and hemorrhagic fevers. Amongthem are the widespread yellow fever virus, theWest Nile virus and the Japanese encephalitisvirus[2]. Flaviviruses are small, enveloped viruses(a50nm in diameter) containing a single-stranded genomic RNA of positive polarity([+]RNA) approximately 11kb in length [3].Since, in the following, we will focus exclusivelyon recent advances in our understanding of

DENV molecular virology, the interested reader

is referred to further references for more detailedand up-to-date reviews of the pathogenesis,transmission and evolution of this medicallyimportant pathogenic agent [48].

The viral replication cycle

The DENV nucleocapsid, which consists of thegenomic (+)RNA and the basic capsid (C) pro-tein, is surrounded by a lipid bilayer into whichthe viral membrane protein (M) and the enve-lope glycoprotein (E) are embedded (Figure1).The E protein is the viral determinant mediating

the binding of the virus to specific receptorsfound on the surface of DENV-permissive hostcells, including dendritic cells (DCs), B cells,T cells, monocytes/hepatocytes and neuronalcells (for further details, see[9]). Several receptorsfor DENV have been identified so far, amongthem heparan sulfate, CD14, 37/67-kDa high-affinity laminin receptor, GRP78/BiP, and thewell-characterized DC-specific intercellularadhesion molecule 3-grabbing nonintegrin(DC-SIGN) [1016]. Upon binding, the virus isinternalized via receptor-mediated endocytosis(Figure1A)

. The low pH of the endosomal path-way induces an irreversible trimerization of theE protein, which thereupon mediates the fusionof the virion envelope with cellular membranes.Following uncoating of the nucleocapsid, theviral genome is released into the cytoplasm of theinfected cells, where it is translated at the roughendoplasmic reticulum (ER), giving rise to apolyprotein of approximately 3400 amino acids.This polyprotein is co- and post-translationallycleaved by a combination of cellular proteasesand the viral nonstructural (NS)2B-3 proteaseinto three structural and seven NS proteins.

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Figure 1. The Dengue virus replication cycle and structure of the viral particle.

(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.

Nucleus

Rough ER

Golgi

1. Binding2. Receptor-mediatedendocytosis

4. Translation

and replication

5. Maturation

6. Virus release

Endosome

Virus-inducedmembranes

3. RNA release

E-dimer

C protein

M protein

pr part of prM protein

Genomic RNAViral proteins

RF

RI

Progeny RNA

Encapsidation

Formation ofreplication complex(membrane bound)

Translation and

processing

(+)

(+)

(+)

(+)

()

()

(+)

(+)

(+)

E-trimer

E-dimer

EprM

heterodimer

(+) RNA

E protein

Intracellular Extracellular

Endosomal (low pH)C proteinM protein

pr part of prM protein


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Following translation and processing, a proteincomplex is assembled that is composed of theNS5 RNA-dependent RNA-polymerase (RdRp),accessory NS proteins, viral (+)RNA and, proba-bly, host cell factors. Within this so-called replica-tion complex (RC), which is associated with virus-induced intracellular membrane structures, viralRNA replication takes place. Replication startswith the synthesis of genome-length (-)RNA,which then serves as a template for the productionof (+)RNA progeny(Figure1B). During completionof transcription, the newly synthesized (-)RNAundergoes base pairing with the (+)RNA template,leading to the formation of an extended RNAduplex called replicative form (RF). It functions asa template for the generation of new (+)RNA via areplicative intermediate (RI) in an asymmetric andsemiconservative manner. The newly produced

(+)RNA strand is released from the RI and eitherattaches to ribosomes to initiate a new translationcycle (dashed line in Figure1B) or assembles intovirions, which most likely form at the ER. Virionmaturation appears to require a passage throughthe Golgi compartment where glycosylation of theviral membrane proteins, as well as proteolyticcleavage of the precursor of mature M (prM)protein into the components pr and M by theGolgi-resident protease Furin or a relatedenzyme, takes place. The uncleaved prM pre-vents E from undergoing an acid-catalyzed tran-

sition into the fusogenic form during the transitthrough the secretory pathway by forming aprME heterodimeric complex. Following cleav-age, the pr-fragment is released, E formshomodimeric complexes and the mature virionsexit the infected cell.

Genome organization & protein functions

The genome of DENV consists of a single-stranded (+)RNA of approximately 11kb in size,which contains a 5 untranslated region (UTR;a100 nucleotides), a single open reading frameencoding for the viral polyprotein and a 3 UTRapproximately 400 nucleotides in length(Figure2A). Attached to the 5 end of the viralgenome is a type I 7-methyl guanosine cap struc-ture, but unlike cellular mRNAs the DENV-genome is not 3 polyadenylated. The 5 and 3UTRs are not well conserved between differentflaviviruses, but in all cases secondary structureshave been identified within these regions.

An important function of the 5 UTR proba-bly resides in the complementary region of thenegative strand, which serves as the initiation sitefor the synthesis of (+)RNA during replication. A

stem-loop (SL) structure near the 5 terminus ofthe genome plays an important role for RNA rep-lication: deletions within this structure ofDENV4 were lethal for replication [17].Furthermore, the 5 UTR influences the trans-lation of the RNA genome[18,19]. The 3 UTRof DENV encompasses three regions: a variableregion (VR) immediately 3 of the stop codonof the long open reading frame, a core regiondownstream of the VR and a 3 -terminalregion. The latter contains a very conserved 3terminal SL, which is absolutely required forreplication [20], and a cyclization sequence (CS)just upstream of the 3 SL. The CS is comple-mentary to a sequence at the 5 -end and medi-ates together with further sequences (seebelow) 5 -3 long-range RNARNA interac-tions, which have been proposed to be necessary

for RNA replication of flaviviruses [21,22]. SinceDENV genomes with mutations in the VR andthe core region of the 3 UTR were shown to beviable in cell culture, their functional impor-tance remains to be established [23,24]. However,even though it has not been possible so far toestablish a clear correlation between a particularDENV-serotype or genotype and the severity ofdisease outcome [4], full-length sequencingexperiments of different DENV genotypes letus assume that sequence differences in the 5and 3 UTRs as well as nucleotide differences

in the coding region of the genome mightplay a role in modulating the severity of DENVinfection [25].

The DENV open reading frame encodes for apolyprotein, which is co- and post-translationallyprocessed into three structural proteins (C, prMand E) and seven NS proteins (NS1, NS2A,NS2B, NS3, NS4A, NS4B and NS5; Figure2B).Processing of the polyprotein into the individualcomponents is carried out by a combination ofdifferent host cell enzymes and the cytoplasmicviral NS2B-3 protease complex (Figure 2B) [2629].The highly basic C protein forms the viral nucle-ocapsid together with the genomic RNA. Theimmature prM prevents the activation of thefusion activity of the E protein during virus mat-uration, and E is important for virus bindingand fusion of viral and cellular membranes dur-ing receptor-mediated endocytosis (see above).Most, if not all, NS proteins are involved in viralRNA replication, which occurs in close associa-tion with cellular membranes within the viralRCs (see below). NS5 is the RdRp and carries inaddition an N-terminal methyltransferasedomain important for the formation of the RNA


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cap structure [3033]. NS3 acts as the viral serineprotease, which requires the cofactor NS2B forfull activity. Furthermore, NS3 contains a RNAhelicase and nucleotide triphosphatase activityimportant for replication of the viral RNA [3439].

The role of the glycoprotein NS1 in viral replica-tion is unknown, but it is assumed that NS1 actsat an early stage in viral RNA replication [4042].Furthermore, it may also be important for thepathogenesis of DHF [43]. Only little is knownabout the functions of the small hydrophobicproteins NS2A, NS4A and NS4B. It has beensuggested that they may serve to anchor the viralreplicase to cellular membranes[44]. In addition,it has been shown that they contribute to theinhibition of the IFN-D/E response of theinfected host cell [45,46]. Recent results indicatethat NS4A plays a role in the induction of

membrane alterations in infected cells, whichmay serve as a scaffold to anchor the viral RC(see below) [47,48]. NS4B seems to be involved inviral replication, for which an interaction withNS3 appears to be required [49,50].

Viral replication

Intracellular sites of viral replication

All (+)RNA viruses investigated so far replicatetheir RNA in close association with virus-induced intracellular membranous structures,which may provide a scaffold for anchoring theviral RC. The biological function of suchstructures is not completely understood, but it isassumed that they help to increase the local con-centration of components required for replica-tion, confine RNA replication to a distinctsubcellular site, and tether viral RNA during

Figure 2. Genome structure and putative topology and functions of the Dengue virus proteins.

(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 5and 3UTRs containing SL structures, are depicted. Complementary sequences in the

5and 3UTRs are highlighted; positions of the VR in the 3UTR, as well as 5and 3UARs 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.

5 UTR

(~ 100 bases)

3 UTR

(~ 400 bases)

Structural region Nonstructural region

Open reading frame

Capsid Binding,fusion

Replication,pathogenesis

IFN

resistance

Proteasecofactor

Protease,

helicase,RNA triphosphatase

Prevention of

premature fusion

IFN resistance,membrane induction

IFN resistance

Methyltransferase,guanylytransferase,

RdRp

3 SL

5 CS

3UAR

VR 3 CS

X X X?

C

Mpr E 1

2A

35

2K

2B 4A 4B

X?

Viral protease (NS2B-3)

Furin

Unknown host cell protease

Host cell signalase

5 SL

5UAR

CAP prM E 1 2A 4B4A2B 3C 5

Single-stranded (+) RNA

Cytoplasm

ER lumen


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unwinding. In addition, the induced membranesmay aid in preventing the activation of dsRNA-induced host defense mechanisms, such as inter-feron-induced pathways in humans. Further-more, the encryption of viral dsRNA in virus-induced membrane alterations could explainwhy DENV is able to efficiently replicate in itsmosquito vector, despite the existence of adsRNA-triggered antiviral RNA interferencepathway in this organism [51].

The best-characterized flavivirus in terms ofinduction of intracellular membrane rearrange-ments is Kunjin virus (KUNV), the Australianvariant of West Nile virus (for a review, see[52]).In KUNV-infected cells, clusters of double-membrane vesicles each vesicle approximately50150nm in diameter have been described.These structures are called vesicle packets (VP)

or smooth membrane structures (SMSs). Fur-thermore, so-called convoluted membranes(CMs) and paracrystalline structures occur,which were found adjacent to the VP/SMS.Since the components of the KUNV-RC werefound by immunolabeling studies to localize toVP/SMS, it is assumed that viral replicationoccurs in this compartment. However, the viralserine-protease colocalizes with the CM, indi-cating that they are the sites of viral polyproteinprocessing. The membrane reorganizationinduced by KUNV might therefore give rise to

adjacent, but distinct, subcellular structureswhere different steps of the viral replicationcycle are carried out [53].

Clusters of double membrane vesicles compa-rable to the VP/SMS of KUNV have also beenobserved for DENV (Figure3) and other flavi-viruses. The detection of viral RNA and severalDENV NS proteins within these structures byelectron and immunofluorescence microscopysuggests that they represent the sites of DENVRNA replication [SMiller, RBartenschlager, UnpublishedData] [47,49,50,54]. Whether there are differentcompartments for polyprotein processing andreplication in DENV-infected cells, as wasdescribed for KUNV, remains to be clarified.

Cyclization of the viral genome

during replica tion

A 25-nucleotide-long region (CS1) just upstreamof the 3 SL within the 3 UTR was found to basepair with a complementary sequence (5 CS) in

the 5 coding region of the capsid gene(Figure2A)[55,56]. Such 5 -3 long-range RNARNA interac-tions across a region of approximately 10kb havebeen proposed to be necessary, but not sufficient,for RNA replication of DENV. In addition, fur-ther sequences, named 5 and 3 UARs (upstreamAUG region), were found to be required for suc-cessful RNARNA complex formation [21].Recent results indicate that RNARNA inter-actions between 5 - and 3 -end sequences of theviral genome enhance DENV RNA synthesis onlyin the presence of an intact 5 SL [57]. In these

excellent studies it was found that the 5 SL func-tions as a promoter for the viral RdRp. Usingatomic force microscopy, Filomatori etal.

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

(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.

Virus-induced structures

Virus particles

200 nm 200 nm


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demonstrated that DENV NS5 the RdRp of thevirus was bound to the viral RNA only when the5 SL was present. A model was developed inwhich interactions between the 5 - and the 3 -ends of the viral RNA position the promoter adja-cent to the 3 -end of the genome (Figure4). TheRdRp binds to the 5 SL and is transferred to thetranscription initiation site at the 3 -end of thegenome, which is made possible by long-rangeRNARNA interactions. These findings provide

an explanation for the strict requirement ofgenome cyclization during DENV replication.Genome cyclization may be an important controlmechanism to ensure that only full-length tem-plates are amplified, to increase RNA stability andto control the levels of minus-strand RNA synthe-sis. Furthermore, cyclization may be importantfor the coordination of translation and RNA syn-thesis by overlapping signals at the 5 - and 3 -ends of the genome and it could localize the viralRdRp or accessory proteins of the RC at theappropriate start site.

Host cell proteins involved in

viral replication

Up to now, only very few host cell factors contrib-uting to the flavivirus replication cycle have beenidentified. One example is the eukaryotic transla-

tion elongation factor eF1-D, which interacts withthe 3 UTR of West Nile virus [58]. Further hostcell proteins possibly involved in the viral replica-tion cycle of flaviviruses are MOV34, which inter-acts with the 3 UTR of Japanese encephalitis virus[33], tubulin and the tumor susceptibility protein101, which interact with Japanese encephalitisvirus NS3[59], and the mosquito La protein, whichbinds to the 3UTR of (+) and (-)RNA of DENV[60]. Although these proteins were shown to inter-act with viral proteins or RNA, their role in theviral replication cycle is not understood thus far.

Taking advantage of large-scale high-throughputscreens, Chu and Yang recently identified the c-Srcprotein kinase as an important host cell factorrequired for DENV assembly [61]. This exampleillustrates the power of such high-content screen-ing approaches and we can assume that more cellu-lar factors and pathways required for flaviviralreplication will be discovered with these methods.

Replication & the cellular nucleus

It is textbook knowledge that the replication offlaviviruses takes place in the cytoplasm of virus-infected cells. However, in several studies someflavivirus structural and NS proteins have beenfound to localize to the nucleus. One prominentexample is DENV NS5, which has been detectedin the nucleus of cells already at very early timepoints after infection (Figures5AC) [62]. Thisresult is perplexing because RdRp activity isrequired in the cytoplasmic RC. Nuclear translo-cation appears to depend on the phosphorylationstatus of NS5 and is modulated by interactionwith NS3. Nuclear hyperphosphorylated NS5was found to be unable to bind to NS3, whereashypophosphorylated NS5 is cytoplasmic and

Figure 4. Proposed model for Dengue virus minus-strand

RNA-synthesis.

5 3UAR and 5 3CS 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].

5 SL

3 SL

5

5

3

Cyclization of genome

53

UAR

3

5

5

5

3

3

53 CS

Binding of NS5

( = RdRp)

Transcription

5

5

(+) RNA

() RNA


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associated with NS3. Likewise, NS5 of therelated flavivirus yellow fever virus was reported

to localize mainly to the nucleus, where it mayexert some function not directly required forRNA replication [63].

The nuclear localization of proteins with amolecular mass higher than 45kDa is an activeprocess that requires the recognition of a nuclearlocalization signal (NLS) within the correspondingprotein by the nuclear transport machinery [64].Previous studies have shown that DENV NS5contains two potential NLS in its central regionthat are recognized by the importin D/E andimportin E1 nuclear transport proteins, respec-tively [65]. Both NLS are highly conserved amongvarious members of the flavivirus genus. Intrigu-ingly, DENV NS5 also possesses the ability to beexported from the nucleus, for which the nuclearexport receptor CRM1 (exportin1) is required[66].The role of nuclear NS5 for DENV replication isunknown, but recent experiments allow us toassume that nuclear NS5 suppresses IL-8 pro-duction, and thus protects against the antiviralactivity of this cytokine[67].

As well as NS5, flavivirus C protein also local-izes to the nucleus, where it is concentrated insmall dot-like structures, which may represent

nucleoli (Figures5DF) [6871]. Similar to NS5, Calso contains a bipartite NLS that is responsible for

nuclear transport. The function of C in thenucle(ol)us remains to be clarified. However, it ispossible that the C protein is not only the buildingblock of nucleocapsids, but is also involved in reg-ulating the DENV replication cycle, for example,by modulating host cell transcription profiles[72].

Apart from NS5 and core, a recent study sug-gests that NS3, and presumably up to 20% ofactive replication complexes, resides in thenucleus of DENV-infected cells [73]. Althoughthis is an interesting observation, nuclear locali-zation of NS3 could not be confirmed by othergroups [49,53] [Miller S, Bartenschlager R, Unpublished Data].The reason for this discrepancy is not clear, but itmay be due to different experimental conditions.Further studies will be required to clarify thepossibility of nuclear DENV RNA replication.

Further studies are required to investigate therole of nuclear localized flavivirus proteins. Onthe one hand, a better understanding could leadto the development of new recombinant vac-cines based on viral proteins that are deficient innuclear trafficking and thus lead to an attenua-tion of the virus. On the other hand, nuclearlocalized viral proteins could be an ideal tool to

Figure 5. Nuclear localization of Dengue virus proteins.

(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.

Merge

MergeDAPI

DAPICapsid

NS5


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unravel the cell biological details of nuclear proc-esses and functions. Finally, nuclear localizedDENV proteins may be important determinantsof pathogenesis, for example, by altering host celltranscriptome profiles, as suggested for the Cprotein, or by subverting innate antiviral defensemechanisms, as discussed for NS5.

Conclusions & future perspective

In recent years, many new important findingshave contributed to a better understanding ofseveral steps of the DENV replication cycle.However, much work remains to be done, inparticular with respect to DENVhost cellinteraction and the pathogenesis of DF, DHFand DSS. Advanced methods, such as genome-wide RNA interference studies, as well asmodern microscopy techniques (e.g., tomo-

graphy or live cell imaging), will help to unravelthe biogenesis of the viral RC, to identify hostcell factors involved in the various steps of theDENV replication cycle and to understand thedynamics of DENVhost cell interaction. Inaddition, 3D reconstruction of virus-inducedmembrane compartments should help to

decipher their interconnection with the sub-cellular environment, the topology of the mem-branous vesicles and eventually definecompartments where distinct steps of theDENV replication cycle occurs (RNA transla-tion, replication and assembly). Finally, morestudies are required to characterize the role ofnuclear localized DENV proteins for viral repli-cation and their possible contribution to patho-genesis. An improved understanding of themolecular mechanisms of the DENV replica-tion cycle may help to develop an effective vac-cine, as well as therapeutic strategies to treatinfections with this insidious pathogen.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial

involvement with any organization or entity with a finan-

cial interest in or financial conflict with thesubject matter ormaterialsdiscussed in themanuscript. Thisincludesemploy-

ment, consultancies, honoraria, stock ownership or options,

expert testimony, grants or patents received or pending,

or royalties.

No writingassistancewasutilized in theproduction of

thismanuscript.

Executive summary

Postgenome: understanding Dengue virus replication

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 theFlaviviridae 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 5untranslated region (UTR), a single open reading frame encoding a viral polyprotein, a

3UTR, 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 5UTR to the appropriate transcription start site at the 3end 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 anddengue haemorrhagic fever.

www.who.int/mediacentre/factsheets/fs117/

en/

Affiliations

Ralf Bartenschlager

Department of Molecular Virology,

ImNeuenheimer Feld 345, University of

Heidelberg, D-69120 Heidelberg, Germany

Tel.: +49 622 156 4569;

Fax: +49 622 156 4570;

[email protected]

Sven Miller3-V Biosciences, c/o Instituteof Biochemistry,

Schafmattstrasse18, ETH Hoenggerberg

HPME 17, CH-8093 Zurich, Switzerland

Tel.: +41 446 336 019;

Fax: +41 446 331 091;

[email protected]


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