Novel cis-Acting Element within the Capsid-Coding Region EnhancesFlavivirus Viral-RNA Replication by Regulating Genome Cyclization

Zhong-Yu Liu,a,b Xiao-Feng Li,a,b Tao Jiang,a,b Yong-Qiang Deng,a,b Hui Zhao,a Hong-Jiang Wang,a Qing Ye,a Shun-Ya Zhu,a,b

Yang Qiu,a,c Xi Zhou,c E-De Qin,a,b Cheng-Feng Qina,b

Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing, Chinaa; State Key Laboratory of Pathogen and Biosecurity, Beijing, Chinab; State KeyLaboratory of Virology, Wuhan University, Wuhan, Chinac

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNAviruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivi-rus. The downstream of 5= cyclization sequence (5=CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, asdemonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhancesvRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealedthat the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PKfunction. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK.Moreover, the function of DCS-PK depends on its position relative to the 5=CS, and the presence of DCS-PK facilitates the for-mation of 5=-3= RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replica-tion by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNAcyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.

The flavivirus genus contains numerous important agents ofhuman infectious diseases, including dengue virus (DENV),

West Nile virus (WNV), Japanese encephalitis virus (JEV), yellowfever virus (YFV), and tick-borne encephalitis virus (TBEV). Hu-man infection by flaviviruses can result in symptoms ranging frommild fever to severe encephalitis and hemorrhagic fever. Due tolack of effective vaccines and specific medicines against many fla-viviruses, they pose a significant threat to human health aroundthe world.

Flaviviruses are enveloped RNA viruses with single-stranded,positive-sense genomes. Their 5= capped viral genome RNA(vRNA) is approximately 10 to 11 kb and contains a single openreading frame (ORF) flanked by 5= and 3= untranslated regions(UTRs). The ORF encodes a polyprotein with more than 3,000residues, which is cotranslationally and/or posttranslationallyproteolytically processed into three structural proteins (capsid,pre-membrane/membrane, and envelope) and seven nonstruc-tural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) byviral and cellular proteases. NS3 and NS5 perform essential en-zyme activities required for vRNA replication. The C-terminaldomain of NS3 has helicase/NTPase (1) and RNA triphosphataseactivities (2, 3). The N-terminal domain of NS5 encodes guany-lyltransferase (4) and methyltransferase activities (5, 6), whereasthe C-terminal domain of NS5 has RNA-dependent RNA poly-merase (RdRp) activity. These enzyme activities are involved invRNA synthesis, 5= capping, and internal adenosine methyl-ation (7).

The 5=UTR, 3=UTR, and capsid-coding sequence are involvedin vRNA replication, translation, and perhaps encapsidation, andmany cis-acting elements have been found in these regions. cis-Acting elements at the 5= end include the 5=-end stem-loop (SLA),which binds viral NS5 RdRp and promotes vRNA replication (8–10); the 5= upstream of AUG region (UAR); the 5= downstream ofAUG region (DAR); and the 5= cyclization sequence (CS) (11–14).The last three are required for genome cyclization by base-pairing

with corresponding complementary sequences in the 3=UTR. Ahairpin in the capsid-coding sequence (cHP) is required for bothstart codon recognition and vRNA replication (15, 16). One of thecritical cis-acting elements at the 3= end is the 3=-terminal stem-loop (3=SL), which is indispensable for vRNA replication (17–19)and is also the binding site for several cellular factors (20–23). Twoconserved elements, DB1 and DB2, which have dumbbell-shapedstructures, are also located within the 3=UTR (24). It should benoted that the 5=UAR can fold into a stem-loop (SLB) (8), andpartial sequences of 3=UAR and 3=DAR can form a small hairpin(sHP) in the linear conformation of vRNA (25); thus, the balancebetween the linear and circular vRNA conformations is critical forflavivirus vRNA replication.

Flavivirus cis-acting elements can be classified into elementsthat are essential for vRNA replication, such as promoters, andelements that are dispensable for vRNA replication but can regu-late this process, for example, enhancers. Most known cis-actingelements of flaviviruses are indispensable for vRNA replication.Enhancer-like elements (DB1 and DB2) had only been found inthe 3=UTR (24, 26) until recently, when a cis-acting enhancer ele-ment, SL6, was discovered in the capsid-coding region of tick-borne flavivirus (27). The results from different groups (28–30)also indicate that the region downstream of the DENV 5=CS reg-ulates vRNA replication, but the mechanism by which this occursis unknown. In this study, using a combination of bioinformatic,biochemical, and reverse genetic approaches, we identified a novelcis-acting element, the downstream of 5=CS pseudoknot (DCS-

Received 28 January 2013 Accepted 1 April 2013

Published ahead of print 10 April 2013

Address correspondence to Cheng-Feng Qin, qincf@bmi.ac.cn.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00243-13

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PK), in the capsid-coding region, which is conserved in mosquito-borne flaviviruses. DCS-PK enhances vRNA replication by regu-lating vRNA cyclization, indicating that they might have played arole in the evolution of mosquito-borne flaviviruses.

MATERIALS AND METHODSCell culture. BHK-21 cells were grown in Dulbecco modification of min-imal essential medium (DMEM; pH 7.5; Gibco) supplemented with 5%fetal bovine serum (FBS; PAA, Morningside, Queensland, Australia) at37°C in 5% CO2.

DNA constructs. The p4-Rluc-Rep replicon was reported previously(31, referred to as DEN-R.luc2A-RP in the original publication). Theinfectious clone p4 (32) and the host Escherichia coli strain for all of the p4and replicon constructs, BD1528, were kindly provided by Stephen S.Whitehead. To construct the p4-cHPstop-SP-IRES-Rluc-Rep replicon,the encephalomyocarditis virus (EMCV) internal ribosome entry site(IRES) sequence was fused upstream of the Renilla luciferase (Rluc) geneby overlapping PCR, and the resulting fragment was then fused down-stream of the spacer sequence (positions 67 to 707 of the enhanced greenfluorescent protein [EGFP] gene ORF). The AscI-XhoI fragment of p4-Rluc-Rep was replaced by an artificial multiple cloning sequence: 5=-GGCGCGCCAAACTTTCCCCTTACGTATTAACATGCATCTTGCGGCCGCGTACAGAGTCTCGAG--3= (restriction sites for AscI, SnaBI, NotI,and XhoI are underlined from the left to right). The resulting constructwas used as a backbone for the assembly of a new replicon. First, a hepatitisD virus ribozyme sequence, followed by a unique AgeI site, was inserteddownstream of the 3= end of DENV cDNA in the backbone construct. Thespacer-IRES-Rluc fragment and the AscI-5=UTR-cHPstop-SnaBI frag-ment, which contains a stop codon in the cHP sequence, were subse-quently subcloned into the backbone to obtain p4-cHPstop-SP-IRES-Rluc-Rep.

A cassette cloning strategy was then used to introduce mutations intop4-cHPstop-SP-IRES-Rluc-Rep. The AscI-5=UTR-cHPstop-SnaB I frag-ment was engineered by overlapping PCR so that its sequence corre-sponding to position 54 to 90 in the capsid ORF was replaced by thefollowing sequence: 5=-CTCCTCACACACTACGTAAATGGGGAGGAG-3=. The underlined sequence indicates the BseRI restriction site. Theresulting construct was named pBseRI-03. Oligonucleotides containingthe desired mutations were annealed and cloned into BseRI-treated pB-seRI-03 to generate AscI-5=UTR-cHPstop-SnaB I fragments containingdifferent mutations. The corresponding fragments were then subclonedinto p4-cHPstop-SP-IRES-Rluc-Rep to obtain replicon mutants.

To introduce mutations into the p4 infectious clone, mutations wereintroduced into the AscI-NheI-XhoI fragment from p4-Rluc-Rep using acassette cloning method similar to that described above. The NheI-XhoIfragments were then replaced by the NheI-XhoI fragment from p4. Theresulting constructs contained AscI-XhoI fragments from p4 with the de-sired mutations. The corresponding fragments were subcloned into the p4plasmid by Eco91I/PauI digestion, and p4 constructs carrying variousmutations in DCS-PK were generated. To generate GVD-containing p4mutants, the SacII-MluI fragment of p4 was substituted with homologousfragments containing GVD mutations. The Eco91I-PauI fragment of thep4-GVD plasmid was substituted with homologous fragments containingmutations. The sequences of the primers and oligonucleotides are avail-able upon request.

RNA structure prediction. RNA secondary structures were predictedusing the mfold online server (33, 34). The pknotsRG (35, 36) and Pseu-doViewer3 (37, 38) online versions were used to predict and illustrate,respectively, the RNA structures containing pseudoknots.

In vitro transcription. The RiboMAX large-scale RNA productionSP6 kit (Promega) was used for in vitro transcription according to themanufacturer’s instructions, with minor changes. For the replicon andinfectious RNA assay, linearized DNA was extracted with phenol-chloro-form, ethanol precipitated, and dissolved in RNase-free water. Topically,2 to 3 �g of linearized DNA, 6 �l of 5� SP6 buffer, and 2.5 �l of SP6

enzyme mix were mixed with 6 mM ATP, 6 mM CTP, 6 mM UTP, 2 mMGTP, and 0.83 mM m7GpppG cap analog (Promega) in a 30-�l reaction.The reactions were incubated at 37°C for 3.5 h, and then RQ1 RNase-freeDNase I (Promega) was added to the reaction and incubated at 37°C for 20min to remove the DNA template. For RNA structure probing and RNAbinding assays, DNA fragments containing the SP6 promoter and respec-tive viral sequences were amplified by high-fidelity PCR and purified fromagarose gels to prepare templates for the transcription of DEN-4 5= 300-ntRNA, DEN-4 3=UTR RNA, JEV 5= 320-nt RNA, and WNV 5= 320-nt RNA.In vitro transcription reactions were topically performed at 37°C for 4.5 hin a 60-�l mixture containing 1 to 2 �g of template DNA, 12 �l of 5� SP6buffer, 6 �l of SP6 enzyme mix, and 5 mM each of the four ribonucleosidetriphosphates, followed by the removal of DNA templates as describedabove. All in vitro-transcribed RNAs were purified using the RNeasy mini-kit (Qiagen) according to the RNA cleanup protocol. Purified RNA wasquantified by an ND-1000 spectrophotometer (NanoDrop) and stored at�80°C before use.

RNase probing. Various 5= end RNAs of flaviviruses were incubated at85°C for 5 min and slowly cooled to room temperature. The RNAs werethen digested by 0.01 U of RNase T1, 0.001 U of RNase V1, or 0.01 ng ofRNase A (Ambion). The reactions were performed at 25°C for 15 min in a10-�l mixture containing 10 mM Tris-HCl (pH 7.0), 100 mM KCl, 10mM MgCl2, 10 �g of yeast RNA, 1 �g of sample RNA, and the respectiveRNase. Control reactions without RNase were performed in parallel.Forty microliters of precipitation/inactivation buffer with ethanol wasadded to stop the reactions. Reactions were incubated at �80°C for 30min, followed by centrifugation at 15,700 � g and 4°C for 15 min. Pelletswere washed with 120 �l of 70% ethanol, air dried, and dissolved in 5 �l ofRNase-free water.

NMIA modification. Various RNAs corresponding to the 5= end se-quences of DENV, JEV, and WNV were modified with N-methylisatoicanhydride (NMIA; Sigma-Aldrich). Experiments were performed as pre-viously described (39). Reactions were incubated at 37°C for 45 min, andthen the modified RNAs were purified by using the RNeasy MinElutecleanup kit (Qiagen).

Primer extension. Purified RNase-treated and NMIA-modified RNAsamples were reverse transcribed into cDNA using carboxyfluorescein(FAM)-labeled primers. The primer sequences were as follows: 5=-FAM-GTGGGATGGAAAGGACTC-3= or 5=-FAM-TTCCCAGAAAAAAGTCC-3= for DENV, 5=-FAM-TCGGGGCTAATGCTGTAAACT-3= for JEV,and 5=-FAM-TGCTGTGAACCTGAAGAAC-3= for WNV. Reverse tran-scription was performed using Superscript II reverse transcriptase (Invit-rogen) in a 20-�l reaction according to the manufacturer’s instructions.The FAM-labeled cDNA samples were analyzed by capillary electropho-resis using a 3730xl DNA analyzer (Applied Biosystems). Dideoxy se-quencing reactions were performed in parallel. The detection of NMIAreactivity by primer extension is generally known as the SHAPE (selective2=-hydroxyl acylation analyzed by primer extension) assay (39).

Replicon assay. A total of 2 � 104 BHK-21 cells were seeded into eachwell of 48-well plates 1 day prior to transfection and incubated at 37°C in5% CO2. Transfection was performed with Lipofectamine 2000 reagent(Invitrogen) in triplicate according to the manufacturer’s instructions. Atotal of 250 ng of RNA was transfected into each well, except for p4-Rluc-Rep, of which 500 ng of RNA was transfected. Cell lysates were collected at6, 24, 48, 72, and/or 96 h after transfection, and Renilla luciferase activitywas measured using the Renilla luciferase assay system (Promega) in aGloMax-96 microplate luminometer (Promega).

Electroporation of full-length vRNA. Portions (5 �g) of each RNAtranscript was electroporated into 400 �l of BHK-21 cells at 107 cells/ml in0.4-cm cuvettes (Bio-Rad) using a Gene Pulser Xcell electroporation sys-tem (Bio-Rad) at 270 V and 975 �F. The cells were resuspended in 14 mlof DMEM containing 5% FBS. A total of 0.5 ml of the cell suspension wasseeded into each well of 24-well plates, and 0.3 ml of the cell suspensionwas seeded into each well of 8-well Lab-Tek II chamber slides (Thermo

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Scientific). Plates and slides were incubated at 37°C in 5% CO2. The me-dium in the 24-well plates was changed 4 h postelectroporation.

qRT-PCR. At 4, 24, 48, 72, and 96 h postelectroporation, electropo-rated BHK-21 cells were washed thoroughly with phosphate-buffered sa-line (PBS), and then total RNA was isolated using the RNeasy minikit(Qiagen) in triplicate except for the experiment accessing the stability of

WT, XT, S1X, S3X, 75.78, and 91.93-GVD RNAs, in which RNA wasisolated in duplicate. Quantitative reverse transcription-PCR (qRT-PCR)was performed on a LightCycler 2.0 (Roche) system with the One StepPrimeScript RT-PCR kit (TaKaRa). Probe (5=-FAM-CAGGAGGATTGT-GACCATAGAGGCCCA-TAMRA-3=) and primers (forward, 5=-CCCCGGAACAACAGTCACA-3=; reverse, 5=-TGCAGTGGTGGTCCTCAAAG-

FIG 1 Structure prediction of DCS-PK among different mosquito-borne flaviviruses. (A) Nucleotide sequences of the downstream of 5=CS region of mosquito-borne flaviviruses. The 5=CSs are boxed, and predicted stem regions of DCS-PK are shown in different colors: red for stem 1, yellow for stem 2, and green for stem3. The numbers at the beginning of the sequences represent the position of the first shown nucleotide in the capsid ORF, and its position in the viral genome (thelatter are shown in parentheses). Abbreviations: DEN-1 to DEN-4, DENV serotypes 1 to 4; SLEV, Saint Louis encephalitis virus; MVEV, Murray Valleyencephalitis virus; USUV, Usutu virus; KOKV, Kokobera virus; TMUV, Tembusu virus. The GenBank accession numbers of representative sequences are asfollows: DEN-1, EU848545; DEN-2, AY702035; DEN-3, EU081188; DEN-4, AF326573; JEV, M55506; WNV, AY490240; SLEV, EU566860; MVEV, AF161266;USUV, EF206350; KOKV, AY632541; TMUV, JQ289550; YFV, AY640589. (B) DCS-PK structures predicted by pknotRG and drawn with PseudoViewer3. Notethat constraints were applied to the stem 1-stem 3 interfaces of several viruses based on sequence comparison shown in panel A and structure probing resultsshown in Fig. 2.

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3=) targeting the NS1 gene were used to detect DENV RNA. Purifiedfull-length RNA transcript was 10-fold serial diluted and used as stan-dards for calculation of RNA copy number.

Immunofluorescence assay (IFA). Electroporated BHK-21 cellsgrown in chamber slides were washed with PBS and fixed with cold ace-tone at 24, 48, 72, and 96 h after electroporation. The fixed cells wereincubated with anti-DENV envelope monoclonal antibody (2D73; 20 �g/ml, 40 �l per well) at 37°C for 1 h, followed by three 10-min PBS washes.Goat anti-mouse IgG (1:200 dilution; Alexa Fluor 488-conjugated [Zs-bio]) was added, and the slides were incubated at 37°C for 1 h, followed bythree 10-min washes with PBS. The cells were then stained with 40 �l of0.5 �g/ml 4=,6-diamidino-2-phenylindole (DAPI) for 5 min and washedwith PBS. The fluorescence images were captured with an Olympus BX51microscope.

Plaque assay. Culture supernatants of electroporated BHK-21 cellswere collected at 48, 72, and 96 h postelectroporation. The virus titers ofthe supernatants were determined by plaque assay as described before(40).

RNA binding assay. A total of 0.3 �g of DEN-4 3=UTR RNA andvarious amounts (0.24, 0.6, 1.2, or 2.4 �g) of different 5= 300-nucleotide(nt) RNAs were mixed and diluted to 14-�l reaction mixes, which alsocontained 5 mM Tris-HCl (pH 8.0) and 0.5 mM EDTA, the 5=:3= RNA

molar ratio in the samples is 1:1, 2.6:1, 5:1, and 10:1, respectively. Thesamples were then heated at 95°C for 2 min and immediately placed in anice bath. Next, 6 �l of 3.3� RNA folding buffer (333 mM HEPES [pH 8.0],20 mM MgCl2, 333 mM NaCl) was added to each sample and mixed well.The RNA samples were incubated at 37°C for 30 min to allow folding intocomplexes, and then 2.5 �l of gel loading solution (Ambion) was added toeach sample. The samples were loaded into 5% native polyacrylamide gels,which had been pre-run in 0.5� Tris-borate-EDTA buffer at 4°C for 30min, and electrophoresis was conducted at 60 V at 4°C for 4 h. The gelswere then stained with SYBR Gold nucleic acid gel stain (Invitrogen) for20 min and directly visualized with a UV transilluminator. The data wereanalyzed using the ImageJ package.

RESULTSConserved three-stem pseudoknot is located downstream of the5=CS in mosquito-borne flavivirus. To identify potential cis-act-ing element(s) downstream of the 5=CS in mosquito-borne flavi-viruses, consensus sequences of the capsid-coding region of eachflavivirus species were generated and subjected to the mfold webserver (33, 34) to obtain structure models. The base-pairing pat-terns of the two most stable structure models implied that they

FIG 2 Biochemical structure analysis of DCS-PK elements from different flaviviruses. (A) A representative SHAPE experiment for the analysis of the structureof DEN-4 5= end RNA. Nucleotide ladders were generated by dideoxy-sequencing. The regions corresponding to DCS-PK and several 5= end RNA elements areindicated. (B) Summary of the RNase probing and NMIA modification results of the DEN-4 5= RNA containing the DCS-PK element. The major RNA elementsof the 5= end are indicated. The symbols used are explained in the right panel. (C) RNase probing and NMIA modification results of the DCS-PK elements fromJEV and WNV. The symbols used are the same in panel B.

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were possibly coexistence by formation of a pseudoknot (data notshown). Sequence comparison also indicated that the down-stream of 5=CS region’s base-pairing potential of folding into apseudoknot is conserved in all mosquito-borne flaviviruses exceptYFV (Fig. 1A). To address the possibility that the cis-acting ele-ment downstream of the 5=CS has a pseudoknot structure, thesequences of downstream of 5=CS regions of different flaviviruseswere subjected to pknotsRG (35, 36). As we hypothesized, similarpseudoknot structures were predicted (Fig. 1B), which were des-ignated DCS-PK. The DCS-PK has three stem regions (Fig. 1B),and stem 1 and loop 2 are highly conserved among mosquito-borne flaviviruses (Fig. 1A), whereas the stem 2-loop 3 hairpin,which links the 3= of stem 1 and 5= of loop 2, has variable sequencesbetween different virus species. The sequences of loop 1 and stem3 are also not conserved. These bioinformatic analyses indicatethat a conserved RNA pseudoknot may be formed in the capsid-coding region of mosquito-borne flavivirus.

Structure probing of the DCS-PK structure. To confirm thephysical existence of the predicted DCS-PK structure, RNase V1/T1/A and NMIA were used to probe the 5= end structure of DENV

serotype 4 (DEN-4), WNV, and JEV (Fig. 2 and data not shown).RNase V1, which cleaves double-stranded or base-stacking re-gions, detected all three DCS-PK stems from DEN-4 and JEV, andstems 1 and 2 from WNV were also RNase V1 sensitive. Single-strand-specific RNase T1/A-sensitive nucleotides were mainly lo-cated in loops and junctions. NMIA selectively adds 2=-O adductsto structural flexible nucleotides, and its reactive sites in DCS-PKwere mainly located in loop regions and loop-stem interfaces,whereas most nucleotides in the stems showed no SHAPE activity.In summary, the structure probing results support the predictedthree-stem pseudoknot structure of DCS-PK.

DCS-PK is required for efficient vRNA replication and viruspropagation. Reverse genetic approaches were used to investigatethe role of DCS-PK in virus life cycle. Silent mutations targetingDCS-PK (Fig. 3A) were introduced into the infectious clone p4 ofDEN-4 (32). The XT mutant was designed to disrupt the overallsecondary structure of DCS-PK. Stem 1 was disrupted in S1X, andS3X included a disrupted stem 3. Mutations were also introducedinto the nonconserved loop 3 to generate mutant 75.78, whichretained the original secondary structure of DCS-PK. The codon

FIG 3 Characterization of DEN-4 mutants containing silent mutations in DCS-PK. (A) Demonstration of mutants containing silent mutations in the DCS-PKelement. Mutations are indicated in the predicted pseudoknot structure of DCS-PK in boldface. The positions of several nucleotides of DCS-PK in the capsid ORFare indicated in the WT structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent theactual folding of these mutants. (B) Replication of vRNA mutants in transfected BHK-21 cells. vRNA levels are expressed as fold changes relative to the RNAcopies 4 h posttransfection. (C) Growth curves of viruses in the supernatants of vRNA-transfected BHK-21 cells. Virus titers were normalized to transfectionefficiency as determined by qRT-PCR at 4 h after transfection. (D) Decay kinetics of various vRNA mutants, which all contained GDD-to-GVD mutations inNS5RdRp. The error bars indicate the standard deviations in all figures.

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usage frequencies in the above mutants were left unchanged toavoid potential effects on viral translation efficiency. Mutant91.93, which targeted the bulged A93 of stem 3 and A91 of loop 2but did not affect DCS-PK secondary interactions, correspondedto a previously reported DEN-2 mutant (28) with reduced vRNAreplication. Detection of viral antigens in vRNA-transfectedBHK-21 cells by IFA demonstrated that all of the above-men-tioned mutant RNAs were infectious. There were fewer DENV-positive cells in XT, 91.93, S1X, and S3X mutant RNA-transfectedcells than in wild-type (WT) or 75.78 mutant RNA-transfectedcells, demonstrating that the disruption of RNA structure inDCS-PK impairs viral replication (data not shown). To directlyassess the effect of DCS-PK on viral genome RNA replication,vRNA levels in transfected cells were determined by qRT-PCR.The vRNA levels in the XT, S1X, and S3X mutant RNA-trans-fected cells were approximately 1 order of magnitude lower thanthose of WT and 75.78 RNA-transfected cells at 48 to 72 h aftertransfection, whereas the latter two exhibited similar vRNA levelsat these time points (Fig. 3B). The vRNA level of 91.93 also showed

a moderate reduction of ca. 50% at 48 to 72 h, which is in agree-ment with previous findings (28). The viral loads in the superna-tants of the WT-, 75.78-, and S3X-transfected BHK-21 cells werecompared. At 48 to 72 h after transfection, the virus titers of S3Xwere significantly lower than those of WT or mutant 75.78(Fig. 3C). To exclude the possible impact of mutations on RNAstability, RNAs containing mutations in the conserved GDD motifof NS5 were electroporated into BHK-21 cells. Monitoring of in-tracellular vRNA levels indicated that the stability of mutant RNAswas similar to that of WT RNA (Fig. 3D). Taken together, theresults described above provide strong evidence that the presenceof DCS-PK structure enhances vRNA replication and thus facili-tates virus propagation. These results also suggest that the func-tion of DCS-PK depends on its secondary structure, as well assome conserved primary sequences.

Secondary structure of DCS-PK is required for its function.The function of DCS-PK and its dependence on the secondarystructure was further investigated with another reverse geneticapproach. For each DCS-PK stem region, a pair of mutants was

FIG 4 Effects of secondary interactions in DCS-PK on vRNA replication. (A) Mutations targeting various stem regions of DCS-PK. The mutations introducedare indicated in boldface. The nucleotide positions of DCS-PK in the capsid ORF are indicated in the S1POS structure. Note that the secondary structures ofDCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B to D) Replication of vRNA mutantscontaining the disrupted or restored DCS-PK stem 1 (B), stem 2 (C), and stem 3 (D), which were determined by qRT-PCR. Metabolism curves of correspondingGVD-containing RNAs are shown in parallel. The data are expressed as fold changes relative to the RNA level 4 h posttransfection.

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constructed to bypass potential influences of changes in the capsidsequence and/or viral translation on vRNA replication (Fig. 4A).One mutant contained a disrupted stem region (S1NEG, S2NEG,and S3NEG), and the corresponding stem region was restored inthe other construct (S1POS, S2POS, and S3POS). The paired mu-tants encoded the same capsid sequence, which differed from theWT capsid by a few residues. The mutations were carefully chosento minimize the changes in conserved residues in the capsid se-quence, and codon usage frequencies of the paired mutants wereremained the same. The vRNA levels of S1POS and S1NEG weresimilar at all time points after transfection (Fig. 4B). In contrast, at48 to 96 h after transfection, the vRNA level in the S2POS-trans-fected cells was significantly higher than that of S2NEG, indicatingthat S2POS replicated more efficiently (Fig. 4C). Similar resultswere observed for S3POS and S3NEG (Fig. 4D). The RNA stabilitywas similar between each pair of mutants, as determined by elec-troporation of NS5 GVD mutant RNAs (Fig. 4B to 4D). IFA de-tection of viral antigens in vRNA-transfected cells (data notshown) agreed with the results of vRNA replication. Taken to-gether, these results indicate that secondary structure is requiredfor DCS-PK function. For stem 2 and stem 3, maintaining theirsecondary structures is most likely sufficient for the function of

DCS-PK, whereas in the case of stem 1, its highly conserved se-quence might be required for DCS-PK function.

The function of stem 2-loop 3 hairpin depends on its second-ary structure. To characterize the structure and function ofDCS-PK in detail, a novel DEN-4 replicon was designed (Fig. 5) tobypass the coding role of DCS-PK sequence. In this replicon, thetranslation of viral protein is under the control of EMCV IRES,and the 0.6-kb spacer sequence separates the 5=UTR-capsid regionand IRES to ensure the correct folding of both (Fig. 5A). To abort5= cap directed translation of capsid ORF, one stop codon wasintroduced into the loop sequence of the cHP element (18) with-out changing its secondary structure. SHAPE analysis showed thatthe stop codon introduced into cHP did not change the overallsecondary structure of DEN-4 5= end (data not shown). The p4-cHPstop-SP-IRES-RLuc-Rep replicated similarly to p4-Rluc-Rep(Fig. 5B) and was suitable for further mutation experiments.

Sequence alignment showed that the primary sequences of thestem 2-loop 3 hairpin are not conserved among flaviviruses, sug-gesting that the secondary structure is crucial in determining thefunction of the stem 2-loop 3 hairpin. To verify this hypothesis, aset of mutations (Fig. 6A) were introduced into p4-cHPstop-SP-IRES-RLuc-Rep, and their replication efficiency was monitored

FIG 5 Design of replicons uncoupling the coding role of DCS-PK from its function in vRNA replication. (A) Organization of replicons used in the present study.In p4-Rluc-Rep, the Rluc ORF was fused in frame with the capsid ORF, whereas in p4-cHPstop-SP-IRES-Rluc-Rep, the EMCV IRES sequence was locatedupstream of the Rluc ORF and directed the translation of the Rluc-DENV polypeptide, and a spacer sequence separated the DENV 5= end and IRES domain toensure the correct folding of both. The foot-and-mouth disease virus 2A sequence was placed after the Rluc ORF in both replicons to cleave the luciferase peptidewith DENV nonstructural proteins. The C terminus of the envelope protein was retained for correct anchoring of DENV polypeptide. The black stars indicate thepositions where artificial stop codons were introduced. (B) Replication kinetics of different replicons. Relative luciferase units are expressed as the ratio of lightunits measured at different time points after transfection to the value at 6 h, which was set to 100%. A replicon containing a mutation in the conserved GDD motifof NS5RdRp (p4-cHPstop-SP-IRES-Rluc-Rep-GVD) is shown as control.

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by a transient luciferase assay. As shown in Fig. 6B, the role of thestem 2-loop 3 hairpin in vRNA replication was confirmed, as de-letion of the entire stem 2-loop 3 hairpin sharply decreased vRNAreplication. Randomization of the stem 2 sequence did not impairvRNA replication, whereas randomization of either the upstreamor downstream strand sequence, which disrupted stem 2 second-ary structure, significantly reduced vRNA replication 48 h post-transfection. Lengthening or shortening stem 2 by 2 bp did notadversely affect vRNA replication. Randomization of 4 of 5 nt inloop 3, lengthening loop 3 by 2 nt, or shortening it by 2 nt also hadno harmful effects on vRNA replication. In summary, the aboveresults demonstrate that the function of stem 2-loop 3 hairpindepends on its secondary structure and is independent of primarysequence.

Sequence requirements of stem 1-loop 2 for efficient vRNAreplication. Loop-helix interactions have been found in manyH-type pseudoknots (41, 42, 43, 44). Generally, loop 2 crosses theminor groove of stem 1, and loop 1 crosses the major groove ofstem 2 (corresponding to stem 3 of DCS-PK), which facilitates theformation of tertiary contacts. Although DCS-PK is not a classicalH-type pseudoknot, the conservation of stem 1 and loop 2 se-

quences (Fig. 1) and the low SHAPE activity of loop 2 (Fig. 2)imply that loop-helix interactions might play roles in its function.To investigate the sequence requirements of stem 1 and loop 2 forvRNA replication, two panels of mutagenesis were performedbased on p4-cHPstop-SP-IRES-RLuc-Rep (Fig. 7). First, random-izing, lengthening, or shortening stem 1 and the disruption ordeletion of the stem 1 secondary structure significantly reducedvRNA replication (Fig. 7A and B), demonstrating the high se-quence dependence on conserved stem 1 for optimal vRNA repli-cation. Next, five base pairs of stem 1 were substituted with differ-ent Watson-Crick base pairs (Fig. 7C). Base pair substitutions(1.2GC, 1.3CG, and 1.4GC) significantly enhanced replicationcompared to single mutations aiming to disrupt the correspond-ing base pairs (G64C, C63G, and C51G) (Fig. 7D), confirming theimportance of secondary interactions in stem 1 for DCS-PK func-tion; however, the replication of all of the base pair-substitutedreplicons was reduced to some extent compared to the WT repli-con. For A48-U65, replacing it with a G-C base pair reducedvRNA replication to ca. 15% relative to the level of the WT repli-con 48 h posttransfection (referred to as the percentage of WTbelow). For G50-C63, the only mutant able to reach 50% of the

FIG 6 Mutagenesis of the stem 2-loop 3 hairpin of DCS-PK. (A) Mutations targeting stem 2-loop 3 hairpin are shown. The mutation sites are indicated in bold.The stem 2-loop 3 hairpin was deleted in �S2. The S2Random mutant replaced stem 2 with a randomized stem sequence. The S2US and S2DS mutants containedthe 5= and 3= halves of the randomized stem, respectively. Stem 2 was lengthened in S2Plus, and it was shortened in S2Min. The L3Random, L3Plus, and L3Minincluded a randomized, lengthened, and shortened loop 3, respectively. This illustration follows the rules outlined in Fig. 3A. (B) Replication of different repliconmutants. Relative luciferase units are expressed as the ratio of light units measured at different time points after transfection to the value at 6 h, which was set to100%.

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FIG 7 Mutagenesis of the stem 1-loop 2 region of DCS-PK. (A) Mutations targeting stem 1 are shown in the pseudoknot structure of DCS-PK. The S1Randommutant replaced stem 1 with a randomized stem sequence. The S1US and S1DS mutants contained the 5= and 3= halves of the randomized stem, respectively. Stem1 was lengthened in S1Plus, and it was shortened in S1Min. The �S1 mutant contained deletion of the A48 to U65 region in capsid ORF. This illustration followsthe rules outlined in Fig. 3A. (B) Replication of replicon mutants shown in panel A. Relative luciferase units are expressed as the percentage of the value 6 hposttransfection, which was set to 100%. (C) Single-base-pair and -nucleotide mutations that were introduced into DCS-PK stem 1-loop 2 region, and theirreplication efficiency. Nucleotides in rectangles or circles were substituted with the nucleotides indicated by arrows. The “�” symbol means deletion. Numbersnear the main DCS-PK structure indicate the positions of the corresponding nucleotides in the capsid ORF. The 1.1GC mutant contained the A48-U65 bpsubstituted with G48-C65, and 1.1UA had a U48-A65 substitution, whereas the A48U mutant, which indicates that A48 was mutated to U, disrupted this basepair. For each base pair, at least three mutants were constructed, and the nomenclature followed the example of the A48-U65 bp. The replication efficiency(mean � the standard deviation) is listed after the name of the corresponding mutant and is expressed as the ratio of a mutant’s relative luciferase units at 48 hafter transfection to the value of the WT replicon, which was set to 100. (D) Replication of mutants shown in panel C. Relative luciferase units are expressed asthe percentage of the value 6 h posttransfection, which was set to 100%.

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level of WT was the C63U mutant (G50-C63 Watson-Crick toG50-U63 wobble), whereas mutants containing other base pairsreplicated poorly, indicating that G50 and the base-pairing inter-actions with its partner are absolutely required for DCS-PK func-tion. All of the G52-C61 base pair substitutions reduced vRNAreplication to ca. 15 to 30% of the WT. Substitutions of the C49-G64 and C51-G62 pairs had less effect on vRNA replication thansubstitutions of the other three base pairs. The above results dem-onstrate that both the base composition and orientation of thebase pairs in stem 1 are important for DCS-PK function.

Loop 2 of DCS-PK has consensus sequence G/AAAGA. Short-ening or lengthening loop 2 significantly reduced vRNA replica-tion (Fig. 7D), indicating the importance of the length of loop 2.All single-base substitutions reduced vRNA replication �2-foldcompared to WT, except for G87A, which replicated to ca. 70% ofWT. These results agree with sequence alignments, which showthat G and A exist at the 5= first position of DCS-PK loop 2 indifferent flaviviruses, whereas the other nucleotides are highlyconserved (Fig. 1). Thus, the loop 2 nucleotide sequence is criticalfor optimal vRNA replication. These results indicate that most ofthe conserved nucleotides in stem 1 and loop 2 are indispensablefor optimal vRNA replication. The above results also agree with

the common loop-helix interaction feature of classical pseudo-knots, suggesting the presence of tertiary interactions betweenDCS-PK stem 1 and loop 2.

Loop 1-stem 3 interactions are important for the structureand function of DCS-PK. To investigate the role of loop 1 inDCS-PK function, mutagenesis was performed based on theabove-mentioned replicon system (Fig. 8A). First, A53G mutationor A53 deletion significantly reduced vRNA replication (Fig. 8B).In contrast, A53U only had little effect on vRNA replication, andthe insertion of 2 nt into loop 1 also did not significantly changevRNA replication (Fig. 8B). Thus, the exact length of loop 1 is notrequired for DCS-PK function, which is different from loop 2. Theresults of the mutagenesis analysis fit well with the sequence com-parison, and loop 1 should contain at least one non-G residue(Fig. 1A).

SHAPE analysis was then performed for the loop 1 mutantsA53G and A53U (Fig. 8C and D). A53G mutation led to a dra-matic increase in the SHAPE activity of stem 3, whereas substitut-ing A53 with U53 did not change the overall SHAPE activity ofDCS-PK. Thus, loop 1 is important for the stability of DCS-PKstructure. These results suggest that, similar to the loop 1-stem 2major groove interactions in classical pseudoknots, loop 1 inter-

FIG 8 Mutagenesis of the loop 1 region of DCS-PK. (A) Mutations introduced into DCS-PK loop 1 region. A53G and A53U replaced A53 with G and U,respectively. The A53�GA mutant increased the length of loop 1 by inserting GA after A53. A53 was deleted in �A53. In the L1J mutant, A53 was mutated to GC,which corresponded to the loop 1 sequence of JEV. In S3J, stem 3 was replaced by the homologous stem region from JEV. In S3L1J, the loop 1 and stem 3sequences were from JEV. (B) The luciferase activity of the replicon mutants shown in panel A 6 to 48 h after transfection. Relative luciferase units are expressedas the percentage of the value 6 h posttransfection, which was set to 100%. (C) Capillary electrophoresis diagrams of a SHAPE experiment with A53U and A53Gmutants are shown. ddG dideoxy sequencing is shown as a ladder. The regions corresponding to the DCS-PK and 5=CS are indicated by braces. (D) Comparisonof the SHAPE activities of WT, A53G, and A53U DCS-PK. Nucleosides reactive with NMIA are indicated by a black square.

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acts with stem 3 to maintain the structure and function ofDCS-PK because the A53G mutation greatly reduced the stabilityof stem 3 without influencing its secondary interactions. Impor-tantly, substitution of the DCS-PK loop 1 sequence of DEN-4 withthe corresponding sequence from JEV (L1J) reduced vRNA repli-cation, whereas replacing loop 1 and stem 3 with homologous

regions from JEV (S3L1J) restored vRNA replication. Although theS3J mutant’s replication efficiency was similar to WT, these data pro-vide functional evidence that loop 1 interacts with stem 3 (Fig. 8A andB). Taken together, the highly consistent results from SHAPEand mutagenesis demonstrate that the interaction between loop 1and stem 3 is critical for the structure and function of DCS-PK.

FIG 9 Mutagenesis of the stem 3 region of DCS-PK. (A) Demonstration of mutations targeting the entire stem 3 region of the DCS-PK element. The mutantS3Random had the 5= helical region of stem 3 replaced by a random double-stranded region, whereas S3US and S3DS served as controls similar to the design ofthe stem 1 and stem 2 mutants. S3Plus had a lengthened stem 3, and S3Min had a stem 3 that was shortened by 1 bp. The 5= helical region of stem 3 was replacedby the corresponding region from JEV in the S3.1.5-J mutant. (B) Replication kinetics of the replicon mutants shown in panel A. Relative luciferase units areexpressed as the percentage of the value 6 h posttransfection, which was set to 100%. (C) Single-base-pair and -nucleotide mutations that were introduced intoDCS-PK stem 3 region. The A59U mutation rendered stem 3 a continuous helix. The �A59, �A93, and �A59.A93 mutants deleted A59, A93, and both A59 andA93, respectively. The A59G and A93G mutants contained the corresponding A substituted with G. The mutant nomenclature and data representation follow therules described in Fig. 7C. (D) Luciferase activity of the mutant replicon RNAs shown in panel C 6 to 48 h after transfection. Relative luciferase units are expressedas the percentage of the value 6 h after transfection, which was set to 100%. (E) Modeling of the cWW_tHS GCA base triple between loop 1 and stem 3. Thisillustration follows the Leontis-Westhof nomenclature. The top nucleotides in the shown triples are from loop 1 and the bottom base-pairing nucleotides arefrom stem 3. Structural files are from the RNA base triples database. Steric clashes are indicated by a cross symbol.

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Structural requirement of stem 3 for efficient vRNA replica-tion. Since the sequence of stem 3 is not conserved among flavi-viruses, mutagenesis was performed to investigate the structuralrequirement of stem 3 for DCS-PK function. As shown in Fig. 9,changing the length of stem 3 by 1 bp or replacing the 5= helix ofstem 3 with the corresponding region from JEV did not affectvRNA replication, but randomization of the 5= helix of stem 3 orhalf of it significantly reduced vRNA replication (Fig. 9B). Mu-tagenesis analysis of bulged A59 and A93, the A55-U97, G56-C96,A58-U94, and C60-G92 bp (Fig. 9C) was performed based on thereplicon system. Deletion of A59 or A93 or the combination ofboth did not affect vRNA replication, and substitution of the A59-A93 bulge with a U-A base pair also had little effect on vRNAreplication, suggesting that the bulges in stem 3 are dispensable for

DCS-PK function; however, the A59G and A93G mutants showedreduced replication compared to WT (Fig. 9D). Substitution ofA55-U97, G56-C96, and A58-U94 with other base pairs did notimpair vRNA replication, suggesting that these base pairs are notstrictly required for DCS-PK function. In contrast, all of the mu-tants that deleted, disrupted, or substituted the C60-G92 bp withother base pairs replicated less efficiently than WT (Fig. 9D).These results suggest that the C60-G92 bp is the most likely deter-minant of stem 3’s ability to form tertiary interactions with loop 1to stabilize the structure of DCS-PK.

Localization of DCS-PK and its distance relative to the 5=CS.In most flaviviruses, the DCS-PK element is located just 2 to 3 ntdownstream of the 5=CS, with two or three adenosine (A) nucle-otides as the spacer sequence between them. Mutagenesis using

FIG 10 Interplay of DCS-PK with 5=CS. (A) Illustration of mutations introduced into the spacer region between the 5=CS and DCS-PK. The conserved AA waschanged to GG and UU in the 46.47GG and 46.47UU mutants, respectively. Seven additional adenosines were inserted into the 46.47pA spacer sequence. pAGGand GGpA contained AAAAAAAGG and GGAAAAAAA spacer sequences, respectively. (B) The organization of replicon mutants, which contained DCS-PKduplications, and mutants, which contained substitutions of DCS-PK with other RNA elements. DCS-PKX contained a mutated DCS-PK. DCS-PKX-PKcontained a mutated DCS-PK followed by a WT DCS-PK, and DCS-PKX-PKX contained two tandem-repeat mutated DCS-PKs. In DCS-SL6, DCS-PK wasreplaced by SL6 of TBEV. DCS-PK was substituted with pseudoknots from the bacteriophage T2 gene 32 mRNA (PDB 2TPK) in the DCS-2TPK mutant. InDCS-JEV, the DCS-PK of DEN-4 was replaced by the DCS-PK of JEV. (C) Replication of the corresponding replicon mutants. The data are presented as describedin Fig. 5 to 9.

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the DEN-4 replicon demonstrated that insertion of more adenos-ines into the spacer sequence had only a slight effect on vRNAreplication, whereas substitutions of adenosines with other nucle-otides (G or U) significantly reduced vRNA replication (Fig. 10Aand C). Inserting either WT or mutated DCS-PK (DCS-PKX-PKor DCS-PKX-PKX) into the downstream region of the mutatedDCS-PK in the original position failed to restore vRNA replication(Fig. 10B and C), confirming that proximity between DCS-PK and5=CS is required for DCS-PK function. Importantly, the DCS-PKof DEN-4 could be functionally replaced by the JEV DCS-PK butnot by the SL6 element of TBEV (27) or the H-type pseudoknot(45) from phage T2 gene 32 mRNA (Fig. 10B and C).

DCS-PK modulates vRNA cyclization. The dependence ofDCS-PK function on its proximity to the 5=CS suggests thatDCS-PK plays a role in vRNA cyclization. Thus, RNA bindingassays were performed to test this hypothesis. The 5= 300-nt RNAsof WT, S1X, S3X, 75.78, S2POS, and S2NEG were used to bind the3=UTR RNA (Fig. 11). The percentage of 5=-3= complexes in-creased and the percentage of free 3= RNA decreased as the con-centration of 5= RNA increased. No difference in complex forma-tion was found between 75.78, S2POS, and WT; however, thepercentages of 5=-3= complexes formed by S1X, S3X, and S2NEG5= RNA mutants were significantly lower than that formed by WTwhen the lowest concentration of 5= RNAs was used (Fig. 11, lane1�). At a higher 5= RNA concentration (Fig. 11, lane 10�), thepercentage of the remaining free 3= RNA was apparently higher forS1X, S3X, and S2NEG than for WT 5= RNA. These results demon-strate that the disrupted DCS-PK structure (Fig. 11, S1X, S3X, andS2NEG) hindered the formation of the 5=-3= complex and that an

intact DCS-PK structure is required for efficient vRNA genomecyclization. The above results agree with the vRNA replicationresults (Fig. 3 and 4), indicating that DCS-PK enhances vRNAreplication by modulating genome cyclization.

DISCUSSION

Accumulating evidence from the last few decades has revealed thatRNA can fold into complex tertiary structures to achieve variousfunctions (46–48), and many RNA regulation elements with spe-cific tertiary structures have been identified, even in mRNAs, suchas frameshifting pseudoknots (49) and riboswitches (50, 51). Inthe case of flaviviruses, several cis-acting elements have been iden-tified in both the 5= and 3=UTRs. The discovery of DCS-PK inmosquito-borne flaviviruses further increases our knowledgeabout the cis-acting RNA elements of flavivirus. The DCS-PK el-ement is located in the capsid-coding region of DENV and Culex-borne flaviviruses, but not YFV. This finding agrees with a phylo-genetics study (52) that indicated that the YFV group divergedearly and evolved independently, whereas DENV and Culex-borne flaviviruses are closer in evolution. The recently identifiedtick-borne flavivirus SL6 element (27) shares homology with stem1 of DCS-PK; however, these two elements are likely to modulatevRNA replication by distinct mechanisms. First, no structural ho-mology was observed between SL6 and DCS-PK; second, the or-ganization of the 5= ends were quite different between the tick-borne and mosquito-borne flaviviruses. It is likely that themosquito-borne and tick-borne flaviviruses acquired enhancer el-ements independently after their separation in evolution and thatthe enhancers perform their functions by distinct mechanisms,most likely due to the differences in the vector species. However,because the mechanism by which SL6 modulates the replication oftick-borne flavivirus is unclear, further studies are needed to un-veil the mechanistic difference between SL6 and DCS-PK.

In the present study, DCS-PK was demonstrated to enhancevRNA replication using biochemical and reverse genetic assays. Arecent report (29) suggested that the DENV CCR1 element, whichis homologous with the stem 1 hairpin of DCS-PK, serves as avRNA packaging signal but not a replication enhancer. Our resultsprovide strong evidence that DCS-PK plays a direct role in thevRNA synthesis phase of virus life cycle. Although whetherDCS-PK is also involved in vRNA encapsidation was not exam-ined in the present study, it will be interesting to investigate themany functions of DCS-PK by various approaches.

Mutagenesis analysis based on a newly designed DEN-4 repli-con indicated the following: (i) the overall secondary structure ofthe stem 2-loop 3 hairpin is needed for vRNA replication, which isconsistent with sequence comparison. (ii) The highly conservedstem 1 and loop 2 sequences are required for DCS-PK function,which is consistent with stem 1-loop 2 interactions in many pseu-doknot tertiary structures. The G52-C61 bp, which is located atthe 3= end of stem 1, is the most mutation-sensitive site in stem 1(Fig. 7), suggesting that more tertiary interactions exist aroundthis site. One possibility is the coaxial stacking with C60-G92 instem 3. G50 is absolutely required for vRNA replication, suggest-ing that it directly participates in tertiary interactions. (iii) Ter-tiary interactions between loop 1 and stem 3 are important forDCS-PK function. Compared to loop 2-stem 1, the loop 1-stem 3interaction involves fewer nucleotides, which renders the loop 1sequence more flexible, for example, its tolerance to insertions.The majority of stem 3 bp are sequence independent, although a

FIG 11 DCS-PK regulates vRNA cyclization. (A) RNA binding assay of 5=RNA containing WT, S1X, or S3X DCS-PK with 3= RNA. 5= RNA and 3= RNAalone were run in parallel as controls. The 5= RNA concentration was increasedas indicated from 1� to 10�. (B) RNA binding assay of 5= RNA containingS2POS, S2NEG, or 75.78 DCS-PK with 3= RNA. The sequences of the DCS-PKmutants are the same as those shown in Fig. 3 and 4.

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few mutants that do not change the secondary structure of stem 3do impair vRNA replication. A possible explanation for this is thatmost of the nucleotides of stem 3 are not directly involved intertiary interactions; however, mutations such as A93G and A59Gpossibly hinder the formation of correct tertiary interactions bysteric effects, thus impairing DCS-PK function. The C60-G92 bpin stem 3 is strictly required for vRNA replication, suggesting thata base triple interaction exists between C60-G92 and loop 1. Weattempted to model this base triple interaction based on the fol-lowing facts: (i) loop 1 interacts with the major groove face of stem3 and forms base triple(s) as in other pseudoknots. (ii) The thirdbase in this base triple interaction can be A, C, or U but not G. Sixbase triple families whose third nucleotide interacts with the ma-jor groove face of a Watson-Crick base pair were identified via theRNA base triples database (http://rna.bgsu.edu/triples/triples.php) (53). Among them, the cWW_tHS GCA (Leontis-Westhofnomenclature [54]) base triple (Fig. 9E) fully satisfies the aboverequirements and explains the loop 1-stem 3 interactions well. Inthis model, the A nucleotide in loop 1 can be substituted with C orU, but not G, due to the steric effects between the 2-exocyclicamino group of G and the 4-exocyclic amino group/5-hydrogenof C.

It has been proposed that the initiation of flavivirus negative-strand RNA synthesis involves three phases: (i) the vRNA switchesto the circular conformation, which is facilitated by 5=-3=UAR,DAR, and CS hybridization, to bring the 5= and 3= ends close andcause the bottom of 3=SL to melt; (ii) the viral RdRp binds to the

SLA element at the 5= end; and (iii) the viral RdRp is translocatedto the 3= end and initiates negative-strand RNA synthesis (8).Friebe and Harris (13) reported that the 5=UAR, 5=DAR, cHP, and5=CS must act together to achieve their functions, and the se-quence downstream of the 5=CS affects vRNA cyclization (30). Inthis report, we further demonstrated that the DCS-PK elementregulates vRNA cyclization, since the disruption of DCS-PK struc-ture hindered the ability of 5= RNA to bind 3= RNA, whereas therestoration of DCS-PK structure also recovered the formation of5=-3= complex (Fig. 11). We also showed that the function ofDCS-PK in vRNA replication depends on interplay with the 5=CS.Based on these results, we propose that the DCS-PK, together withthe 5=UAR, 5=DAR, cHP, and 5=CS, form a functional domain thatmodulates the conformational change of vRNA ends (Fig. 12).The 5=UAR, 5=DAR, cHP, and 5=CS constitute the core of thiscyclization domain, whereas DCS-PK helps the core fold into aspecific conformation that favors genome cyclization by potentialtertiary interactions. More efficient genome cyclization improvesthe overall efficiency of the vRNA replication process. Furtherinvestigation of the structure and function of this cyclization do-main will facilitate the in-depth understanding of flavivirus repli-cation and provide new targets for the design of anti-flavivirusdrugs.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation ofChina (31270196, 31000083, and 30972613), National 973 project ofChina (2012CB518904), and the Open Research Fund Program of theState Key Laboratory of Virology of China (no. 2012005).

We thank Stephen S. Whitehead (National Institute of Allergy andInfectious Diseases, National Institutes of Health) for the p4 infectiousclone and Xiao-Yan Che (Zhujiang Hospital, Southern Medical Univer-sity, China) for 2D73 monoclonal antibody.

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