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Journal of Virology, August 2005, p. 10278-10288, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10278-10288.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Structure of the Dengue Virus Helicase/Nucleoside Triphosphatase Catalytic Domain at a Resolution of 2.4 Å

Ting Xu,1,{dagger} Aruna Sampath,2,{dagger} Alex Chao,2 Daying Wen,2 Max Nanao,4 Patrick Chene,3 Subhash G. Vasudevan,1,2 and Julien Lescar1*

School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive Singapore 637551,1 Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670,2 Novartis Institute for Biomedical Research, Oncology Department, CH-4002 Basel, Switzerland,3 EMBL, Grenoble outstation, Grenoble, France, 380434

Received 11 March 2005/ Accepted 24 April 2005


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ABSTRACT
 
Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3' single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.


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INTRODUCTION
 
Dengue fever is a viral infection transmitted by Aedes mosquitoes to humans, with 50 to 100 million cases occurring annually. The growing urbanization of populations combined with the failure to control the mosquito vector has resulted in the spread of the disease. Today, about two and a half billion people live in areas where viral transmission of one of the four serotypes of Dengue virus occurs, making dengue an increasingly important public health concern (19). The clinical features of infection range from a febrile flu-like illness to more severe and potentially lethal forms called Dengue hemorrhagic fever and Dengue shock syndrome. The Dengue virus belongs to the Flavivirus genus, which includes other important human pathogens such as yellow fever, Japanese encephalitis, and West Nile viruses within the Flaviviridae family of enveloped viruses (29). There is no effective specific antiviral therapy available to treat Dengue or any other infection caused by flaviviruses, and the control of Dengue fever through vaccination is currently elusive.

The Dengue virion contains a single-stranded, positive-sense RNA genome of approximately 11 kb which is translated into a large polyprotein during the infectious life cycle. This polyprotein is processed by cellular and viral proteases into three mature structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Replication of flaviviruses occurs in the cytoplasm of infected cells within membrane-associated replication complexes composed of viral RNA and nonstructural proteins as well as poorly identified host factors (47). NS3, a large multifunctional protein of 618 amino acids endowed with protease, helicase, nucleoside 5'-triphosphatase (NTPase), as well as 5'-terminal RNA triphosphatase activities, plays an important role in viral polyprotein processing and genome replication (29).

The N-terminal 180 amino acids of NS3 comprises a serine protease domain, with the protein NS2B acting as a membrane-anchoring cofactor, necessary for proteolytic activity (46, 8, 28, 49). Its C-terminal domain is involved in viral RNA replication (2, 3). The region spanning residues 180 to 618 of the Dengue virus NS3 amino acid sequence comprises two motifs named Walker A, GK(S/T), and Walker B, DEx(D/H). These motifs are present in a vast family of nucleotide binding proteins that participate in a wide variety of cellular functions by coupling NTP hydrolysis with directional movement, nucleic acid duplex destabilization, RNA processing, and DNA recombination and repair (44, 37).

The presence of five additional conserved motifs places NS3 in superfamily 2 of RNA helicases/NTPases, according to the classification of helicases into three major superfamilies (17). Functionally, the helicase and NTPase activities of the NS3 protein have been characterized for several members of the Flaviviridae, including hepatitis C virus (20), Dengue virus (3), West Nile virus (4), yellow fever virus (45), and Japanese encephalitis virus (42). Dengue viruses and bovine viral diarrhea virus with impaired helicase activity are not able to replicate, demonstrating the importance of NS3 in the Flaviviridae life cycle (30, 18).

Several helicase crystal structures have been reported, including the DNA helicases PcrA from Bacillus stearothermophilus (39) and Rep from Escherichia coli (25) that are representatives of superfamily 1 and the RNA helicase from HCV (48) and UvrB, an enzyme involved in nucleotide excision repair (31), which are members of superfamily 2. A "core" {alpha}/ß structural motif of about 150 amino acids is conserved across these two superfamilies and bears structural similarity with the RecA protein involved in homologous DNA recombination (38; reviewed in reference 7). In the helicase structures determined so far, this structural core is visible as a tandem of parallel {alpha}/ß structures that has probably arisen through gene duplication. Amino acids from the most conserved motifs, Walker A and B (motifs I and II, respectively; see Fig. 3), belong to the amino-terminal {alpha}/ß domain and interact with the NTP substrate and Mg2+, respectively (7).



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  		FIG. 3. Structural alignment (produced using program T-Coffee) (33) of Flaviviridae helicase domains. After cleavage with enterokinase, the mature Dengue virus NS3:171-618 protein which was crystallized has three additional amino acids, Ala, Met, and Ala (in italics) at its N-terminal end that come from cloning. The sequences of Japanese encephalitis virus (JEV, strain JaOArS982, NP_059434), West Nile virus (WNV, strainB956, AAT02759, tick-borne encephalitis virus (TBV, strain western subtype, NP_043135), yellow fever virus (YFV, strain 17D, NP_041726), and hepatitis C virus (HCV, strain H77, NP_671491) were obtained from GenBank. Secondary structure elements of Dengue virus NS3:171-618 are displayed above the sequence alignment. The conserved motifs among superfamily 2 helicases are indicated with purple (motif I), pink (motif II), orange (motif III), and yellow background (Ia, IV, V, and VI) and labeled.

However, several issues still remain unanswered regarding the precise mechanism that allows the conversion of chemical energy obtained through ATP hydrolysis with the strand separation activity of superfamily 2 helicases. Dengue virus NS3 provides an attractive model to study such questions.

Here we report a functional characterization of the Dengue virus serotype 2 NTPase/helicase domain as well as its refined three-dimensional structures in two crystal forms, to 2.4 Å and 2.8 Å resolution. The three-lobed structure displays an asymmetric distribution of charges on its surface and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 hepatitis C virus helicase. The presence of a sulfate ion bound at the NTPase active site in one crystal form reveals residues implicated in the catalytic mechanism assisted by a divalent metal ion. Superposition with the hepatitis C virus helicase domain bound to RNA (24) suggests a number of conserved residues of NS3 likely to contact nucleic acid and also a possible mechanism for strand separation.


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MATERIALS AND METHODS
 
Cloning and expression. The catalytic domain of the Dengue virus 2 helicase (residues 171 to 618) (accession no. AY037116; nucleotides 5032 to 6301) was amplified by PCR using the forward primer 5'-TAATTCCATGGCAAGCATCGAAGACAATCC-3' and the reverse primer 5'-TAATAAGCTTTTACTTTCTTCCAGCTGCAAA-3'. The underlined regions correspond to NcoI and HindIII sites, respectively. The pET32b plasmid containing the DNA insert between the NcoI and HindIII sites permits the expression of Dengue virus NS3 residues 171 to 618 fused at its N terminus to thioredoxin and a (His)6 tag followed by an enterokinase cleavage site. Transformed Escherichia coli BL21-CodonPlus (Stratagene) clones were grown at 37°C in LB medium containing 100 µg ml–1 ampicillin and 50 µg ml–1 chloramphenicol to an optical density at 600 nm of 0.6 to 0.8. Protein expression was induced at 14°C by adding isopropyl-ß-D-thiogalactopyranoside (IPTG) with a final concentration of 0.4 mM. After overnight growth, cells were harvested by centrifugation at 8,000 x g for 10 min at 4°C and stored at –20°C. The NS3FL gene was cloned and expressed as described (2). Expression of the selenomethionine (SeMet)-substituted protein was carried out as described (13).

Protein purification. Cells resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 5% glycerol) were lysed by sonication and the lysate clarified by centrifugation at 30,000 x g for 30 min at 4°C. The supernatant was purified by metal affinity using a HisTrap HP column (Amersham Bioscience) equilibrated with buffer A (20 mM Na3PO4, pH 7.4, 0.5 M NaCl, 40 mM imidazole). Proteins were eluted using a linear Imidazole concentration gradient in buffer A. After concentration by ultrafiltration and dilution in buffer B (20 mM Tris-HCl, pH 8.0), the protein was loaded onto a HiPrep 16/10 Q Sepharose Fast Flow (Amersham Bioscience) preequilibrated with buffer B and eluted using a linear NaCl concentration gradient. Fractions containing Trx-NS3:171-618 were pooled and concentrated by ultrafiltration and the buffer changed to 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM CaCl2 in order to remove the Trx-(His)6 fusion partner by enzymatic cleavage.

The NS3:171-618 protein was obtained by enterokinase digestion (substrate/enzyme ratio of 200:1) at 4°C for approximately 12 h. The enzymatic reaction was stopped by adding a protease inhibitor cocktail (Sigma) and the cleavage mixture was loaded onto an Econo-column (Bio-Rad) containing 2 ml of Ni-nitrilotriacetic acid resin in buffer C (20 mM Na3PO4, pH 7.4, 0.5 M NaCl) in order to remove the Trx-(His)6 protein from the mixture. Concentrated NS3:171-618 proteins were further purified using a HiPrep Sephacryl S-100 column in buffer D (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 1 mM EDTA, 1 mM dithiothreitol). Fractions containing NS3:171-618 were pooled and concentrated to 20 mg ml–1 in buffer E (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol). The purification of SeMet NS3:171-618 was similar to the native protein except for the presence of 10 mM dithiothreitol to prevent oxidation.

Helicase activity assay. The double-stranded RNA substrate was generated (28). Plasmid pGEM4Z, linearized with PvuII, was used as a template for SP6 RNA polymerase and transcribed in the presence of [{alpha}-32P]GTP (3,000 Ci/mmol) to generate a labeled single-stranded RNA of 34 nucleotides. Plasmid pGEM3Z linearized with XbaI was transcribed to generate a nonlabeled single-stranded RNA of 179 nucleotides with 37-nucleotide and 113-nucleotide tails at its 5' and 3' ends, respectively. The double-stranded DNA substrate was prepared as described (16). Briefly, primer 1 (5'-GCCTCGCTGCCGTCGCCA-3') was labeled at its 5' end using T4 polynucleotide kinase and [{gamma}-32P]ATP (Amersham Biosciences) for 1 h at 37°C and annealed with the complementary primer 2 (5' TGGCGACGGCAGCGAGGCTTTTTTTTTTTTTTTTTTTT-3').

The unlabeled and radiolabeled nucleic acids were mixed in a molar ratio of 5:1 and annealed in 25 mM HEPES, pH 7.5, 1 mM EDTA, 0.5 M NaCl, 0.1% sodium dodecyl sulfate in a thermocycler (95°C for 5 min, 55°C for 30 min, and 25°C overnight), precipitated with ethanol, and purified from a 10% native polyacrylamide gel electrophoresis. The reaction mixture contained 25 mM HEPES, pH 7.5, 5 mM ATP, 3 mM MnCl2, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, 5 U RNasin (for the RNA substrate), and 0.25 pmol of double-stranded substrate and either NS3:171-618 or NS3FL in a final volume of 20 µl. The mixture was incubated for 30 min at 37°C and the reaction was terminated by the addition of 5 µl of 5x loading dye (100 mM Tris-HCl, pH 7.5, 50 mM EDTA, 0.1% Triton X-100, 0.5% sodium dodecyl sulfate, 50% glycerol, 0.1% bromophenol blue). The helicase assay mixtures were resolved on a 10% native polyacrylamide gel electrophoresis and analyzed on a Typhoon Phosphorimager using the ImageQuant software (Amersham Biosciences). The percentage of duplex unwinding was calculated by comparing the intensities of the two bands. The background from the negative control was subtracted.

NTPase activity assay. The assay was carried out as described (26). Purified NS3:171-618 (or NS3FL) at a concentration of 5 nM was preincubated in a 96-well plate (Nunc, Immunoplate F96 MaxiSorp) for 5 min at 37°C in 90 µl reaction buffer (50 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 1.5 mM dithiothreitol, 0.05% Tween 20, 0.25 ng/µl bovine serum albumin [Sigma]). The reaction was initiated by the addition of 10 µl ATP and carried out for 10 min at 37°C. The Malachite green reagent (200 µl) was then added and the absorbance was immediately measured at 630 nm. The Km of the enzyme was determined from measurements of the initial rates at different ATP concentrations with the GraphPad Prism Software.

Crystallization and data collection. Crystals of Dengue virus NS3:171-618 were grown by the hanging drop vapor diffusion method over wells containing 0.1 M MES (morpholineethanesulfonic acid, pH 6.5), 0.2 M (NH4)2SO4, 14% polyethylene glycol 8000. A volume of 2 µl of well solution was mixed with an equal volume of NS3:171-618 at a concentration of 10 mg ml–1. The drop was equilibrated against a reservoir containing 1 ml of the precipitating solution at 18°C. After macroseeding, crystals of the native (as well as the SeMet) protein grew as thin elongated plates over 2 to 5 days to dimensions of approximately 0.02 by 0.3 by 0.1 mm3. For data collection, crystals were soaked in a cryoprotecting solution of 0.1 M MES, pH 6.5, 0.2 M (NH4)2SO4, 14% polyethylene glycol 8000, 25% glycerol before being mounted and cooled to 100 K in a nitrogen gas stream (Oxford cryosystems). Diffraction intensities were recorded on beamline ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) on an ADSC charge-coupled device detector, using an attenuated beam of 0.125 by 0.050 mm2. Integration, scaling, and merging of the intensities were carried out using programs from the CCP4 suite (10).

Phasing, model building, structure refinement, and analysis. The structure was solved using single anomalous dispersion data collected at the peak of the Selenium absorption edge (Table 1) from one selenomethionine derivatized NS3:171-618 crystal. Out of the 32 selenium atoms present within the two molecules of the asymmetric unit, 28 could be located using program SOLVE (40). An initial map was calculated and modified with program RESOLVE (40), using the heavy atom positions to locate the noncrystallographic symmetry axis relating the two molecules present in the asymmetric unit. The program SHARP (15) was used to locate three other selenium atoms and a new set of single anomalous dispersion phases were calculated and modified with the program SOLOMON/DM (10). The resulting map was of extremely good quality, allowing the tracing of about 400 residues.


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  		TABLE 1. Data collection and phasing statistics

Many aromatic residues could be clearly identified in the electron density map and the 15 SeMet residues per molecule were used as markers to assist amino acid sequence assignment using program O (23). The SeMet structure was partially refined with ncs restraints, using experimental single anomalous dispersion phases as additional restraints (program CNS) (6). The best defined of the two independent molecules in the asymmetric unit was constructed, and after modifications were made to the model, the second molecule was generated by applying the ncs operator. This partial model was subsequently placed into the native crystal form using molecular replacement (32) and structure building and refinement was carried out using difference Fourier maps. Finally this model was placed back into the SeMet crystal form and used as a guide to complete refinement (Table 2).


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  		TABLE 2. Refinement statistics

The coordinates have been submitted to the PDB with accession numbers 2BHR and 2BMF for the SeMet and native structures, respectively. Figures 4 to 7 were drawn using program PyMOL (11). Superpositions of structures and root mean square (RMS) deviation calculations were carried out using program LSQKAB from the CCP4 suite (10).



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  		FIG. 4. (A) Overall fold of Dengue virus NS3:171-618 helicase. Secondary structural elements are labeled. The same color code (and the relative orientation between the three domains) is also used in the topology diagram. The sulfate ion bound in the ATPase catalytic site is represented as sticks. (B) Folding diagram of the Dengue virus NS3:171-618 helicase using the same color code. The location of the conserved superfamily 2 helicases motifs is indicated. The missing segment connecting strands ß2A and ß3 in domain I comprising residues 244 to 251 is drawn as a broken line.



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  		FIG. 7. Left panel: Surface representation of the Dengue virus NS3:171-618 protein in the same orientation as in Fig. 4A, illustrating the presence of a tunnel across this face of the protein and the presence of several basic patches able to accommodate nucleic acid substrates (see text). Positive electrostatic potentials are colored blue and negative are colored red. The putative interaction between Dengue virus NS3:171-618 and a single-stranded nucleic acid substrate (represented as sticks) is shown. The model was obtained by superposition with the hepatitis C virus helicase in complex with a dU8 oligonucleotide (PDB code 1A1V) (24). The right panel is a view with the molecule rotated by 180° around a vertical axis. An excess of negative charges is visible on this face of Dengue virus NS3:171-618.


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RESULTS AND DISCUSSION
 
Functional characterization. The helicase unwinding activity was assayed using radiolabeled substrates having a 3' single-stranded tail, in the presence of Mn2+, ATP, and various concentrations of enzyme. At relatively high concentrations, NS3:171-618 displays strand displacement activity using both double-stranded DNA and double-stranded RNA as substrates (Fig. 1A and 1B). This is in contrast with the hepatitis C virus NS3 helicase, which unwinds DNA duplexes more efficiently and whose RNA unwinding activity is enhanced by the presence of the NS4A cofactor and the intact protease domain (16, 34). Replacement of the Mn2+ divalent metal ion with Mg2+ did not alter the unwinding activity (not shown).



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  		FIG. 1. Helicase activity of Dengue virus NS3. The values represent an average of three experiments. (A) Unwinding activity of Dengue virus NS3:171-618 using a radiolabeled double-stranded DNA substrate. The first lane is the negative control in the absence of enzyme. Enzyme concentrations are indicated. (B) Unwinding activity of Dengue virus NS3:171-618 with a double-stranded RNA substrate. The second lane is the positive control (heat-denatured duplex). (C) Unwinding activity of the full-length NS3 protein with a double-stranded RNA substrate.

In order to check whether the Dengue virus helicase behaves similarly, we compared the unwinding activity of NS3:171-618 with that of NS3FL. Interestingly, based on the enzyme concentrations required to achieve 50% unwinding of a double-stranded RNA substrate, NS3FL shows a 30-fold higher unwinding activity compared to NS3:171-618 (Fig. 1). A similar increase in activity was obtained using double-stranded DNA as a substrate.

Determination of the kinetic parameters for ATP hydrolysis for both NS3:171-618 and NS3FL was carried out by monitoring the amount of inorganic phosphate released in a colorimetric assay (Fig. 2) (26). The results show that NS3:171-618 has a higher affinity for ATP (Km = 34 ± 3 µM) than NS3FL (Km = 297 ± 34 µM). Both enzymes have similar turnover numbers (kcat = 6.9 s–1 and 5.8 s–1 for NS3:171-618 and NS3FL, respectively).



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  		FIG. 2. Comparison of the ATPase activity of NS3FL and NS3:171-618. The ATPase assay (see Materials and Methods) was carried out with 5 nM of enzyme in the presence of the indicated concentrations of ATP. The amount of inorganic phosphate released during catalysis was measured with the malachite green reagent and the initial rates were calculated (pmol PO4 s–1). The results were fitted using the Michaelis-Menten equation (Vo = [Vmax [S]/(Km + [S])].

Taken together, our results demonstrate that the NS3 fragment we crystallized is enzymatically active in both NTP hydrolysis and duplex unwinding. In addition, a strong influence of the N-terminal protease domain of Dengue virus NS3 on both its helicase and ATPase activity is observed, comparing well with the previously published helicase and NTPase activities of similar Dengue virus 2 NS3 fragments, comprising residues 169 to 618 (3) and 161 to 618 (28). By decreasing the rate of ATP hydrolysis and simultaneously increasing its unwinding activity, domain fusion has provided the virus replication machinery with an optimized bifunctional enzyme. These enzymatic activities could be further regulated by additional protein-protein interactions in the context of the viral replication complex.

Structure determination and quality of the model. The structure of Dengue virus NS3:171-618 was determined using one crystal of SeMet with data collected to 2.8 Å resolution at the selenium absorption edge. A native data set was collected to 2.4 Å resolution. The native and SeMet proteins both crystallized in space group P21, with a slight variation in one unit cell dimension (Table 1). Both crystal forms contain two molecules of Dengue virus NS3:171-618 per asymmetric unit with a similar crystal packing. A summary of the data collection, phasing, density modification, and structure refinement statistics is shown in Table 1 and 2.

Overall, the path of the main chain is unambiguously defined in the electron density maps of each crystal form. One of the two molecules in the asymmetric unit has a lower average temperature factor and is better ordered. After superposition, the r.m.s. deviations between 428 equivalent C{alpha} atoms of the two best-ordered copies in the two crystal forms is 0.51 Å. Differences between the four molecules present in the two asymmetric units are located at their N-terminal ends, within the phosphate binding loop and in the loops exposed to the solvent. Residues 244 to 251 within domain 1 are missing in our model (see Fig. 4). The equivalent residues form the solvent accessible strand ß4 of the seven-stranded ß-barrel in the hepatitis C virus NS3 structure (24). However, this stretch of sequence is predicted to form a solvent-exposed loop in Dengue virus NS3 and could be mobile. A number of charged side chains on the surface of the protein are also not visible in the electron density map and have been omitted from the final model.

Overall architecture. The structure of Dengue virus NS3:171-618 depicted in Fig. 4 together with its topology diagram reveals a three-lobed flattened structure, comprising a large number of loops and with overall dimensions of about 60 Å by 60 Å by 35 Å. A significant structural feature is a long tunnel that runs across the center of one face of the protein (see Fig. 7).

An amino acid sequence alignment of the NS3 helicases from several members of the Flaviviridae is shown in Fig. 3. In contrast to SF1 helicases, which comprise four structural domains, two being present as insertions within the tandem core structure (39, 25), the Dengue virus NS3:171-618 helicase reveals three structural domains of about 150 amino acids each, separated by a series of clefts that follow each other in sequence, like in the hepatitis C virus NS3 helicase structure (9, 24). Domain I (residues 181 to 326) and domain II (residues 327 to 481) show little sequence identity with each other, but are structurally similar, being composed of a large central six-stranded parallel ß-sheet with a pronounced twist, flanked by four {alpha}-helices (Fig. 4B).

Domain III (residues 482 to 618) is predominantly {alpha}-helical and could be described as a bundle of four approximately parallel {alpha}-helices ({alpha}1", {alpha}3", {alpha}4", and {alpha}7"), surrounded by three shorter helices ({alpha}2", {alpha}5", and {alpha}6"), and augmented by two antiparallel ß-strands largely exposed to the solvent. The major contacts between domains include a long ß-hairpin (ß4A' and ß4B') that extends largely from domain II into domain III and interactions between helix {alpha} 3 of domain I with {alpha}1" and {alpha}2" of domain III (Fig. 4). A superposition of the four independent molecules in the two crystal forms reveals no hinge motion between domains, suggesting a rather rigid structure in the absence of nucleic acid or ATP.

A comparison with the hepatitis C virus NS3 structure is shown in Fig. 5, where the two Flaviviridae helicase domains are displayed side by side. Domains I and II of dengue virus NS3:171-618 share, respectively, 19% and 20% sequence identity with their counterparts in hepatitis C virus NS3 and have a similar structure. After superposition of the individual domains, the r.m.s. deviations are 2.3 Å and 1.8 Å for domain I (114 equivalent C{alpha} atoms) and II (132 equivalent C{alpha} atoms), respectively. Domain III (residues 482 to 618) differs most between Dengue virus and hepatitis C virus, an observation consistent with the lack of detectable sequence identity between the C-terminal ends of these two proteins.



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  		FIG. 5. Side-by-side views of Dengue virus NS3:171-618 (left) and hepatitis C virus (right) helicases (9, 24, 48) displayed in the same orientation. Only the {alpha}-carbon atoms traces are shown. The two molecules were superimposed based on their structurally conserved domains I and II, colored grey. Their domains III (top of the figure) colored blue (Dengue virus NS3: 171-618) and red (hepatitis C virus) bear no significant structural similarity. The N- and C-terminal ends of the polypeptide chains are indicated.

A search through the Protein Data Bank, using the DALI server (22), did not return any significantly homologous structure, indicating that domain III of Dengue virus NS3 has a unique fold. This further illustrates that during evolution, the common structural core formed by the RecA-like tandem structures has been augmented with additional domains present as C-terminal additions for superfamily 2. Domain III, however, influences both the NTPase and helicase activities, as demonstrated by the mutation of a single Arg residue within helix {alpha}2" (Arg-513-Ala) which slightly decreases NTPase activity and produces a defective helicase (3). Recently, residues 303 to 618 of Dengue virus NS3 were shown to bind to the RNA-dependent RNA polymerase NS5 (5). This interaction might involve the C-terminal domain III of Dengue virus NS3. It will be interesting to determine whether the structural differences observed in domain III between the hepatitis C virus and Dengue virus NS3 helicases are correlated with a diverging mode of interaction with their RNA-dependent RNA polymerases.

Noncrystallographic dimer. In both crystal forms, the N terminus of one monomer adopts an extended conformation, making an intermolecular sheet with strand ß"1 of domain III in the neighboring molecule. Owing to the presence of the protease domain at its N terminus, a similar interaction could not be formed by the full-length NS3 protein. The other main intermolecular interaction involves residues 393 to 399 from domain II of one monomer with residues from domain III of the other monomer. Oligomerization has been proposed as a means to provide helicases with multiple nucleic acid binding sites, which would facilitate translocation of the protein along the strands (9, 25, 43). The hepatitis C virus NS3 helicase could oligomerize (27, 36). However, the dimer observed in our crystal differs from the one reported (9). Thus, it is unclear whether the dimer present in our crystal structure merely derives from packing constraints or from a specific interaction. The relatively small surface area (1,050 Å2) buried in this interaction, however, would rather favor the first possibility (1). This would be consistent with our observation using size exclusion chromatography that in the absence of nucleic acids, the NS3:171-618 native protein is a monomer.

NTPase active site. In one of the two crystal forms (SeMet, see Table 2), a sulfate ion from the crystallization buffer is located in a pocket next to the N-terminal end of helix {alpha}1 (Fig. 6), making close contacts with Arg-460, Arg-463 of motif VI, and residues protruding from the P-loop (motif I) and motif II. A network of solvent molecules is also buried in this pocket. One solvent molecule or a counterion makes hydrogen bonds with Asp-284 and Glu-285 of the DExH motif II. Overall, the orientations of the residues in this cavity match very well with equivalent residues of the PcrA helicase bound to a nonhydrolyzable ATP analogue (43) with the sulfate ion being located at a position between the ß- and {gamma}-phosphates of the adenylyl imidodiphosphate substrate (Fig. 6).



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  		FIG. 6. View of the ATP binding pocket of Dengue virus NS3:171-618 (yellow sticks) with the bound sulfate ion. Residues of Dengue virus NS3:171-618 are labeled in black. The PcrA helicase structure in complex with ADPNP (residues in pink) is overlaid (43). The water molecule (possibly the divalent Mg2+ or Mn2+) coordinated by residues Glu-285 and Asp-284 of Dengue virus NS3:171-618 (motif II) is represented as a brown sphere.

As shown in Fig. 6, the base and ribose moieties bulge out from the NTP binding cavity. As a consequence, there are minimal structural constraints for their recognition by the enzyme. This is consistent with a lack of specificity for the base, which is in agreement with the reported similar efficiency for either purine or pyrimidine triphosphate hydrolysis and also with the capacity of NS3 to hydrolyze short oligoribonucleotides regardless of the nature of the base moiety (3). Thus, residues Lys-37, Thr-38, Asp-223, Glu-224, Gln-254, Arg-610, and Arg-287 of PcrA (43) superimpose on their counterparts in Dengue virus NS3:171-618: Lys-199, Thr-200 of the P-loop (motif I), Asp-284, Glu-285 (motif II) and Gln-456, Arg-460, and Arg-463 of Dengue virus NS3 (motif VI), respectively. These residues are therefore likely to play similar roles during NTP hydrolysis with the side chain of Lys-199 contacting the ß-phosphate of the nucleotide, during transition state stabilization, and the strictly conserved Asp-284 and Glu-285 residues coordinating the divalent cation (Mg2+ or Mn2+) (43).

In the native crystal form, a peak is also visible at this location in the Fourier density map, but modeling it as a sulfate ion yields very high temperature factors in the range of 75 to 100 Å2, suggesting weak occupancy or high mobility. Indeed, a comparison of the four molecules in the two crystal forms shows that the presence of the sulfate ion stabilizes the conformation of the P-loop. Conversely, in the absence of the sulfate ion (native crystal), the glycine-rich P-loop is more mobile. This is accompanied by a slight displacement of helix {alpha}1 in one molecule of about 1.8 Å. As suggested previously (24, 38), a number of residues surrounding the NTP binding pocket are likely to function as sensors, by coupling nucleoside 5'-triphosphate binding and hydrolysis with nucleic acid recognition and duplex unwinding, through concerted allosteric conformational changes.

In this respect, both His-287 (motif II) and Gln-456 (motif VI) might play important roles. In our structure, the imidazole side chain of His-287 forms a hydrogen bond with the carbonyl oxygen of Glu-412 (motif V). It is also at the right distance to form polar contacts with the side chains of Glu-285 (motif II), Thr-317 (motif III), and Gln-456. In the Japanese encephalitis virus and hepatitis C virus NS3 helicases, substitution of this histidine residue by an alanine (yielding altered motifs II with sequences DEAA and DECA, respectively) dramatically reduces the helicase activity while most of the NTPase activity is retained (41, 21).

Mutagenesis studies performed on Gln-254 (motif III in PcrA) (12) which is spatially equivalent to Gln-456 of Dengue virus NS3 (Fig. 6) have demonstrated a correlation between the nature of the charge carried by this residue and the coupling between the ATPases and helicase activities, leading to the proposal that this residue might act as a {gamma}-phosphate sensor (12, 38). We also note in the same segment of the polypeptide chain (motif VI) the presence of two buried Arg residues, Arg-457 and Arg-458, which point away from the NTP binding site. The strictly conserved residue Arg-457 makes a hydrogen bond with the carbonyl oxygen of Arg-427 at the base of the ß4A' ß4B' hairpin. This polar interaction might also play a role by transmitting conformational changes between the NTP binding site and the long ß4A' ß4B' hairpin, which abuts onto domain III and is located in the vicinity of a putative nucleic acid binding site (see below).

Nucleic acid binding sites. The structure of Dengue virus NS3:171-618 reveals a tunnel at its center surrounded by residues emanating from the three domains. This tunnel is lined with a number of basic residues and is wide enough to accommodate a single-stranded nucleic acid substrate of about six nucleotides, but not a duplex (Fig. 7). Large conformational changes, like interdomain hinge motions, would be necessary to accommodate a double-stranded substrate at this position. Dengue virus-NS3:171-618 contains an unusually high proportion of charged residues and the distribution of these residues on its surface is asymmetric (Fig. 7). The face lined by the tunnel bears an excess of positively charged residues, with several basic patches able to accommodate nucleic acid duplexes, while the other face is more negatively charged. This suggests that electrostatic repulsion might play an important role, possibly by propelling the protein along the polymeric substrate and by preventing the reannealing of unwound nucleic acid strands.

We generated a model for single-stranded nucleic acid binding by superimposing Dengue virus NS3:171-618 to the hepatitis C virus NS3 helicase bound to a deoxyuridylate octamer (dU8) oligonucleotide (24). In this experimental complex, the dU8 ligand lies in a groove between the three domains and makes interactions with residues from motifs Ia, IV, and V. In our model, residues interacting with the phosphodiester backbone would include Arg-225 (motif Ia), Lys-366 (motif IV), Arg-387, Lys-388, and Arg-538 and Arg-599 from domain III. Interestingly, this model places two basic residues of motif VI, Arg-457 and Arg-458, which are well conserved across the Flaviviridae, more than 10 Å away from the nearest phosphate of the single-stranded nucleic acid ligand, suggesting that these basic residues are not directly involved in nucleic acid binding. When these two residues are mutated to alanine, Dengue virus ATPase activity is reduced and the helicase activity is lost (30). Thus, as proposed earlier, these residues might be involved in the coupling of the two enzymatic activities (30). Alternatively, this effect could be due to the modifications of the surface electrostatic potential.

Implications for Dengue virus NS3 function. Given their overall structural similarity and a common 3'-> 5' directionality for translocation along the polymeric substrate, the mechanisms for nucleic acid duplex unwinding by Dengue virus and hepatitis C virus NS3 proteins could be similar (24). In essence, translocation of the polynucleotide would result from interdomain movements triggered by the hydrolysis of a nucleotide at the NTP binding site. Translocation of the Dengue virus NS3 protein would then occur in the 3' to 5' direction along a single-stranded nucleic acid substrate trapped in the tunnel. Several locations are possible for nucleic acid duplex binding on the basic patches at the protein surface, but the polarity of the single-stranded substrate in the tunnel, if correct, would more likely place the duplex in contact with domains II and III (on the left side of the molecule in Fig. 4A), possibly in the concave surface between these two domains or at the surface of domain II (see Fig. 7, left panel).

Interestingly, this model puts the duplex in contact with several basic residues protruding from domain II: Arg-342, Lys-366; Arg-376, and Lys-377 (helix {alpha}1'), Lys-381; Lys-396 and Arg-398 (helix {alpha}2'); and Lys-418 and pointing towards the N-terminal domain, where the NS3 protease domain could provide additional interactions. Thus, our structure provides an experimental basis to carry out site-directed mutagenesis to probe the function of charged residues for nucleic acid binding and duplex unwinding. A number of residues lining the tunnel (e.g., Ile-365, Leu-443, and Arg-599) could play a role similar to Trp-501 of hepatitis C virus NS3 (24), or Trp-259 and Phe-626 of PcrA (43) by intercalating between consecutive bases to assist translocation along the single-stranded nucleic acid substrate.

One implication of our model is that a 3' single-stranded tail of a minimum of about 8 to 10 nucleotides would be required for initiation of the unwinding reaction. This requirement is in agreement with our preliminary observation of the absence of unwinding activity of Dengue virus NS3:171-618 on a blunt-ended duplex DNA substrate (not shown). The origin of the 3'-> 5' directionality (as opposed to 5'-> 3'), however, is more difficult to interpret. Detailed mechanistic studies as well as experimental structures of complexes with nucleic acid substrates are required to resolve these issues.

In summary, the three-dimensional structure of the Dengue virus NTPase/helicase catalytic domain should inform biochemical experiments to dissect the enzymatic mechanism and accelerate ongoing research programs aimed at developing compounds with specific antiviral activity.


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ACKNOWLEDGMENTS
 
We thank Félix Rey and Marc Delarue for useful comments and encouragements. We are grateful to Janet Smith and Richard Kuhn for discussions prior to joint submission of our manuscripts.

Financial support via grants from N.T.U. (SUG 14/02), the Singapore Biomedical Research Council (03/1/21/20/291 and 02/1/22/17/043), and the Singapore National Medical Research Council (NMRC/SRG/001/2003) to the laboratory of J.L. are acknowledged as well as provision of excellent beam time and support by the E.S.R.F. (Grenoble, France).


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive, Singapore 637551. Phone: (65) 6316 2859. Fax: (65) 6791 3856. E-mail: julien{at}ntu.edu.sg. Back

{dagger} These two authors have contributed equally to this work. Back


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Journal of Virology, August 2005, p. 10278-10288, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10278-10288.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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