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Journal of Virology, July 2006, p. 6686-6690, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02215-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structure-Based Mutational Analysis of the NS3 Helicase from Dengue Virus

Aruna Sampath,1,{dagger} Ting Xu,2,{dagger} Alex Chao,1 Dahai Luo,2 Julien Lescar,2* and Subhash G. Vasudevan1*

Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670, Singapore,1 School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive, Singapore 637551, Singapore2

Received 20 October 2005/ Accepted 10 April 2006


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ABSTRACT
 
We performed a mutational analysis of the NS3 helicase of dengue virus to test insights gleaned from its crystal structure and identified four residues in the full-length protein that severely impaired either its RTPase and ATPase (Arg-457-458, Arg-460, Arg-463) or helicase (Ile-365, Arg-376) activity. Alanine substitution of Lys-396, which is located at the surface of domain II, drastically reduced all three enzymatic activities. Our study points to a pocket at the surface of domain II that may be suitable for the design of allosteric inhibitors.


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TEXT
 
The dengue virus NS3 protein contains protease, RNA helicase, 5'-nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities (1, 2, 5, 15, 20, 21). NS3 proteins from the four dengue virus serotypes share a minimum of 67% amino acid sequence identity. The enzymatic activities of NS3 proteins from several members of the Flaviviridae have been studied, including hepatitis C virus (HCV) (9), yellow fever virus (YFV) (19), Japanese encephalitis virus (18, 19), and West Nile virus (4). The unwinding of duplex RNA structures yielding individual RNA strands is thought to be required for an efficient viral genomic RNA synthesis by the NS5 RNA-dependent RNA polymerase. The essentiality of the helicase activity of NS3 in viral replication has been demonstrated through site-directed mutagenesis (8, 17); hence, it is an attractive target for the design of antiviral compounds. Two three-dimensional (3D) structures of active flavivirus helicase/NTPase catalytic domains from dengue virus (23) and yellow fever virus (22), respectively, were recently reported. As with the HCV NS3 helicase (12), the structure can be divided into three domains. The seven sequence motifs characteristic of superfamily 2 helicases (7) are present in domains I and II, situated at the N-terminal end of the protein. The NTPase site resides between these two domains. The C-terminal domain III differs most with its HCV counterpart (22, 23) and was suggested to bind NS5 (10). A tunnel that runs across the interface between domain III and the tip of domains I and II was proposed to accommodate a single-stranded nucleic acid tail along which the enzyme could translocate, following interdomain motions triggered by NTP hydrolysis (22, 23). Interestingly, recent studies on HCV helicase (14) suggest that the energy derived from nucleic acid binding may be used to unwind several base pairs at the fork essentially without ATP. However, the unidirectional translocation of the enzyme along a long stretch of DNA is derived from ATP hydrolysis.

Matusan et al. (17) reported mutations in the conserved motifs of dengue virus NS3 helicase. Here, based on their evolutionary conservation and structural insights (22, 23), we targeted 14 residues within the NS3 helicase (Fig. 1) using site-directed mutagenesis. The mutant proteins were tested for their involvement in the RNA-stimulated NTPase, RTPase, and double-stranded RNA (dsRNA) unwinding activity. While the truncated carboxyl-terminal two-thirds of NS3 used to determine the 3D structure displays helicase activity, we performed the mutational studies on full-length NS3 protein (NS3FL), since its dsRNA unwinding activity is approximately 30-fold greater (23). The wild-type NS3FL gene from dengue virus 2 (TSV01 strain, accession number AY037116, nucleotides 4522 to 6375) and each individual mutant was cloned and expressed in Escherichia coli as a fusion with thioredoxin reductase (Trx) with an N-terminal hexahistidine tag, as described previously (23). The TrxNS3FL fusion proteins were soluble, with yields of 4 to 6 mg of enzyme per liter of culture. The structural integrity of each mutant protein was assessed by measuring its far-UV circular dichroism spectrum and found to be similar to the wild-type enzyme (data not shown).


Figure 1
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  		FIG. 1. Diagram depicting the conserved amino acid sequence motifs of dengue virus 2 NS3FL helicase (labeled I to VI) and the mutations performed in this study. The boundaries of the NS3 protease and helicase catalytic domains are indicated.

NTPase and RTPase activities of mutants. We tested the RNA-stimulated ATPase and RTPase activities of the mutants as previously described (1, 23). The kinetic parameters for ATP hydrolysis of wild-type TrxNS3FL and mutants are summarized in Table 1. Single alanine substitutions of the "arginine fingers" Arg-460 and Arg-463 in motif VI (corresponding to Arg-464 and Arg-467 in YFV helicase [22]) resulted in residual ATPase activities of 26% and 29%, respectively, and a comparable decrease in RTPase activity (Fig. 2A). This suggests that these two basic residues may be involved in transition-state stabilization via charge neutralization of the {gamma}-phosphate of either a bound NTP or an RNA 5'-triphosphate oligonucleotide. By contrast, three strictly conserved residues, Glu-230, Asn-329, and Gly-414, which are also within a distance of 5 to 6 Å from the ATP substrate, showed only a slight decrease in ATPase activity, implying a lack of any direct involvement in the catalytic mechanism. Interestingly, the single mutation Lys-396-Ala completely abrogated ATPase activity (Table 1). The other mutant proteins studied retained an enzymatic activity comparable to that of the wild-type enzyme or displayed a slight increase (up to twofold) (Table 1). Overall, a good correlation exists between the ATPase and RTPase activities for the various mutants studied, and the small differences observed may be attributed to the fact that the RTPase assay uses an RNA template whose binding may influence the measured activity (e.g., residues Gly-414 and Gln-456). Alanine substitution of Lys-396, Arg-457-458, Arg-460, or Arg-463, which has impaired ATPase activity, also shows poor RTP hydrolysis, further suggesting that these two activities probably share a common catalytic site (see Table 1 and references 1 and 2). Since the Lys-396-Ala mutant did not show any ATPase activity, we determined whether it could still bind ATP. Incubation with 32P-ATP followed by short UV irradiation at various time points up to 10 min was carried out (Fig. 2B). While the wild-type enzyme showed a time-dependent binding of [{alpha}32-P]ATP, no ATP binding was observed for the Lys-396-Ala mutant (Fig. 2B).


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  		TABLE 1. Summary of ATPase, helicase, and RTPase activities of NS3 mutant proteinsa


Figure 2
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  		FIG. 2. ATP binding, RTPase, and helicase activities of the various TrxNS3FL mutants. Autoradiograms were analyzed with a phosphorimager (GE Healthcare Biosciences) and quantified with the ImageQuant software. (A) RTPase activities of NS3 proteins. The reaction was carried out using 10 nM {gamma}-32P-labeled substrate and 500 nM purified TrxNS3FL. Samples were subjected to electrophoresis on a 24% native polyacrylamide gel. (B) UV cross-linking of [{alpha}32P]ATP binding to NS3 protein. Cross-linking reactions were carried out in microtiter tubes in a volume of 10 µl containing 2 µM protein and 8.8 µCi [{alpha}32-P]ATP (~400 Ci/mmol) in binding buffer (20 mM HEPES at pH 7.9, 25 mM KCl, 0.1 mM EDTA, and 5% glycerol). The mixture was kept on ice for 15 min, followed by irradiation in an HL-2000 Hybrilinker (UVP Laboratory Products) instrument at the indicated times in minutes (1,000 µJ/min). A volume of 2.5 µl 5x dye (100 mM Tris-HCl at pH 7.5, 50 mM EDTA, 0.1% Triton X-100, 0.5% sodium dodecyl sulfate, 50% glycerol, 0.1% bromophenol blue) was added, and samples were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel at 200 V for 35 min and subjected to autoradiography. The corresponding silver-stained gels are shown below each autoradiogram. The doublet seen corresponds to a nick in the NS3 protein, but the protein migrates as a single band in a native polyacrylamide gel. (C) Schematic representation of the dsRNA substrate used in the unwinding assay. (D) RNA helicase activity of wild-type and mutant NS3FL fusion proteins. Reactions were performed using a concentration of 12.5 nM of {alpha}32P-labeled dsRNA substrate and 400 nM enzyme. To prevent reannealing of unwound ssRNA, a 50-fold excess of unlabeled ssRNA was added to the termination mixture. The reaction was resolved on an 8% native polyacrylamide gel. WT represents the wild-type full-length fusion protein TrxNS3FL, and Trx is the thioredoxin reductase tag (negative control). (E) Dissociation of the dsRNA protein complex. The reaction was performed as for panel D but with a 100-fold excess of unlabeled ssRNA in the termination mixture. The products were subjected to electrophoresis on a native gel with 0.1% sodium dodecyl sulfate in the buffer.

Helicase activities of mutants. Several basic or hydrophobic residues (Ile-365, Arg-342, Arg-376, Lys-381, Lys-396, Arg-398, and Lys-418) protruding from domain II of the dengue virus NS3 helicase structure (23) could make contacts with an RNA substrate. We mutated these residues to assess their role in helicase activity using a 32P-labeled dsRNA substrate containing both 5' and 3' overhangs (Fig. 2C) (23). The dsRNA unwinding activities of the mutants are shown in Fig. 2D. Two single mutations (Ile-365-Ala and Arg-376-Ala) completely abolished unwinding activity. In the Lys-396-Ala mutant, the activity is reduced by 80% compared to the wild-type enzyme. Interestingly, mutants with severely impaired helicase activity appeared to interact more tightly with the dsRNA substrate, as shown by the presence of supershifts visible in Fig. 2D. This suggested the formation of a dsRNA mutant enzyme complex more stable than the wild-type protein. To rule out the possibility that any single-stranded RNA (ssRNA) produced in the assay might comigrate with the shifted bands on the gel in Fig. 2D, we added a 100-fold excess of unlabeled competitor ssRNA to the reaction mixture and used mild denaturing conditions. As a result, these complexes could be fully (Arg-396-Ala mutant) or partially (Ile-365-Ala and Arg-376-Ala mutants) dissociated (Fig. 2E) and the dsRNA substrates released from the complex. The other mutants showed activities similar to that of the wild-type protein or an increase in dsRNA unwinding.

Structural and mechanistic interpretation. Lys-396-Ala is the only mutation identified in this study which completely abrogates the RTPase, ATPase, and helicase activity, yet the 3D structure does not suggest an obvious explanation. Lys-396 belongs to helix {alpha}2' and is located at the surface of domain II approximately midway between the ATP binding pocket and the putative single-strand binding tunnel (Fig. 3A). Its side chain is too distant to make direct contact with the phosphate groups of the polynucleotide in the tunnel but could interact with a nucleic acid segment bound at the surface of domain II. Interestingly, the segment corresponding to dengue virus NS3 helix {alpha}2' is disordered in the YFV helicase structure (22), indicating dynamic properties that might be important for NS3 enzymatic functions.


Figure 3
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  		FIG. 3. Mapping of residues that disrupt the ATPase, RTPase, and/or helicase activities onto the three-dimensional structure of the dengue virus helicase catalytic domain and a proposed model of action. (A) The main residues identified in this study that appear to be important for the ATPase, RTPase, or helicase enzymatic activity of NS3 (shown as a ribbon diagram) are shown as sticks. (B) Proposed "inchworm" model of Den NS3 translocation and unwinding activity (based in part on the work of Dumont et al. [6]). Arrows indicate possible hinge interdomain motion occurring during the catalytic steps. The concave face created by the interface of domain II and domain III is proposed to bind dsRNA and act as the "translocator." The region at the tip of domain II is proposed to participate in disrupting hydrogen bonds between bases at the fork.

A double alanine NS3FL mutant at position Arg-457-Arg-458 (motif VI) only displays 27% residual ATPase activity. Using a truncated helicase domain, Matusan et al. (17) reported that alanine substitution of this conserved motif abolishes the helicase activity in vitro, whereas we observe a twofold increase in the helicase activity of the corresponding NS3FL mutant. The release of constraints formed by the buried Arg-457 and Arg-458 could lead to structural rearrangements in domain II, resulting in repositioning of the neighboring "arginine fingers" in a conformation less favorable for NTP hydrolysis (16). This residual ATPase activity, however, appears sufficient for duplex unwinding. Mutation of the "arginine fingers" abolished both the ATPase and helicase activities of the enzyme from HCV (11). In our study, in spite of a severe reduction in ATPase activity, only a limited reduction in helicase activity was observed for the corresponding mutants. Conversely, the substitution of either Ile-365 or Arg-376 to alanine almost completely abolished the helicase activity while fully preserving ATPase activity. This challenges the assumption that ATPase inhibitors can be used as antivirals to prevent RNA unwinding (3).

Ser-364 and Ile-365 are well-conserved residues located at the N terminus of helix {alpha}1' within a solvent-exposed pocket at the tip of domain II, which could make contact with an ssRNA tail trapped in the tunnel or with bases at the fork. Interestingly, Arg-376 and Lys-396 belong to helices {alpha}1' and {alpha}2' of domain II, respectively (Fig. 3A [23]). Whether movements in these two helices within domain II (23) are coupled with helicase activity remains to be determined. Recent single-molecule studies (6) support an inchworm-like translocation mechanism for the HCV NS3 helicase with two spatially distinct RNA binding sites. Extending their model, we propose that the pocket next to Ile-365 could act as the "helix opener" and disrupts hydrogen bonds at the fork. The basic concave face between domains II and III would act as "the translocator" by binding dsRNA ahead of the fork (Fig. 3B). The pocket next to Ile-365 appears to be an attractive region for the design of small molecules that would lock the helicase in a nonfunctional conformation.


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FOOTNOTES
 
* Corresponding author. Mailing address for Julien Lescar: School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive, Singapore 637551, Singapore. E-mail: julien{at}ntu.edu.sg. Mailing address for Subhash Vasudevan: Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670, Singapore. Phone: 65 67222909. Fax: 011-65-67222916. E-mail: subhash.vasudevan{at}novartis.com. Back

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


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Journal of Virology, July 2006, p. 6686-6690, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02215-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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