Conclusion

We reported two mutations (Ile365Ala, Arg376Ala) for which the helicase activity is completely abolished despite an intact ATPase activity. Interestingly, a shallow

FIG. 5. Surface representation of the Den NS3 helicase domain in the same orientation as in Fig. 1. The putative interaction between the enzyme and a nucleic acid substrate is shown. The model was generated in part by superposition with the HCV helicase in complex with a dU8 oligonucleotide (PDB code: 1A1V; Kim et al 1998).

FIG. 5. Surface representation of the Den NS3 helicase domain in the same orientation as in Fig. 1. The putative interaction between the enzyme and a nucleic acid substrate is shown. The model was generated in part by superposition with the HCV helicase in complex with a dU8 oligonucleotide (PDB code: 1A1V; Kim et al 1998).

pocket is located in the vicinity of Ile365, lined by residues protruding from domain II (Thr 408, Asp 409, Leu 443) and domain III (Asp 603, Leu 605). This previously unidentified region could be a regulatory site controlling structural transitions between closed and open forms of the enzyme. Taken together, our results point to the helicase activity for the design of antiviral therapeutics: compounds binding to allosteric pockets might achieve both specificity and activity by hindering enzyme movements required for nucleic acid strand separation. Ongoing structural work on the NS2B/NS3 protease, NS3 helicase domain, NS5 methyltransferase and polymerase catalytic regions hold much promise for the future of rational drug design against flaviviruses.

Our hope is that the development of specific compounds with antiviral activity against flaviviruses will parallel the successes that recently followed structural determinations of key viral proteins from HCV or HIV.

FIG. 6. Results of the site-directed mutagenesis study. Left panel shows the residual ATPase (lower histogram) and helicase activity (upper histogram) for each residue which was mutated into alanine in our study. The activities are expressed as a percentage of wild-type which was arbitrarily set to 100%. Residues which drastically disrupt either of the two enzymatic functions when substituted by alanine are circled. Right panel shows the mapping of these residues onto the 3D structure of NS3 helicase.

FIG. 6. Results of the site-directed mutagenesis study. Left panel shows the residual ATPase (lower histogram) and helicase activity (upper histogram) for each residue which was mutated into alanine in our study. The activities are expressed as a percentage of wild-type which was arbitrarily set to 100%. Residues which drastically disrupt either of the two enzymatic functions when substituted by alanine are circled. Right panel shows the mapping of these residues onto the 3D structure of NS3 helicase.

Financial support via Grants from N.T.U. (RG29/05), 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 J.L. laboratory is acknowledged as well as provision of excellent beam-time and support by the E.S.R.F. (Grenoble, France).

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