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

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 singlestranded 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.

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

“…ATPase Activity of DENV4 NS2B 18 NS3 and Mutants-The ATPase activity of DENV4 NS2B 18 NS3 carrying the introduced mutations was measured using the Malachite green assay as reported previously (6). Initially, we determined the activities of wild type DENV4 NS2B 18 NS3 and its helicase domain (residues 172-618), and we found that the helicase domain was only about 30% as active as the full-length protein (the catalytic efficiency, k cat /K m , for NS2B 18 NS3 was taken as 100%).…”

“…The NS3 protein (618 amino acids in DENV4) contains a serine-protease domain at its N terminus (whose activity requires the formation of a noncovalent complex with the central 40-residue hydrophilic segment of the membrane-bound NS2B protein cofactor) and an ATP-driven helicase and RNA triphosphatase at its C-terminal end. Atomic structures for the active NS3 protease domain (NS2B 47 NS3pro) (2)(3)(4), the ATPase/helicase domain (NS3hel) (5)(6)(7)(8)(9), and the full-length NS3 molecule fused to 18 residues of the NS2B cofactor (NS2B 18 NS3) (10) have been reported and reviewed (11). Together, these structures provide an explanation for the lack of protease activity and solubility observed for NS3pro domains expressed in Escherichia coli (6,12,13).…”

“…5 By contrast, both in terms of substrate specificity and helicase and ATPase activity, differences exist between NS2B 47 NS3, the proteolytically inactive NS2B 18 NS3 protein, the full-length NS3 protein, and the isolated NS3 helicase domain (16). Thus, several studies (6,10,16) point to an influence of the protease domain on the activities performed by the helicase domain. Together, these data suggest that coupling and subsequent co-evolution of these two domains within a single polypeptide chain are functionally important, but a clear mechanistic and structural explanation remains elusive.…”

“…Atomic structures for the active NS3 protease domain (NS2B 47 NS3pro) (2)(3)(4), the ATPase/helicase domain (NS3hel) (5)(6)(7)(8)(9), and the full-length NS3 molecule fused to 18 residues of the NS2B cofactor (NS2B 18 NS3) (10) have been reported and reviewed (11). Together, these structures provide an explanation for the lack of protease activity and solubility observed for NS3pro domains expressed in Escherichia coli (6,12,13). We also reported crystal structures for NS3hel from DENV4 in complex with single strand RNA and a complete set of ligands detailing the RNA-stimulated ATP hydrolysis pathway (14).…”

“…ATPase Activity Assay-The ATPase activity assay was performed as described previously (6,18). Preincubation of 90 l of reaction buffer (50 mM Tris-HCl, pH 7.4, 2 mM MgCl 2 , 1.5 mM dithiothreitol, 0.05% Tween 20, 0.25 ng/l bovine serum albumin, and 1 g average length 200 -250 bases poly(U)) containing 4.8 nM protein was carried out in a 96-well plate (Nunc, Immunoplate F96 MaxiSorp) for 5 min.…”