Crystal Structure of the NS3 Protease-Helicase from Dengue Virus

Abstract

Several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. During infection, the flavivirus RNA genome is translated into a polyprotein, which is cleaved into several components. Nonstructural protein 3 (NS3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease N-terminal domain, with a segment of 40 residues from the NS2B protein acting as a cofactor. The ATPase/helicase domain is located at the C terminus of NS3. Atomic structures are available for these domains separately, but a molecular view of the full-length flavivirus NS3 polypeptide is still lacking. We report a crystallographic structure of a complete NS3 molecule fused to 18 residues of the NS2B cofactor at a resolution of 3.15 Å. The relative orientation between the protease and helicase domains is drastically different than the single-chain NS3-NS4A molecule from hepatitis C virus, which was caught in the act of cis cleavage at the NS3-NS4A junction. Here, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab initio using small-angle X-ray scattering experiments in solution, suggesting a stable molecular conformation. We propose that a basic patch located at the surface of the protease domain increases the affinity for nucleotides and could also participate in RNA binding, explaining the higher unwinding activity of the full-length enzyme compared to that of the isolated helicase domain.Several members of the flaviviruses are important human pathogens, including yellow fever virus (YFV), Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus (WNV), and dengue virus (25). For the latter virus, neither a specific therapy nor a vaccine exists, and treatment is currently limited to the use of analgesics and fluid replacement. Since dengue virus is endemic in most tropical and subtropical areas, causing several hundreds of thousands of severe cases (dengue shock syndrome or dengue hemorrhagic fever), with approximately 30,000 deaths per year, compounds with antiviral activity are actively sought (21, 46). The positive-sense flavivirus RNA genome of 11 kb forms a single open reading frame that is translated into a polyprotein precursor of ca. 370 kDa consisting of the structural proteins C, prM, and E and seven nonstructural proteins, nonstructural protein 1 (NS1), NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During viral maturation, this polyprotein is cleaved by host cell proteases in the endoplasmic reticulum and by the NS3 protein in the cytoplasm (Fig. 1A) (25). Cleavage of the polyprotein is mediated by the serine protease N-terminal domain of NS3, with a hydrophilic segment of 40 residues from the transmembrane NS2B protein acting as a cofactor necessary for this activity. The domain required for ATPase/helicase ...

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

“…1A) due to an ϳ161°rotation of the protease relative to the helicase domain (calculated using DynDom) (25). This new structural state is also consistent with the envelope determined ab initio using small angle x-ray scattering experiments in solution (10). The structures of individual domains remain intact, with root mean square deviations of 1.11 Å (146 C␣ atoms) for the protease and 0.97 Å (439 C␣ atoms) for the helicase domain, respectively.…”

“…Cloning and Site-directed Mutagenesis of DENV4 NS2B 18 NS3-The construct encoding the wild type DENV4 NS2B 18 NS3 protein was described before (10). Site-directed mutagenesis was carried out using the QuikChange site-directed mutagenesis kit (Stratagene) as described previously (13).…”

“…Although this crystal form was obtained using similar crystal growth conditions, the relative orientations between the protease and helicase domains differ markedly compared with the crystallographic structure of the NS2B 18 NS3 protein we reported previously and named "conformation I" (10). In its second conformation, the protease domain has rotated by an angle of ϳ161°relative to the helicase domain.…”

“…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.…”