The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180 -618) by an 11-amino acid linker (169 -179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B 18 NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ϳ161°with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn 2؉ refined to a resolution of 2.2 Å . The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu 173 and Pro 174 or replacing Pro 174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro 176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.
Dengue virus (DENV)4 is an important vector-borne virus that causes a spectrum of illnesses in humans ranging from asymptomatic to severe disease, including dengue hemorrhagic fever and dengue shock syndrome. More than half of the ϳ70 members of the flavivirus genus that includes the four serotypes of DENV (DENV1-4), yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and West Nile virus, are important human pathogens (1). The positive-sense flavivirus RNA genome of ϳ11 kb contains a single open reading frame that is translated into a polyprotein precursor, consisting of the structural proteins C, prM, and E and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During viral replication, the polyprotein is cleaved by host proteases in the endoplasmic reticulum lumen and viral proteases at the cytoplasmic face (1). 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) (2-4), the ATPase/helicase domain (NS3hel) (5-9), 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 explana...