Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Abstract

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an ''induced fit'' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.Keywords: protein structure/folding; conformational changes; enzymes; active sites; viral proteases; crystallography; enzyme inhibitors; mechanism-enzymes; protein structures-new Supplemental material: see www.proteinscience.org Flaviviruses, such as the closely related West Nile (WNV) and Dengue (DV) viruses, are members of a larger family called the Flaviviridae (Rice 1996; Burke and Monath 2001), which include the more distantly related hepaciviruses (e.g., Hepatitis C Virus [HCV]) and pestiviruses (e.g., Bovine virus diarrhea). WNV, DV, and HCV are recognized as major health threats that affect millions of people worldwide, with no specific countermeasures to treat them. WNV is transmitted to animals, including humans, by mosquito bites. It is encoded by a singlestrand, positive-sense, 11-kb RNA genome, which serves as mRNA for synthesis of the polyprotein precursor and as a template for genome replication in the host cell. The genome encodes three structural proteins found in the mature virion (C, prM, and E) and seven ''nonstructural'' (i.e., not part of the virion architecture) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) ( Abbreviations: NS, nonstructural; NS3pro, NS3 protease domain; WNV, West Nile Virus; DV, Dengue Virus; HCV, Hepatitis C Virus; JEV, Japanese Encephalitis Virus; KV, Kunjin Virus; YFV, Yellow Fever Virus; TM, transmembrane (helix); RSMD, root mean square deviation; NSLS, Brookhaven National Synchrotron Light Source; ALS, Berkeley Advanced Light Source; MAD, multiwavelength anomalous dispersion; DTT, dithiothreitol.Article published online ahead of print. Article and publication date are at

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

“…62 It was withdrawn from the market due to side effects in 2008. 63 Various polypeptides composed of D-arginine residues have been reported as WNV Comp.…”

“…51 In one of the known structures the WNV protease is inhibited by the natural inhibitor aprotinin. In contrast to small molecule inhibitors, aprotinin occupies with its P3-P4'-segment Pro-Cys-Lys(P1)-Ala-Arg-Ile-Ile all major binding pockets of the protease (2ijo.pdb) 63 ( Figure. 1.11).…”

“…In both inhibitor-free and -bound structures, the β1 strand of the NS2B have the same conformation, while the residues following Trp62 adopt a completely new conformation in the inhibitor-free protease. 63 In complex with inhibitor, the NS2B cofactor forms a belt that wraps the NS3 protein (Figure 1.12). In contrast, a new turn-and-a-half helix (α 1 ) followed by an unexpected reversal of the protein chain is found for the NS2B in the inhibitor-free structure forming an enzymatically inactive open conformation 63 ( Figure. 1.12).…”

“…63 In complex with inhibitor, the NS2B cofactor forms a belt that wraps the NS3 protein (Figure 1.12). In contrast, a new turn-and-a-half helix (α 1 ) followed by an unexpected reversal of the protein chain is found for the NS2B in the inhibitor-free structure forming an enzymatically inactive open conformation 63 ( Figure. 1.12). .…”

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

“…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).…”