Ligand-Bound Structures of the Dengue Virus Protease Reveal the Active Conformation

Abstract

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic ␤-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4= to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease. Dengue virus (DENV) is a member of the flavivirus genus, which includes several viruses that are important human pathogens, including yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). The four serotypes of DENV are estimated to cause 50 to 100 million human infections worldwide every year in tropical and subtropical regions (16). There are currently no clinically approved vaccines or therapeutics for DENV. Understanding the molecular details of DENV infection is essential for vaccine and antiviral development.Flaviviruses contain a single strand of positive-sense RNA that encodes three structural and seven nonstructural (NS) proteins that are translated as a single polypeptide chain. NS3 contains two functions, an N-terminal serine protease and a C-terminal RNA helicase. The catalytic triad (His51, Asp75, and Ser135) is within the NS3 protease domain, but a region of NS2B is also required for catalytic activity (15). NS2B contains three predicted transmembrane helices, ensuring that the NS2B-NS3 protease is bound to the endoplasmic reticulum. The viral polyprotein is cleaved into the individual proteins by a combination of host proteases and the viral NS2B-NS3 protease (6, 30). The proteolytic activity of the viral protease is essential for viral replication, making it an excellent antiviral target (18).Ligand-free structures of flavivirus proteases have been solved for DENV-1 and -2 and a WNV active-site mutant (1, 7, 13). In addition, the structure of Mur...

Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

“…Residues Gln106 and Arg133 of the protease directly interacted with policresulen via hydrogen bonding, which is an interaction that is very different from that used by the previously reported inhibitors, which bind to the catalytic triad residues (His51, Asp75, and Ser135) of the protease [57,61] . Therefore, this newly determined binding arrangement between policresulen and the protease is expected to provide useful hints for designing Notably, policresulen is an organic acid with hemostatic and antimicrobial activities, and it is used as a clinical medication in gynecology for the treatment of vaginitis [29] .…”

Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

“…To explore the drugs' potential modes of actions, we docked temoporfin, niclosamide, and nitazoxanide to the crystal structure of the ligand-bound NS3 proteases of DENV3 (PDB ID: 3U1I) [43] and ZIKV (PDB ID: 5LC0) [44] after removing the NS2B peptides. We first employed a SiteMap [45] calculation on NS3pro of DENV3 to identify potential pockets on NS3 for binding of these drugs.…”

“…The NS2B-bound crystal structures of the Dengue NS3 protease (PDB ID: 3U1I [43]) and of the Zika NS3 protease (PDB ID: 5LC0 [44]) were used as docking targets to predict the possible bound conformations of these compounds. The program Autodock Vina [47] was used to dock the molecules into two putative binding sites corresponding to two NS2B residues that bind to NS3: L51/M51 and V53/I53.…”

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

“…We compared the 1 H-15 N HSQC spectra of the unlinked protease complex in the absence and presence of the Bz-nKRR-H inhibitor (7,16). Chemical shift perturbation was observed, but no new peaks appeared (Fig.…”

“…Crystal structures of the DENV protease in the absence and presence of inhibitors have been determined recently (6,7). The NS2B fragment in these structures adopts very different conformations, i.e.…”