No abstract

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.Flaviviridae are enveloped viruses with positive-strand RNA genomes that have been grouped into three genera, Hepacivirus, Pestivirus, and Flavivirus (11,59). Several members of the Flavivirus genus, e.g., dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus, and West Nile virus (WNV), are medically important arthropod-borne pathogens afflicting humans. DENV infects 50 to 100 million people each year, with ϳ500,000 patients developing the more severe disease dengue hemorrhagic fever, leading to hospitalizations and resulting in approximately 20,000 deaths, mainly in children (24,26,27,29). Based on serological studies, DENVs are further classified into four distinct serotypes, DENV 1 to 4, whose respective genomes share ϳ60% sequence identity, with ϳ90% sequence identity within a serotype (7, 26). The DENV RNA genome spans about 10.7 kb and contains a type I methyl guanosine cap structure at its 5Ј end but is devoid of a polyadenylate tail. The genomic RNA is translated into a single polyprotein (58), which is cleaved into three structural (C-prM-E) and seven nonstructural (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) proteins by both the viral and cellular proteases (28). The viral serine protease is within the N-terminal region of NS3, and recent structural studies show that part of its catalytic site is formed by the viral cofactor NS2B upon substrate binding (18). The C-terminal region of NS3 forms the RNA helicase domain, which is thought to either separate a double-stranded RNA template into individual strands or disrupt secon...

Together with the NS5 polymerase, the NS3 helicase has a pivotal function in flavivirus RNA replication and constitutes an important drug target. We captured the dengue virus NS3 helicase at several stages along the catalytic pathway including bound to single-stranded (ss) RNA, to an ATP analogue, to a transition-state analogue and to ATP hydrolysis products. RNA recognition appears largely sequence independent in a way remarkably similar to eukaryotic DEAD box proteins Vasa and eIF4AIII. On ssRNA binding, the NS3 enzyme switches to a catalyticcompetent state imparted by an inward movement of the P-loop, interdomain closure and a change in the divalent metal coordination shell, providing a structural basis for RNA-stimulated ATP hydrolysis. These structures demonstrate for the first time large quaternary changes in the flaviviridae helicase, identify the catalytic water molecule and point to a b-hairpin that protrudes from subdomain 2, as a critical element for dsRNA unwinding. They also suggest how NS3 could exert an effect as an RNA-anchoring device and thus participate both in flavivirus RNA replication and assembly.

Regulated proteolysis by the two-component NS2B/ NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1 and P3. In contrast, the P2 and P4 substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.Dengue virus is the etiologic agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is the most prevalent arthropod-transmitted infectious disease in humans. Dengue consists of four closely related but antigenically distinct viral serotypes (DEN1-4), 1 of the genus Flavivirus (1, 2).Following primary infection, lifelong immunity develops that prevents repeated assault by the same serotype but does not provide protection from a virus of a different serotype (3). Dengue diseases are endemic in the tropics and subtropics, and the viruses are maintained in a cycle that involves humans and the Aedes aegypti mosquito. Infection with dengue viruses produces a spectrum of clinical illness ranging from a nonspecific viral syndrome to severe and fatal hemorrhagic disease (1, 2). Currently there is no antiviral drug or vaccine available against dengue viruses, and the pathogenesis of the disease is poorly understood.As with other members of the Flaviviridae family, the genomes of the dengue viruses consist of a positive singlestranded RNA of ϳ10,700 bases in length (4). Co-translational processing and post-translational processing of the polyprotein give rise to three structural proteins and at least seven nonstructural proteins (4). The correct processing of these proteins is essential for virus replication and requires host proteases such as signalase and furin (5) and a two-component viral protease, NS2B/NS3 (4). Previous studies have shown that the N-terminal part of NS3 contains trypsin-like protease domain (6) and that the activity of NS3 was dependent on at least 40 amino ...

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.