Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution.
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-A 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.
Yap
Thai Leong
TL
Novartis Institute for Tropical Diseases, 05-01 Chromos, 10 Biopolis Road, Singapore 138670, Singapore.
Xu
Ting
T
Chen
Yen-Liang
YL
Malet
Helene
H
Egloff
Marie-Pierre
MP
Canard
Bruno
B
Vasudevan
Subhash G
SG
Lescar
Julien
J
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003461:Crystallography; D017931:DNA Primers; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure*; D013054:Spectrophotometry, Atomic
D017931:DNA Primers; D012324:RNA-Dependent RNA Polymerase