Crystal Structure of Catalytic Domain of Japanese Encephalitis Virus NS3 Helicase/Nucleoside Triphosphatase at a Resolution 1.8 angstrom

Research Institute for microbial diseases, Department of molecular virus, Osaka University* Institute for Protein Research, Research Center for structual and function proteomics, Osaka University, Japan** Institute for Protein Research, Department of Protein crystallography, Osaka University, Japan***
â—‹Tetsuo Yamashita* Hideaki Unno** Mori Yoshio* Moriishi Kohji* Tsukihara Tomitake*** Matsuura Yoshiharu*

Mosquito-borne flaviviruses have a single stranded and positive-sense RNA as a genome and are classified into two serocomplexes, Japanese encephalitis virus (JEV) and dengue virus (DEN), and unclassified yellow fever virus (YFV). The flavivirus NS3 proteins are composed of two domains, the N-terminal protease and the C-terminal helicase/nucleoside triphosphatase catalytic domains, which are essential for viral replication. The crystal structures of the helicase domains of DEN and YFV have been recently elucidated. To obtain more insight in the structure of the flavivirus helicases, we determined the crystal structure of JEV helicase domain. The purified samples were crystallized by a hanging drop vapour-diffuision method. A single crystal was obtained by the reservoir solution containing 15% ethanol, 100mM Tris-HCl (pH7.0) and 4% pentaerythriol etoxylate (3/4 EO/OH). Data obtained by the beamline BL44XU of Spring-8 (Harima, Japan) using Mac Science DIP6040 imaging plate were processed with the program HKL2000 and the crystal structure was determined by a molecular replacement method. The 3D structures of JEV NS3 helicase domain at 1.8 angstrom were well conserved with those of DEN and YFV. In the comparison of flavivirus helicases, the active site topology of helicase of JEV, DEN and YFV is conserved well, but only HCV helicase is distant away from Walker motif A of other flavivirus helicase. In addiction, the detail analysis of the active site residues of JEV NS3 helicase domain by using mutagenesis will be discussed.