title: Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 A.
abstract: The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199), Lys(200) and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(457), Arg(461) and Arg(464)), and also Arg(458) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamashita
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Mori
authors.forename: Hideki
authors.initials: H
authors.lastname: Tani
authors.forename: Kohji
authors.initials: K
authors.lastname: Moriishi
authors.forename: Akihisa
authors.initials: A
authors.lastname: Takamizawa
authors.forename: Masanobu
authors.initials: M
authors.lastname: Agoh
authors.forename: Tomitake
authors.initials: T
authors.lastname: Tsukihara
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004664:Encephalitis Virus, Japanese / Q000201:enzymology* / Q000235:genetics; D018067:Flaviviridae / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D055672:Static Electricity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases