title: An efficient screening system for influenza virus cap-dependent endonuclease inhibitors.
abstract: The synthesis of influenza virus mRNA is primed by capped (m(7)GpppNm-) short RNAs that are cleaved from RNA polymerase II transcripts by a virally encoded endonuclease. This cap-dependent endonuclease activity called "cap-snatching" may provide a unique target for novel anti-viral agents. To screen candidate inhibitors, it is essential to establish a method for producing efficiently a capped RNA substrate and a convenient assay for the cap-snatching activity. A 3'-biotinylated short RNA was prepared by in vitro transcription, purified by C18 reverse-phase column chromatography, and subjected to a capping reaction involving three recombinant capping enzymes. This capped RNA was shown to be an efficient substrate for the cap-snatching assay. Cap-snatching activity was then measured with the novel pull-down assay developed in this study, which is based on the streptavidin-biotin interaction. A known inhibitor for the cap-snatching reaction was evaluated by the pull-down assay, demonstrating the efficacy of the established screening system.
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address: shibagakiy@pharm.kitasato-u.ac.jp.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Shibagaki
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Ikuta
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Iguchi
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Takaki
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kaihotsu
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Masuda
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Maeyama
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Kiyohisa
authors.initials: K
authors.lastname: Mizumoto
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Seisuke
authors.initials: S
authors.lastname: Hattori
mesh_terms: D000998:Antiviral Agents / Q000302:isolation & purification*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004722:Endoribonucleases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006801:Humans; D009975:Orthomyxoviridae / Q000187:drug effects*; D014764:Viral Proteins / Q000037:antagonists & inhibitors*
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D014764:Viral Proteins; D004722:Endoribonucleases; C043026:methylated cap-dependent endonuclease
keywords: Antiviral agent; Cap-snatching; Influenza virus; mRNA capping