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J Virol Methods. 2014 Jun;202:8-14. doi: 10.1016/j.jviromet.2014.02.005. Epub 2014 Mar 11.

An efficient screening system for influenza virus cap-dependent endonuclease inhibitors.

Author information

1
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.
2
Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

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.

KEYWORDS:

Antiviral agent; Cap-snatching; Influenza virus; mRNA capping

PMID:
24613941
DOI:
10.1016/j.jviromet.2014.02.005
[Indexed for MEDLINE]
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