tetano
Editor, Senior Moderator
J Virol Methods. 2014 Mar 7. pii: S0166-0934(14)00048-2. doi: 10.1016/j.jviromet.2014.02.005. [Epub ahead of print]
An efficient screening system for influenza virus cap-dependent endonuclease inhibitors.
Shibagaki Y1, Ikuta N2, Iguchi S2, Takaki K2, Watanabe S2, Kaihotsu M2, Masuda C2, Maeyama K2, Mizumoto K2, Hattori S2.
Author information
Abstract
The synthesis of influenza virus mRNA is primed by capped (m7GpppNm-) 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.
Copyright ? 2014. Published by Elsevier B.V.
KEYWORDS:
Antiviral agent, Cap-snatching, Influenza virus, mRNA capping
PMID:
24613941
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24613941
An efficient screening system for influenza virus cap-dependent endonuclease inhibitors.
Shibagaki Y1, Ikuta N2, Iguchi S2, Takaki K2, Watanabe S2, Kaihotsu M2, Masuda C2, Maeyama K2, Mizumoto K2, Hattori S2.
Author information
Abstract
The synthesis of influenza virus mRNA is primed by capped (m7GpppNm-) 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.
Copyright ? 2014. Published by Elsevier B.V.
KEYWORDS:
Antiviral agent, Cap-snatching, Influenza virus, mRNA capping
PMID:
24613941
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24613941