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

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

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.

Publication types

  • Evaluation Study

MeSH terms

  • Antiviral Agents / isolation & purification*
  • Drug Evaluation, Preclinical / methods*
  • Endoribonucleases / antagonists & inhibitors*
  • Enzyme Inhibitors / isolation & purification*
  • Humans
  • Orthomyxoviridae / drug effects*
  • Viral Proteins / antagonists & inhibitors*

Substances

  • Antiviral Agents
  • Enzyme Inhibitors
  • Viral Proteins
  • Endoribonucleases
  • methylated cap-dependent endonuclease