High throughput screening assay for negative single stranded RNA virus polymerase inhibitors

J Virol Methods. 2005 Sep;128(1-2):29-36. doi: 10.1016/j.jviromet.2005.03.012. Epub 2005 Apr 18.

Abstract

The Paramyxoviridae form a large family of viruses containing many human and veterinary pathogens for which a need for antiviral treatment is emphasized, particularly following the recent emergence of new viruses. The viral RNA-dependent RNA polymerase constitutes an obvious target for antiviral compounds. An in vitro assay was developed that allows high throughput screening of compounds potentially inhibiting the Sendai virus RNA-dependent RNA polymerase. Screening relies on the detection of the Photinus pyralis luciferase produced in a transcription/translation coupled assay using a mini-replicon virus. It contains an internal control for possible adverse effects of the tested compounds on translation or on luciferase activity. It is estimated that the mini-replicon template produced in one fertilized egg is sufficient to run 5000-10,000 reactions. This assay constitutes a simple, sensitive and easily automated method to perform high throughput screening of Paramyxoviridae RNA-dependent RNA polymerase inhibitors.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antiviral Agents / pharmacology*
  • Enzyme Inhibitors / pharmacology*
  • Fireflies / enzymology
  • HeLa Cells
  • Humans
  • Luciferases / metabolism
  • Microbial Sensitivity Tests / methods*
  • Paramyxoviridae / drug effects
  • Paramyxoviridae / enzymology
  • Paramyxoviridae / genetics
  • Protein Biosynthesis
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA-Dependent RNA Polymerase / antagonists & inhibitors*
  • RNA-Dependent RNA Polymerase / metabolism*
  • Sendai virus / drug effects
  • Sendai virus / enzymology*
  • Sendai virus / genetics
  • Sendai virus / metabolism
  • Transcription, Genetic

Substances

  • Antiviral Agents
  • Enzyme Inhibitors
  • RNA, Messenger
  • Luciferases
  • RNA-Dependent RNA Polymerase