High throughput screening assay for negative single stranded RNA virus polymerase inhibitors.
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.
Pelet
Thierry
T
Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CMU, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
Miazza
Vincent
V
Mottet
Geneviève
G
Roux
Laurent
L
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D048888:Fireflies / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D010252:Paramyxoviridae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D029082:Sendai virus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D008156:Luciferases; D012324:RNA-Dependent RNA Polymerase