Polyadenylation-dependent screening assay for respiratory syncytial virus RNA transcriptase activity and identification of an inhibitor.
RNA-dependent RNA polymerase from respiratory syncytial virus (RSV) is a multi-subunit ribonucleoprotein (RNP) complex that, in addition to synthesizing the full 15 222 nt viral genomic RNA, is able to synthesize all 10 viral mRNAs. We have prepared crude RNP from RSV-infected HEp-2 cells, based on a method previously used for Newcastle disease virus, and established a novel polyadenylation-dependent capture [poly(A) capture] assay to screen for potential inhibitors of RSV transcriptase activity. In this homogeneous assay, radiolabeled full-length polyadenylated mRNAs produced by the viral RNP are detected through capture on immobilized biotinylated oligo(dT) in a 96-well streptavidin-coated FlashPlate. Possible inhibitors identified with this assay could interfere at any step required for the production of complete RSV mRNAs, including transcription, polyadenylation and, potentially, co-transcriptional guanylylation. A specific inhibitor of RSV transcriptase with antiviral activity was identified through screening of this assay.
Mason
Stephen W
SW
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd, Laval, Québec H7S 2G5, Canada. smason@lav.boehringer-ingelheim.com
Lawetz
Carol
C
Gaudette
Yvon
Y
Dô
Florence
F
Scouten
Erika
E
Lagacé
Lisette
L
Simoneau
Bruno
B
Liuzzi
Michel
M
D002460:Cell Line; D005821:Genetic Techniques; D006801:Humans; D026723:Polyadenylation; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000201:enzymology*; D018894:Reverse Transcriptase Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D014158:Transcription, Genetic
D012333:RNA, Messenger; D018894:Reverse Transcriptase Inhibitors; D012194:RNA-Directed DNA Polymerase