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Nucleic Acids Research 2004 32(16):4758-4767; doi:10.1093/nar/gkh809
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Published online 8 September 2004

Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved

Polyadenylation-dependent screening assay for respiratory syncytial virus RNA transcriptase activity and identification of an inhibitor

Stephen W. Mason*, Carol Lawetz, Yvon Gaudette, Florence Dô, Erika Scouten, Lisette Lagacé, Bruno Simoneau1 and Michel Liuzzi

Department of Biological Sciences and 1 Department of Chemistry, Boehringer Ingelheim (Canada) Ltd, Laval, Québec H7S 2G5, Canada

* To whom correspondence should be addressed. Tel: +450 682 4640; Fax: +450 682 4642; Email: smason{at}lav.boehringer-ingelheim.com
Present address: Michel Liuzzi, Cooperative Laboratory Idenix—Universita di Cagliari, Sesta Strada Ovest, Zona Industriale Macchiareddu, 09010 UTA (CA), Italia

Received May 13, 2004; Revised July 15, 2004; Accepted August 17, 2004

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 FlashPlateTM. 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.


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