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Journal of Virology, October 2005, p. 13105-13115, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.13105-13115.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inhibitors of Respiratory Syncytial Virus Replication Target Cotranscriptional mRNA Guanylylation by Viral RNA-Dependent RNA Polymerase

Michel Liuzzi,1* Stephen W. Mason,1* Mireille Cartier,1 Carol Lawetz,1 Robert S. McCollum,1 Nathalie Dansereau,1 Gordon Bolger,1 Nicole Lapeyre,1 Yvon Gaudette,1 Lisette Lagacé,1 Marie-Josée Massariol,1 Florence Dô,1 Paul Whitehead,1 Lyne Lamarre,1 Erika Scouten,1 Josée Bordeleau,2 Serge Landry,2 Jean Rancourt,2 Gulrez Fazal,2 and Bruno Simoneau2

Department of Biological Sciences,1 Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Laval, Quebec, Canada2

Received 4 March 2005/ Accepted 13 July 2005

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5' and 3' ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5' cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

* Corresponding author. Present address for M. Liuzzi: Cooperative Laboratory Idenix-Universita di Cagliari, Sesta Strada Ovest, Zona Industriale Macchiareddu, 09010 UTA-Cagliari, Italy. Phone: 39 070 254021. Fax: 39 070 247360. E-mail: Liuzzi.Michel{at}idenix.com. Mailing address for S. Mason: Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 rue Cunard, Laval, Quebec H7S 2G5, Canada. Phone: (450) 682-4640. Fax: (450) 682-4642. E-mail: smason{at}lav.boehringer-ingelheim.com.

Journal of Virology, October 2005, p. 13105-13115, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.13105-13115.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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