Respiratory Syncytial Virus Inhibitor AZ-27 Differentially Inhibits Different Polymerase Activities at the Promoter

Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesiz...

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Bibliographic Details
Published in:Journal of virology 2015-08, Vol.89 (15), p.7786-7798
Main Authors: Noton, Sarah L, Nagendra, Kartikeya, Dunn, Ewan F, Mawhorter, Michael E, Yu, Qin, Fearns, Rachel
Format: Article
Language:English
Subjects:
Antiviral Agents - pharmacology
Benzazepines - pharmacology
Enzyme Inhibitors - pharmacology
Humans
Niacinamide - analogs & derivatives
Niacinamide - pharmacology
Promoter Regions, Genetic - drug effects
Respiratory syncytial virus
Respiratory Syncytial Virus Infections - virology
Respiratory Syncytial Viruses - drug effects
Respiratory Syncytial Viruses - enzymology
Respiratory Syncytial Viruses - genetics
RNA Replicase - antagonists & inhibitors
RNA Replicase - genetics
RNA Replicase - metabolism
Transcription, Genetic - drug effects
Vaccines and Antiviral Agents
Viral Proteins - antagonists & inhibitors
Viral Proteins - genetics
Viral Proteins - metabolism
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Summary:Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA and add and methylate a cap on each of the viral mRNAs. Previous studies (H. Xiong et al., Bioorg Med Chem Lett, 23:6789-6793, 2013, http://dx.doi.org/10.1016/j.bmcl.2013.10.018; C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14) had identified a small-molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to inhibit equally both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both of these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3' end of the genome to a greater extent than those from the 5' end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase also can utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on the addition of up to three nucleotides by back-priming but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter. Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an attractive target for drug development, but this large enzymatic complex is poorly characterized, hampering drug development efforts. AZ-27 is a small-molecule inhibitor previously shown to target the RSV large polymerase subunit (C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14), but its inhibitory mechanism was unknown. Understanding this would be valuable both for characterizing the polymerase and for further development of inhibitors. Here, we show that AZ-27 inhibit
ISSN:0022-538X
1098-5514
DOI:10.1128/JVI.00530-15