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

Abstract

Respiratory syncytial virus (RSV) isIn 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 backprimed 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. -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 inhibits an early stage in mRNA transcription, as well as genome replication, by inhibiting initiation of RNA synthesis from the promoter. However, the compound does not inhibit back priming, another RNA synthesis activity of the RSV polymerase. These findings provide insight into the different activities of the RSV polymerase and will aid further development of antiviral agents against RSV. W orldwide, respiratory syncytial virus (RSV) is the major cause of respiratory disease in infants under the age of one, and it is the leading cause of infant hospitalization in the United States (1, 2). RSV also is recognized as a significant cause of morbidity and mortality in the elderly (3). Significant efforts to develop a safe and effective vaccine against RSV are ongoing, but this has proven difficult, and currently none is licensed (4, 5). The only effective antiviral drug is palizivumab, a humanized monoclonal antibody against the viral fusion protein, but this drug is costly and effective only if administered prophylactically (6). Currently there are no licensed, effective antiviral treatments. However, studies in human subjects showed that there is a correlation between virus load and disease severity, suggesting that administration of effective RSV inhibitors early in the disease course would reduce morbidity (7-9), and a recent human trial of a candidate RSV drug confirmed that a small-molecule inhibitor of viral replication ameliorated RSV-induced disease (10). Thus, there is a window during infection in which it is possible to treat RSV with antiviral drugs. This highlights the need to develop a detailed understan...

The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

“…The location of YM-53403 resistance mutations in variable region 2 of the L gene, combined with structural domain studies of the RSV RNA polymerase indicating a dimerization function in this area (16,28,29), suggests that the compound may allosterically inhibit a tertiary complex essential for transcription or replication. Our findings are consistent with a recent publication showing the ability of a closely related compound, AZ-27, to inhibit a common step in the initiation of RSV transcription and replication (30). The attenuation of RIG-I-like pathways (Fig.…”

Respiratory Syncytial Virus: Infection, Detection, and New Options for Prevention and Treatment

“…Elucidation of viral transcriptional processes is essential to develop viral replicon systems that can be used to screen inhibitors and develop antiviral drugs (101). RSV replicons have been used already to develop RSV polymerase inhibitors to the point of clinical trials (102), as we discuss in further detail in "Nucleoside Analogues and Small-Molecule Inhibitors.…”

Novel diversity-oriented synthesis-derived respiratory syncytial virus inhibitors identified via a high throughput replicon-based screen

“…The minigenome assay was used to determine if the compound inhibited a step specific to transcription, or replication, or if it inhibited both. Two minigenome templates were used, one optimized for measuring mRNA transcription, and the other for measuring RNA replication, as described previously (Noton et al, 2015) (Fig. 6A and B, respectively).…”

“…These possibilities were investigated by testing BRD3969 in an in vitro RNA synthesis assay (Noton et al, 2015). Purified recombinant RSV polymerase (L/P complex) was incubated in a transcription buffer containing an oligonucleotide RNA template, consisting of nucleotides 1–25 of the RSV tr promoter sequence, NTPs and a trace amount of [α -32 P]-GTP.…”

“…The mutation conferring resistance to YM53403 has been shown to also confer resistance to a related analog, AZ-27 (Noton et al, 2015). The partial resistance of this mutant to BRD3969 in the CPE assay (Table 3) suggests that this inhibitor may share the same target with YM53403 and AZ-27.…”