The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

Noton, Sarah L.; Fearns, Rachel

doi:10.1261/rna.2813411

Cited by 29 publications

(47 citation statements)

References 44 publications

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“…3D). This finding is consistent with results from a previous minigenome study, which showed that deletion or substitution of nt 1 and 2 of the Le did not inhibit RNA synthesis from the ϩ3 position (29). To date, we have been unable to detect initiation from position ϩ1 of the Le region in the in vitro RNA synthesis assay, suggesting that either initiation at ϩ1 is much less efficient or that additional sequence and/or different conditions are required.…”

Section: Discussionsupporting

confidence: 92%

“…To date, we have been unable to detect initiation from position ϩ1 of the Le region in the in vitro RNA synthesis assay, suggesting that either initiation at ϩ1 is much less efficient or that additional sequence and/or different conditions are required. Our previous studies have indicated that replication initiation at position ϩ1 can occur by an unusual nontemplated initiation mechanism, and we have suggested that the RdRp might utilize a primer to accomplish this (29,32). A recent paper showing that the dengue virus RdRp can self-generate a primer in a template-independent manner to initiate from the ϩ1 position on a template lends credence to this hypothesis (35).…”

Section: Discussionmentioning

confidence: 68%

“…We had previously noted that nt 3 to 12 of the RSV Le sequence contains a C/U-rich sequence with strong similarity to the conserved GS sequence found at the start of the each RSV gene (29). This similarity was even more pronounced if the promoter sequence was aligned with the L GS signal, which differs slightly from the canonical GS sequences but appears to function in a similar way (7) (Fig.…”

Section: Resultsmentioning

confidence: 88%

“…Recent studies using the RSV minigenome system identified RNA initiated from position ϩ1 of the Le and also at position ϩ3 (29). Initiation at position ϩ1 was expected, as this is the initiation site for antigenome synthesis, but identification of RNA initiated at position ϩ3 was a surprising finding.…”

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confidence: 99%

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Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

Tremaglio

Noton

Deflubé

et al. 2013

J Virol

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103

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The mechanisms by which the respiratory syncytial virus (RSV) RNA-dependent RNA polymerase (RdRp) initiates mRNA transcription and RNA replication are poorly understood. A previous study, using an RSV minigenome, suggested that the leader (Le) promoter region at the 3= end of the genome has two initiation sites, one at position ؉1, opposite the 3= terminal nucleotide of the genome, and a second site at position ؉3, at a sequence that closely resembles the gene start (GS) signal of the RSV L gene. In this study, we show that the ؉3 initiation site of the Le is utilized with apparently high frequency in RSV-infected cells and yields small RNA transcripts that are heterogeneous in length but mostly approximately 25 nucleotides (nt) long. Experiments with an in vitro assay in which RSV RNA synthesis was reconstituted using purified RdRp and an RNA oligonucleotide showed that nt 1 to 14 of the Le promoter were sufficient to signal initiation from ؉3 and that the RdRp could access the ؉3 initiation site without prior initiation at ؉1. In a minigenome assay, nucleotide substitutions within the Le to increase its similarity to a GS signal resulted in more-efficient elongation of the RNA initiated from position ؉3 and a reduction in RNA initiated from the NS1 gene start signal at ؉45. Taken together, these data suggest a new model for initiation of sequential transcription of the RSV genes, whereby the RdRp initiates the process from a gene start-like sequence at position ؉3 of the Le. R espiratory syncytial virus (RSV) is the leading cause of pneumonia and viral deaths in infants worldwide and is increasingly recognized as a significant pathogen of the elderly and immunocompromised (1). Treatment for RSV infection is limited to supportive care, and at present there are no approved vaccines. RSV is a member of the family Paramyxoviridae in the order Mononegavirales, the nonsegmented negative-strand (NNS) RNA viruses. Like other viruses in this order, the RSV genome serves as the template for mRNA transcription and genome replication, carried out by the viral RNA-dependent RNA polymerase (RdRp) (reviewed in reference 2).The RSV genome is 15.2 kb and encodes mRNAs from 10 sequentially arranged genes (reviewed in reference 3). Two short extragenic regions border the genome, a 3= 44-nt leader region (Le) and a 5= 155-nt trailer (Tr) region (4, 5). The Le region contains promoter sequences that are required for transcription of all RSV genes and genome replication (4). Transcription occurs in an obligatorily sequential, polarized manner to generate 10 subgenomic mRNAs (6). During this process, the RdRp is controlled by cis-acting gene start (GS) and gene end (GE) sequences that flank each of the genes (7, 8). The GS sequences direct initiation of RNA synthesis. They are highly conserved in RSV, with 9 of 10 GS sequences being identical and the tenth having only slight differences (9). In related viruses, the GS signals have been shown to be multifunctional, directing the RdRp to cap and methylate the nascent transcript (10-12...

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 88%

mentioning

confidence: 99%

See 2 more Smart Citations

Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

Tremaglio

Noton

Deflubé

et al. 2013

J Virol

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103

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“…By stopping and restarting RNA synthesis at the gene junctions, the RdRp is able to sequentially synthesize the 10 individual mRNAs from the single le promoter. To perform RNA replication, the RdRp binds to the same promoter sequence, but in this case it initiates RNA synthesis opposite position 1 (29,30). Replicative RNA becomes encapsidated with N protein as it is synthesized, and this is thought to cause the RdRp to become superprocessive, enabling it to override the ge signals and elongate the RNA to the end of the genome to generate a complementary antigenome RNA (31,32).…”

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confidence: 99%

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

Noton

Nagendra

Dunn

et al. 2015

J Virol

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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...

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Respiratory Syncytial Virus: Virology, Reverse Genetics, and Pathogenesis of Disease

Collins

Fearns

Graham

2013

Current Topics in Microbiology and Immunology

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Human respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus of family Paramyxoviridae. RSV is the most complex member of the family in terms of the number of genes and proteins. It is also relatively divergent and distinct from the prototype members of the family. In the past 30 years, we have seen a tremendous increase in our understanding of the molecular biology of RSV based on a succession of advances involving molecular cloning, reverse genetics, and detailed studies of protein function and structure. Much remains to be learned. RSV disease is complex and variable, and the host and viral factors that determine tropism and disease are poorly understood. RSV is notable for a historic vaccine failure in the 1960s involving a formalin-inactivated vaccine that primed for enhanced disease in RSV naïve recipients. Live vaccine candidates have been shown to be free of this complication. However, development of subunit or other protein-based vaccines for pediatric use is hampered by the possibility of enhanced disease and the difficulty of reliably demonstrating its absence in preclinical studies.

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The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

Cited by 29 publications

References 44 publications

Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

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

Respiratory Syncytial Virus: Virology, Reverse Genetics, and Pathogenesis of Disease

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