RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end.

ABSTRACT The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3′ ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3′ genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3′-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3′ end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancingcis-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3′ end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3′ and 5′ ends. The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.

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Cryo-EM structure of the respiratory syncytial virus RNA polymerase

Nature Communications ◽

10.1038/s41467-019-14246-3 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 11

Author(s):

Dongdong Cao ◽

Yunrong Gao ◽

Claire Roesler ◽

Samantha Rice ◽

Paul D’Cunha ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Polymerase ◽

Virus Rna ◽

Syncytial Virus

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Synthesis of Respiratory Syncytial Virus RNA in Cell-free Extracts

Journal of General Virology ◽

10.1099/0022-1317-70-3-755 ◽

1989 ◽

Vol 70 (3) ◽

pp. 755-761 ◽

Cited By ~ 8

Author(s):

R. C. Herman

Keyword(s):

Respiratory Syncytial Virus ◽

Virus Rna ◽

Syncytial Virus

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Unravelling the complexities of respiratory syncytial virus RNA synthesis

Journal of General Virology ◽

10.1099/vir.0.81786-0 ◽

2006 ◽

Vol 87 (7) ◽

pp. 1805-1821 ◽

Cited By ~ 61

Author(s):

Vanessa M. Cowton ◽

David R. McGivern ◽

Rachel Fearns

Keyword(s):

Respiratory Syncytial Virus ◽

Vaccine Development ◽

Virus Genome ◽

Genome Replication ◽

Promoter Regions ◽

Genome Expression ◽

Virus Rna ◽

A Genome ◽

Genome Rnas ◽

Syncytial Virus

Human respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral- and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 3′ ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed.

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Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0913065107 ◽

2010 ◽

Vol 107 (22) ◽

pp. 10226-10231 ◽

Cited By ~ 22

Author(s):

S. L. Noton ◽

V. M. Cowton ◽

C. R. Zack ◽

D. R. McGivern ◽

R. Fearns

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Replication ◽

Syncytial Virus

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

Nucleic Acids Research ◽

10.1093/nar/gkh809 ◽

2004 ◽

Vol 32 (16) ◽

pp. 4758-4767 ◽

Cited By ~ 33

Author(s):

S. W. Mason

Keyword(s):

Respiratory Syncytial Virus ◽

Screening Assay ◽

Transcriptase Activity ◽

Virus Rna ◽

Syncytial Virus

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Mapping the Transcription and Replication Promoters of Respiratory Syncytial Virus

Journal of Virology ◽

10.1128/jvi.76.4.1663-1672.2002 ◽

2002 ◽

Vol 76 (4) ◽

pp. 1663-1672 ◽

Cited By ~ 50

Author(s):

Rachel Fearns ◽

Mark E. Peeples ◽

Peter L. Collins

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Replication ◽

Saturation Mutagenesis ◽

Common Element ◽

Independent Events ◽

Naturally Occurring ◽

A Genome ◽

Syncytial Virus ◽

Overlapping Sets ◽

Transcription And Replication

ABSTRACT An important, unresolved issue in mononegavirus biology is whether or not transcription is initiated by the same promoter as RNA replication. In this study, residues important for respiratory syncytial virus (RSV) transcription and RNA replication were identified by subjecting the first 26 nucleotides of genome RNA to saturation mutagenesis. This analysis was performed using a genome analog that allowed transcription and RNA replication to be dissociated from each other and monitored as independent events in an intracellular assay. This analysis showed that nucleotides 3C, 5C, 8U, 9U, 10U, and 11U were important for transcription and RNA replication. Additional nucleotides (1U, 2G, 6U, and 7U) were important for RNA replication, but not transcription. At position 4, G versus C or U augmented transcription and decreased replication, showing that the naturally occurring assignments in the genomic (4G) and antigenomic (4U) promoters are optimal for transcription and RNA replication, respectively. These data show that RSV transcription and RNA replication each involve a cis-acting signal at the very 3" end of the genome. This signal appears to contain a minimum, common element that functions in both transcription and RNA replication, defined by those substitutions that had similar effects on the two processes. Apart from these common nucleotides, other positions were involved in RNA replication but not transcription or had different effects on the two processes. This indicates that the promoters for transcription and replication involve overlapping sets of nucleotides at the very 3" end of the genome and provides evidence that the nucleotide preferences for the two processes are not identical.

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