Identification of Internal Sequences in the 3′ Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity

Abstract

Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3 end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di-and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.

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

“…This template was based on a dicistronic minigenome similar to that used for a number of studies of RSV transcription and replication (Fearns et al 2002;Cowton and Fearns 2005;McGivern et al 2005;Noton et al 2010). The minigenome is generated from a T7 promoter, and its 39 terminus is generated by ribozyme cleavage.…”

“…The NNS RNA viruses replicate via a positive-strand RNA replication intermediate called the antigenome, and both the antigenome and genome RNAs are encapsidated with viral nucleoprotein (N) as they are synthesized to form a helical nucleocapsid (Whelan et al 2004;Albertini et al 2006;Green et al 2006;Tawar et al 2009). Concurrent encapsidation of the nascent replication product is believed to be necessary for polymerase processivity and so is a key feature of RNA replication, although it is not yet clear if it is a component of the replication initiation complex or becomes associated after initiation has occurred (Vidal and Kolakofsky 1989;Gubbay et al 2001;Qanungo et al 2004;McGivern et al 2005). In RSV, as in the other NNS viruses, there are two promoter regions for RNA replication: the leader (Le) region at the 39 end of the genome and the trailer-complement (TrC) region at the 39 end of the antigenome (Mink et al 1991).…”

“…Functional mapping of the Le region showed that the first 11 nucleotides are sufficient to recruit the RSV polymerase and direct initiation of RNA synthesis (Fearns et al 2002;Cowton and Fearns 2005), while nucleotides up to position 36 are required in addition for encapsidation of the replication product ( Fig. 1A; McGivern et al 2005). The first 36 nt of the TrC promoter are also sufficient to signal synthesis of an encapsidated replication product (Fearns et al 2000).…”

“…Studies of RSV RNA replication initiation have been greatly aided using a minigenome system in which RNA replication is reconstituted in cells using plasmid-expressed RNA and polymerase proteins (Grosfeld et al 1995;Yu et al 1995;Fearns et al 1997Fearns et al , 2000Fearns et al , 2002Peeples and Collins 2000;Marriott et al 2001;Cowton and Fearns 2005;McGivern et al 2005;Noton et al 2010). Of particular significance is the use of a minigenome (or mini-antigenome) in which the template RNA provided to the polymerase is limited to a single step of replication so that the products reflect what is produced from mutant templates, rather than a template that is selected over multiple replication cycles (Fearns et al 2002;Cowton and Fearns 2005;McGivern et al 2005;Noton et al 2010).…”

“…Of particular significance is the use of a minigenome (or mini-antigenome) in which the template RNA provided to the polymerase is limited to a single step of replication so that the products reflect what is produced from mutant templates, rather than a template that is selected over multiple replication cycles (Fearns et al 2002;Cowton and Fearns 2005;McGivern et al 2005;Noton et al 2010). This system has allowed analysis of replication initiation in an intracellular setting using defined mutant templates.…”