Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus

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-enhancing cis-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.Human respiratory syncytial virus (RSV) is a member of the family Paramyxoviridae of the order Mononegavirales, the nonsegmented negative-strand RNA viruses (35). The genome of RSV (strain A2) is 15,222 nucleotides (nt) in length and encodes 11 proteins. Three proteins are associated with the nucleocapsid: the major RNA-binding nucleocapsid N protein, the P phosphoprotein, and the major polymerase subunit L (21, 32, 42). The RSV N, P, and L proteins together with the RNA genome are the virus-specific components required for RNA replication (19,43). Processive transcription requires, in addition, the transcription antitermination protein, M2-1 (11,15,20).In some aspect...

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

“…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).…”

“…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). In their natural contexts, the two promoter regions have functional differences, with the Le region being able to signal both transcription and RNA replication (Fearns et al 2002), while the TrC region signals a higher level of RNA replication than the Le (Fearns et al 2000;Hanley et al 2010). The RSV Le and TrC promoter regions are identical for 10 of the first 11 nucleotides and share significant similarity for the first 36 nucleotides, but then diverge (Mink et al 1991).…”

“…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).…”

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

“…Antigenome and genome RNAs are encapsidated with N protein as they are synthesized, and it is thought that concurrent encapsidation increases polymerase processivity, allowing RNA synthesis to continue through the gene junctions (17,38). RSV follows the same strategy of RNA synthesis as do the prototype viruses, described above, although at a more detailed level there are differences between RSV and various other mononegaviruses in the structure of the gene junction regions (7), adherence to the "rule of six" (33), and organization of cis-acting elements at the genome termini (8,14). In addition, RSV requires a transcription elongation factor, M2-1, for processive transcription (9) and encodes a second factor, M2-2, which appears to increase the efficiency of replication at the expense of transcription (2).…”

“…Initiation of replication and transcription occurs at or near the 3Ј end of the genome (11) and is directed by cis-acting sequences located within a 3Ј extragenic region known as the leader (Le) (4,14,15,20,27,39). For members of the heterologous Paramyxovirinae subfamily of Paramyxoviridae, the genome promoter is more complex and extends into the first gene (20,21,29,37).…”