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

Abstract

RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3' terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at approximately 60% of WT levels, and its product was found to be initiated at the WT position (-1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved.

Fig. 1.

Schematic diagram (not to scale) illustrating the minireplicon template used in this study. The 3′ end of the minireplion is created by a ribozyme (curved arrow) and consists of the first 36 nucleotides of TrC sequence. Note that this promoter sequence lacks transcription-specific signals and so is optimized for detection of replication products. The 5′ end of the template contains the RSV Tr region, with a 22-nucleotide deletion at the 5′ terminus, indicated with a dotted line. This modification prevents the primary replication product from acting as a template for new minireplicons, as depicted by the arrow with a cross. Sites where the Northern blot riboprobes hybridize to the minireplicon template and replication product, and where reverse transcription primers for primer extension (P.E.) and 5′ rapid amplification of cDNA ends (5′ RACE) bind the replication product, are indicated.

Fig. 2.

Impact of mutating the 3′ terminal nucleotide of the template on RSV RNA replication. (A) Quantitation of encapsidated template generated in transfections in which plasmid encoding L was present (+L) or absent (-L). (B) Quantitation of replication products generated from the mutant minireplicons. Black bars represent the replication product in the total RNA samples; white bars represent replication product measured in the MCN-treated RNA samples. (C) Direct comparison of the amounts of total and MCN-resistant replication product generated in the WT and Δ1U reactions. Each RNA value was determined based on Northern blot analysis, such as that shown in Fig. S1. Levels were calculated relative to the respective WT value for A and B, or relative to the WT or Δ1U total RNA values for C (set at 1.0 in each case). Each error bar represents the standard error of the mean from at least three independent experiments.

Fig. 3.

Effect of the 3′ terminal mutations on initiation site selection. Primer extension analysis of the RNA generated from the 1U-C, 1U-A, and Δ1U mutant minireplicons (A, B, and C, respectively). In each case, the mutant RNA was compared with RNA generated from a WT minireplicon. In i and ii, the primer hybridized at positions 24–48 relative to the +1 initiation product. Lane 4 or 5 (as indicated) is a negative control of RNA from cells transfected with plasmid encoding the relevant mutant minireplicon but no L polymerase plasmid. Molecular weight markers present in lanes 1–3 or 1–4 are end-labeled primers, representing products initiated from positions +3 to +1 or +4 to +1 of the template, respectively. In each case, ii is a longer exposure of the gel shown in i to show the +3 initiations. (B iii and iv) Primer extension analysis of the same RNA samples as shown in i and ii, using primers that hybridized at positions 74–100 (iii) or 124–148 (iv) relative to the 5′ end of the +1 initiation product.

Fig. 4.

Sequence analysis of RNA produced from mutant templates. 5′ RACE analysis of the RNA generated from the 1U-C, 1U-A, and Δ1U mutants (A, B, and C, respectively). In each case, i shows agarose gel electrophoresis of the 5′ RACE product generated from RNA from cells transfected with plasmid encoding the relevant mutant minireplicon and either lacking or containing the L polymerase plasmid or lacking MVA-T7, as indicated; ii shows sequence analysis of the input minireplicon plasmid DNA isolated from the transfected cells, and iii shows the corresponding sequence trace derived from the bulk 5′ RACE product (tailed with dGTP). The sequence traces are presented as template sense DNA; TrC sequence is underlined, with position 1 relative to the WT template indicated by an arrow. Sequencing of 5′ RACE reactions tailed with dCTP and of clonal isolates (Fig. S4) showed that the G peak at position 1 in iii is a sequencing artifact.

Fig. 5.

Conserved mechanism for replication initiation. (A) Schematic diagram depicting a proposed model for RSV replication initiation (detailed in text). White box depicts active site of polymerase. Black dots represent putative interactions between the polymerase and the 5′ terminal ATP and the template. Asterisk represents a putative stacking interaction between the ATP and CTP residues. Black bar represents hydrogen bonding. The 3′ terminal position of the template (1U) is shown in gray to illustrate that it is not required for initiation. (B) Alignment of the 5′ terminal trailer sequences of representatives of the family Paramyxoviridae. Alternative terminal sequences were found in the databank but appear to be incomplete.