Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct;17(10):1895-906.
doi: 10.1261/rna.2813411. Epub 2011 Aug 30.

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

Sarah L Noton  1 , Rachel Fearns
Affiliations

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

Sarah L Noton et al. RNA. 2011 Oct.

Abstract

There is limited knowledge regarding how the RNA-dependent RNA polymerases of the nonsegmented negative-strand RNA viruses initiate genome replication. In a previous study of respiratory syncytial virus (RSV) RNA replication, we found evidence that the polymerase could select the 5'-ATP residue of the genome RNA independently of the 3' nucleotide of the template. To investigate if a similar mechanism is used during antigenome synthesis, a study of initiation from the RSV leader (Le) promoter was performed using an intracellular minigenome assay in which RNA replication was restricted to a single step, so that the products examined were derived only from input mutant templates. Templates in which Le nucleotides 1U, or 1U and 2G, were deleted directed efficient replication, and in both cases, the replication products were initiated at the wild-type position, at position -1 or -2 relative to the template, respectively. Sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence. These data indicate that the RSV polymerase is able to select the first two nucleotides of the antigenome and initiate at the correct position, even if the 3'-terminal two nucleotides of the template are missing. Substitution of positions +1 and +2 of the template reduced RNA replication and resulted in increased initiation at positions +3 and +5. Together these data suggest a model for how the RSV polymerase initiates antigenome synthesis.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
(A) Diagram showing the sequence of the RSV Le nucleotides 1–36, with the regions required for polymerase recruitment and initiation, and encapsidation indicated. Positions +1, +3, and +5, which were found to be used as initiation sites in this study, are marked. Nucleotides that are different from those in the TrC promoter are underlined. (B) Schematic diagram (not to scale) depicting the dicistronic minigenome template. The 3′ end of the minigenome consists of the first 36 nt of Le sequence, which directly abuts a CAT-specific sequence that has the RSV N-P gene junction inserted, resulting in two segments, CAT 1 (552 nt) and CAT 2 (190 nt). The 3′ terminus of the Le sequence is generated by a delta ribozyme (curved arrow), which creates a 2′,3′ cyclic phosphate group at the end of the template RNA, while the 5′ terminus of the minigenome is created by a hammerhead ribozyme. Note that CAT 1 lacks a transcription-specific gene start signal but possesses a gene end signal, as indicated by a black box. CAT 2 is separated from CAT 1 by a short intergenic region and is flanked by gene start (gray box) and gene end signals. The 5′ terminus of the minigenome consists of the RSV Tr, with a 22-nt deletion at the 5′ end, as indicated by a dotted line. This deletion removes the promoter from the mini-antigenome replication product such that it is unable to act as a template for new minigenomes, as depicted by the arrow with the cross. The positions on the replication product where reverse transcription primers for primer extension (P.E.) and 5′ rapid amplification of cDNA ends (5′-RACE) hybridize are shown.
FIGURE 2.
FIGURE 2.
Impact of deleting the 3′ terminal 1–3 nt of the minigenome template on RSV RNA replication in HEp-2 cells. (A) Northern blot analysis of minigenome RNAs: nuclease-resistant negative-sense minigenome templates expressed by MVA-T7 polymerase (i); positive-sense replication products expressed by the RSV polymerase from the minigenome templates in total RNA samples (ii); and positive-sense, nuclease-resistant replication products (iii). (Lane 2) A negative control in which the L plasmid, which encodes the enzymatic activity of the RSV polymerase complex, was omitted from the wild-type (wt) minigenome transfection. (B) Primer extension analysis of RNA generated from wt, Δ1, Δ2, and Δ3 templates (lanes 6,8–10). (Lane 7) A negative control in which L was omitted from the wt minigenome transfection. The band that can be detected at the top of the gel in these lanes (and in subsequent figures) is a nonspecific background band. Molecular weight markers in lanes 1–5 are end-labeled oligonucleotides, representing products initiated from position +5 to +1 of the wt template, respectively. (C) Quantitation of total (black bars) and nuclease-resistant RNA (white bars) expressed from each minigenome, as determined by Northern blotting (i); and quantitation of RNA initiated at the +1 position (relative to the wt minigenome) from each minigenome, as determined by primer extension (ii). Each value was calculated relative to the wt value, and each error bar represents standard error of the mean of at least three independent experiments.
FIGURE 3.
FIGURE 3.
Impact of deleting nucleotides 1 and 2 of the minigenome template on RSV RNA replication in BSR-T7 cells. (A,B) The panels are arranged as in Figure 2. (C) 5′-RACE sequence analysis of the RNA products generated from the Δ1 and Δ2 mutant templates: sequence analysis of the input minigenome plasmid DNA isolated from Δ1 (i) and Δ2 (iv) transfected cells. 5′-RACE sequence trace of the corresponding Δ1 (ii) and Δ2 (v) replication products (tailed with dCTP). All sequence traces are shown as template sense DNA. The Le sequence (underlined); sites of mutation in the templates (arrows); and the reintroduced nucleotides in the replication products. Note that the minor sequence that can be detected (beneath the 5′-RACE sequence trace) corresponds to the sequence of input plasmid DNA. Agarose gel electrophoresis of the 5′-RACE products generated from Δ1 and Δ2 mutant RNA samples from transfections that were lacking or containing the L polymerase plasmid, as indicated (iii and vi).
FIGURE 4.
FIGURE 4.
Impact of substituting nucleotide 1U of the minigenome template on RSV RNA replication. Minigenome replication was reconstituted in HEp-2 cells simultaneously infected with MVA-T7, and the resulting RNA was analyzed. Panels are as described for Figure 2, except that panel Cii shows quantitation of RNA initiated at positions +1 and +3; each value was calculated relative to the wt +1 initiation value, and each error bar represents range of the mean of two independent primer extension analyses.
FIGURE 5.
FIGURE 5.
Impact of substituting nucleotides 2G and 3C of the minigenome template on RSV RNA replication. Minigenome replication was reconstituted in HEp-2 cells simultaneously infected with MVA-T7, and the resulting RNA was analyzed. (A–C) The panels are arranged similarly to Figure 2 except that in B, the primer extensions of the RNA produced from the position 2 and position 3 substitution mutants are separated into two panels, i and ii, respectively, and (C) quantitation of the RNA initiated at +1 and +3 from the position 2 and position 3 substitution mutant templates are separated into two panels, ii and iii, respectively. (Aiv) Northern blot analysis of positive-sense RNA generated from a transcription-competent minigenome, to act as a molecular weight marker (MW), and the 2C mutant template, as indicated. The positions of the antigenome, CAT 1, and CAT 2 mRNAs are indicated, and the subgenomic RNA generated from the 2C minigenome is indicated by an asterisk in panels Aii and Aiv. In Bi the position of the +2 band detected from the 2C minigenome is indicated with a dot. (D) Sequence alignment showing the similarity between Le nucleotides 3–12 and the gene start (GS) signal sequence. (E) 5′-RACE sequence analysis of the RNA products generated from the 3U mutant template; arranged as Figure 3C.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albertini AA, Wernimont AK, Muziol T, Ravelli RB, Clapier CR, Schoehn G, Weissenhorn W, Ruigrok RW 2006. Crystal structure of the rabies virus nucleoprotein–RNA complex. Science 313: 360–363 - PubMed
    1. Buchholz UJ, Finke S, Conzelmann KK 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73: 251–259 - PMC - PubMed
    1. Butcher SJ, Grimes JM, Makeyev EV, Bamford DH, Stuart DI 2001. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410: 235–240 - PubMed
    1. Chen D, Patton JT 2000. De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation. RNA 6: 1455–1467 - PMC - PubMed
    1. Chetverin AB, Chetverina HV, Munishkin AV 1991. On the nature of spontaneous RNA synthesis by Qβ replicase. J Mol Biol 222: 3–9 - PubMed

Publication types

MeSH terms

LinkOut - more resources