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The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter.

Noton SL, Deflubé LR, Tremaglio CZ, Fearns R - PLoS Pathog. (2012)

Bottom Line: The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism.Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection.These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT
Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1-25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3' terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

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Related in: MedlinePlus

Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ CCGCGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.
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ppat-1002980-g008: Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ CCGCGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.

Mentions: Having shown that nts were added to the 3′ end of the TrC RNA in vitro with some specificity, it was of interest to determine if this occurred during RSV infection. In the context of an RSV infection, the TrC sequence is at the 3′ end of the antigenome. To our knowledge, no one has previously identified additional sequences at the 3′ terminus of the RSV antigenome. However, antigenome 3′ terminal sequences are rarely determined directly, but instead are inferred from the genome sequence [7], [46]–[48]. In one paper in which antigenome RNA was analyzed, only a small number of individual clones were sequenced [49]. Thus, prior sequencing analyses did not exclude the possibility that nts are added to the TrC region of a subpopulation of antigenome RNAs during RSV infection. To examine this possibility, antigenome RNA from RSV infected cells was tailed with either A or C residues, transcribed into cDNA by 3′ rapid amplification of cDNA ends (3′ RACE) and sequenced. Direct sequence analysis of the cDNA population showed that there was a mixed population of sequences, with a significant proportion of antigenomes containing additional nts of G, U and/or C at the −1, −2, and −3 positions relative to the 3′ end of the TrC promoter, respectively (Figure 8B, left panels). Sequencing of individual cDNA clones showed that while 10/19 clones contained wt antigenome sequence with no additional nts, 7/19 clones contained a 3′ G, 3′ UG, or 3′ CUG at the end of the antigenome (Figure 8C; note that 2/19 clones did not fall into either category). These sequence additions are consistent with a back-priming event involving interaction of nts 1, 2 and 13, 14 of the TrC RNA and extension by 1–3 nts in a template dependent manner, as illustrated in Figure 8A (left panel). Examination of the Le promoter sequence at the 3′ end of the genome showed that it also has the potential to form a secondary structure that could be used to direct back-priming. Indeed, in this case, a significantly stronger secondary could be formed than by the TrC sequence (Figure 8A, right panel). However, analysis of the same RNA preparation using Le specific probes showed that there was no additional sequence at the 3′ end of the Le promoter in the genome RNA (Figure 8B, right panels), demonstrating that the 3′ end of the Le is unmodified. These findings suggest that in addition to being able to use the TrC RNA as a promoter, the RdRp also facilitates a back-priming event to allow a precise sequence of nts to be added to the 3′ end of the antigenome.


The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter.

Noton SL, Deflubé LR, Tremaglio CZ, Fearns R - PLoS Pathog. (2012)

Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ CCGCGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475672&req=5

ppat-1002980-g008: Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ CCGCGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.
Mentions: Having shown that nts were added to the 3′ end of the TrC RNA in vitro with some specificity, it was of interest to determine if this occurred during RSV infection. In the context of an RSV infection, the TrC sequence is at the 3′ end of the antigenome. To our knowledge, no one has previously identified additional sequences at the 3′ terminus of the RSV antigenome. However, antigenome 3′ terminal sequences are rarely determined directly, but instead are inferred from the genome sequence [7], [46]–[48]. In one paper in which antigenome RNA was analyzed, only a small number of individual clones were sequenced [49]. Thus, prior sequencing analyses did not exclude the possibility that nts are added to the TrC region of a subpopulation of antigenome RNAs during RSV infection. To examine this possibility, antigenome RNA from RSV infected cells was tailed with either A or C residues, transcribed into cDNA by 3′ rapid amplification of cDNA ends (3′ RACE) and sequenced. Direct sequence analysis of the cDNA population showed that there was a mixed population of sequences, with a significant proportion of antigenomes containing additional nts of G, U and/or C at the −1, −2, and −3 positions relative to the 3′ end of the TrC promoter, respectively (Figure 8B, left panels). Sequencing of individual cDNA clones showed that while 10/19 clones contained wt antigenome sequence with no additional nts, 7/19 clones contained a 3′ G, 3′ UG, or 3′ CUG at the end of the antigenome (Figure 8C; note that 2/19 clones did not fall into either category). These sequence additions are consistent with a back-priming event involving interaction of nts 1, 2 and 13, 14 of the TrC RNA and extension by 1–3 nts in a template dependent manner, as illustrated in Figure 8A (left panel). Examination of the Le promoter sequence at the 3′ end of the genome showed that it also has the potential to form a secondary structure that could be used to direct back-priming. Indeed, in this case, a significantly stronger secondary could be formed than by the TrC sequence (Figure 8A, right panel). However, analysis of the same RNA preparation using Le specific probes showed that there was no additional sequence at the 3′ end of the Le promoter in the genome RNA (Figure 8B, right panels), demonstrating that the 3′ end of the Le is unmodified. These findings suggest that in addition to being able to use the TrC RNA as a promoter, the RdRp also facilitates a back-priming event to allow a precise sequence of nts to be added to the 3′ end of the antigenome.

Bottom Line: The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism.Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection.These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT
Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1-25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3' terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

Show MeSH
Related in: MedlinePlus