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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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Effect of NTP concentration on RNA synthesis and 3′ nt addition.Reactions contained 0.2 µM TrC RNA with wt (lanes 2–4) or mutant (lane 5) RdRp, [α-32P]GTP and varying concentrations of NTPs, from 200 µM to 1 mM of each NTP, as indicated. Lane 1 shows the molecular weight ladder.
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ppat-1002980-g003: Effect of NTP concentration on RNA synthesis and 3′ nt addition.Reactions contained 0.2 µM TrC RNA with wt (lanes 2–4) or mutant (lane 5) RdRp, [α-32P]GTP and varying concentrations of NTPs, from 200 µM to 1 mM of each NTP, as indicated. Lane 1 shows the molecular weight ladder.

Mentions: To determine if similar results were obtained with a different NTP label, reactions were performed, as described above, using [α-32P]GTP rather than [α-32P]ATP. In this case, the in vitro RNA synthesis reaction also resulted in products of 8–10 and 21 nts in length (Figure 2A, lane 2; note that these bands are faint in this experiment due to the relatively low NTP concentration; see Figure 3). However, dominant products of 26, 27 and 28 nts were also detected, specifically in reactions containing wt RSV RdRp and the TrC RNA. The fact that these products were larger than the input template suggested that they might have been generated as a result of the RdRp adding nts to the 3′ end of the template RNA, as has been shown for a number of other viral RdRps in in vitro reactions [33]–[43]. To test this possibility, reactions were performed with GTP as the only NTP source, to prevent de novo RNA synthesis from the TrC promoter. Under these conditions, a 26 nt band was observed (Figure 2B, lane 3). This result indicated that the 26 nt band was the result of nt addition to the 3′ end of the TrC template and was not a product of de novo RNA synthesis. In addition, RNA containing 3′ puromycin (PMN) in place of the 3′ hydroxyl group was tested in a reaction containing all four NTPs. The presence of 3′ PMN should abrogate 3′ terminal nt addition, while not preventing the ability of the RdRp to use the RNA as a template. The 3′ PMN TrC RNA generated significant levels of the RNAs≤23 nts, but the 26–28 nt RNA products were not detected (Figure 2C, lane 3). These results show that the RNA products smaller than 25 nts were generated by de novo RNA synthesis from the promoter, whereas the products longer than 25 nts were generated by addition of nts to the 3′ end of the template. In summary, the data presented in Figures 1 and 2 show that the RSV RdRp had two distinct RNA synthesis activities in vitro: one in which it used the TrC RNA as a template for de novo synthesis of RNA products, yielding a dominant product of 21 nts, minor products of 22 and 23 nts, and a series of smaller RNAs, and another in which it added additional nts to the 3′ end of the TrC RNA to generate products of 26–28 nts in length. Having identified these activities, we set out to examine the mechanisms by which they occurred.


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

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

Effect of NTP concentration on RNA synthesis and 3′ nt addition.Reactions contained 0.2 µM TrC RNA with wt (lanes 2–4) or mutant (lane 5) RdRp, [α-32P]GTP and varying concentrations of NTPs, from 200 µM to 1 mM of each NTP, as indicated. Lane 1 shows the molecular weight ladder.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002980-g003: Effect of NTP concentration on RNA synthesis and 3′ nt addition.Reactions contained 0.2 µM TrC RNA with wt (lanes 2–4) or mutant (lane 5) RdRp, [α-32P]GTP and varying concentrations of NTPs, from 200 µM to 1 mM of each NTP, as indicated. Lane 1 shows the molecular weight ladder.
Mentions: To determine if similar results were obtained with a different NTP label, reactions were performed, as described above, using [α-32P]GTP rather than [α-32P]ATP. In this case, the in vitro RNA synthesis reaction also resulted in products of 8–10 and 21 nts in length (Figure 2A, lane 2; note that these bands are faint in this experiment due to the relatively low NTP concentration; see Figure 3). However, dominant products of 26, 27 and 28 nts were also detected, specifically in reactions containing wt RSV RdRp and the TrC RNA. The fact that these products were larger than the input template suggested that they might have been generated as a result of the RdRp adding nts to the 3′ end of the template RNA, as has been shown for a number of other viral RdRps in in vitro reactions [33]–[43]. To test this possibility, reactions were performed with GTP as the only NTP source, to prevent de novo RNA synthesis from the TrC promoter. Under these conditions, a 26 nt band was observed (Figure 2B, lane 3). This result indicated that the 26 nt band was the result of nt addition to the 3′ end of the TrC template and was not a product of de novo RNA synthesis. In addition, RNA containing 3′ puromycin (PMN) in place of the 3′ hydroxyl group was tested in a reaction containing all four NTPs. The presence of 3′ PMN should abrogate 3′ terminal nt addition, while not preventing the ability of the RdRp to use the RNA as a template. The 3′ PMN TrC RNA generated significant levels of the RNAs≤23 nts, but the 26–28 nt RNA products were not detected (Figure 2C, lane 3). These results show that the RNA products smaller than 25 nts were generated by de novo RNA synthesis from the promoter, whereas the products longer than 25 nts were generated by addition of nts to the 3′ end of the template. In summary, the data presented in Figures 1 and 2 show that the RSV RdRp had two distinct RNA synthesis activities in vitro: one in which it used the TrC RNA as a template for de novo synthesis of RNA products, yielding a dominant product of 21 nts, minor products of 22 and 23 nts, and a series of smaller RNAs, and another in which it added additional nts to the 3′ end of the TrC RNA to generate products of 26–28 nts in length. Having identified these activities, we set out to examine the mechanisms by which they occurred.

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