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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

A semi-specific sequence of nts is added to the 3′ terminus of the TrC RNA.RNA synthesis reactions were performed with either [α-32P]ATP, [α-32P]CTP, [α-32P]GTP, or [α-32P]UTP (panels A–D, respectively). In each case, isolated wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template (lanes 2–4), or its complement Tr 1–25 (lane 5), in a reaction containing either all four NTPs (each at 500 µM; lane 2), or a single NTP (at 500 µM, lanes 3–5). Lane 1 of each panel shows the molecular weight ladder. It should be noted that to avoid confusion the marker indicators are aligned to the outermost part of the molecular weight ladder band, which in each case migrated somewhat more slowly than the rest of the gel. The position of bands representing TrC RNA containing an additional 1, 2, or 3 nts at the −1, −2, and −3 positions relative to the template, respectively, are indicated.
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ppat-1002980-g006: A semi-specific sequence of nts is added to the 3′ terminus of the TrC RNA.RNA synthesis reactions were performed with either [α-32P]ATP, [α-32P]CTP, [α-32P]GTP, or [α-32P]UTP (panels A–D, respectively). In each case, isolated wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template (lanes 2–4), or its complement Tr 1–25 (lane 5), in a reaction containing either all four NTPs (each at 500 µM; lane 2), or a single NTP (at 500 µM, lanes 3–5). Lane 1 of each panel shows the molecular weight ladder. It should be noted that to avoid confusion the marker indicators are aligned to the outermost part of the molecular weight ladder band, which in each case migrated somewhat more slowly than the rest of the gel. The position of bands representing TrC RNA containing an additional 1, 2, or 3 nts at the −1, −2, and −3 positions relative to the template, respectively, are indicated.

Mentions: The data presented in Figure 2 show that in addition to generating newly synthesized RNA, the RSV RdRp could add nts to the 3′ end of the TrC RNA. Experiments were performed to determine which nts could be added, and to establish if they were added in a specific order. Reactions were performed containing each NTP label, either alone, or in combination with the other unlabeled NTPs. As described above, incubation with GTP in the absence of other NTPs showed strong incorporation into a 26 nt band, but no detectable incorporation into longer RNAs (Figure 6C, lane 3). If other NTPs were included in the reaction, a 27 nt band could be detected (Figure 6C, lane 2). This indicated that a different nt was added after the G to generate the 27 nt RNA. Labeled CTP was also incorporated into a 26 nt band in the absence of other NTPs, and yielded dominant bands of 26 and 28 nts when all four NTPs were present (Figure 6B, compare lane 3 with lane 2). In contrast, when UTP was used as a label, no incorporation was detected with UTP alone, but a 27 nt band was dominant when the other NTPs were present and a 28 nt band could be faintly detected (Figure 6D, compare lane 3 with 2). Similarly to the results shown in Figures 1 and 4, ATP showed only very weak incorporation into RNA longer than 25 nts, either in the presence or absence of other NTPs (Figure 6A). These data suggest that nts were incorporated onto the 3′ end of the TrC RNA with some specificity. Based on these data it can be deduced that either a G or C residue could be added to the −1 position at the 3′ end of the TrC RNA; a U residue could only be efficiently added after G, resulting in the 27 nt bands detected with either the GTP or UTP label, but not detectable with a CTP label; a C residue could then be added to the U to generate the 28 nt band, detected with CTP, and UTP, and to a lesser extent with a GTP label (see also Figures 2, 3 and 4). Thus, the sequence of nts most frequently added to the 3′ end of the TrC RNA was G, GU, GUC, or C only; other nt sequences, such as an A tract, might also have been added to a lesser extent. This experiment also revealed that ATP and CTP could be incorporated into the 3′ end of the Tr sense RNA also (Figure 6A and B, lane 5), but the CTP label showed that this occurred less frequently than addition to the 3′ end of the TrC RNA (Figure 6B, compare lanes 3 and 5).


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

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

A semi-specific sequence of nts is added to the 3′ terminus of the TrC RNA.RNA synthesis reactions were performed with either [α-32P]ATP, [α-32P]CTP, [α-32P]GTP, or [α-32P]UTP (panels A–D, respectively). In each case, isolated wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template (lanes 2–4), or its complement Tr 1–25 (lane 5), in a reaction containing either all four NTPs (each at 500 µM; lane 2), or a single NTP (at 500 µM, lanes 3–5). Lane 1 of each panel shows the molecular weight ladder. It should be noted that to avoid confusion the marker indicators are aligned to the outermost part of the molecular weight ladder band, which in each case migrated somewhat more slowly than the rest of the gel. The position of bands representing TrC RNA containing an additional 1, 2, or 3 nts at the −1, −2, and −3 positions relative to the template, respectively, are indicated.
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Related In: Results  -  Collection

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

ppat-1002980-g006: A semi-specific sequence of nts is added to the 3′ terminus of the TrC RNA.RNA synthesis reactions were performed with either [α-32P]ATP, [α-32P]CTP, [α-32P]GTP, or [α-32P]UTP (panels A–D, respectively). In each case, isolated wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template (lanes 2–4), or its complement Tr 1–25 (lane 5), in a reaction containing either all four NTPs (each at 500 µM; lane 2), or a single NTP (at 500 µM, lanes 3–5). Lane 1 of each panel shows the molecular weight ladder. It should be noted that to avoid confusion the marker indicators are aligned to the outermost part of the molecular weight ladder band, which in each case migrated somewhat more slowly than the rest of the gel. The position of bands representing TrC RNA containing an additional 1, 2, or 3 nts at the −1, −2, and −3 positions relative to the template, respectively, are indicated.
Mentions: The data presented in Figure 2 show that in addition to generating newly synthesized RNA, the RSV RdRp could add nts to the 3′ end of the TrC RNA. Experiments were performed to determine which nts could be added, and to establish if they were added in a specific order. Reactions were performed containing each NTP label, either alone, or in combination with the other unlabeled NTPs. As described above, incubation with GTP in the absence of other NTPs showed strong incorporation into a 26 nt band, but no detectable incorporation into longer RNAs (Figure 6C, lane 3). If other NTPs were included in the reaction, a 27 nt band could be detected (Figure 6C, lane 2). This indicated that a different nt was added after the G to generate the 27 nt RNA. Labeled CTP was also incorporated into a 26 nt band in the absence of other NTPs, and yielded dominant bands of 26 and 28 nts when all four NTPs were present (Figure 6B, compare lane 3 with lane 2). In contrast, when UTP was used as a label, no incorporation was detected with UTP alone, but a 27 nt band was dominant when the other NTPs were present and a 28 nt band could be faintly detected (Figure 6D, compare lane 3 with 2). Similarly to the results shown in Figures 1 and 4, ATP showed only very weak incorporation into RNA longer than 25 nts, either in the presence or absence of other NTPs (Figure 6A). These data suggest that nts were incorporated onto the 3′ end of the TrC RNA with some specificity. Based on these data it can be deduced that either a G or C residue could be added to the −1 position at the 3′ end of the TrC RNA; a U residue could only be efficiently added after G, resulting in the 27 nt bands detected with either the GTP or UTP label, but not detectable with a CTP label; a C residue could then be added to the U to generate the 28 nt band, detected with CTP, and UTP, and to a lesser extent with a GTP label (see also Figures 2, 3 and 4). Thus, the sequence of nts most frequently added to the 3′ end of the TrC RNA was G, GU, GUC, or C only; other nt sequences, such as an A tract, might also have been added to a lesser extent. This experiment also revealed that ATP and CTP could be incorporated into the 3′ end of the Tr sense RNA also (Figure 6A and B, lane 5), but the CTP label showed that this occurred less frequently than addition to the 3′ end of the TrC RNA (Figure 6B, compare lanes 3 and 5).

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