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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

RNA products are generated from the +3 site on the TrC template.(A and B). Effect of omitting UTP from the RNA synthesis reaction. RNA synthesis reactions were performed with all four NTPs (lane 1) or with UTP omitted (lane 2). Reactions contained 2 µM TrC template RNA, wt RdRp, and 1 mM each NTP including either [α-32P]ATP (A), or [α-32P]GTP (B). (C) The 21 nt product is initiated with GTP. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions contained 2 µM TrC template RNA, 10 µM cold GTP and 1 mM ATP, CTP and UTP, and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. (D) [γ-32P]GTP is incorporated into 11 and 13 nt products if UTP is omitted from the reaction. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions included 2 µM TrC template RNA, 50 µM cold GTP and 1 mM ATP, and CTP and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. Note that the 25 nt bands in panel D, lanes 2 and 3 could be due to kinase activity (either in the RSV RdRp or a contaminant of the preparation) phosphorylating the TrC template RNA. The long products detected with [α-32P]GTP in lanes 1 of panels C and D might be due to extensive 3′ nt addition, or repeated stuttering of the RdRp on the U tracts in the template.
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ppat-1002980-g004: RNA products are generated from the +3 site on the TrC template.(A and B). Effect of omitting UTP from the RNA synthesis reaction. RNA synthesis reactions were performed with all four NTPs (lane 1) or with UTP omitted (lane 2). Reactions contained 2 µM TrC template RNA, wt RdRp, and 1 mM each NTP including either [α-32P]ATP (A), or [α-32P]GTP (B). (C) The 21 nt product is initiated with GTP. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions contained 2 µM TrC template RNA, 10 µM cold GTP and 1 mM ATP, CTP and UTP, and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. (D) [γ-32P]GTP is incorporated into 11 and 13 nt products if UTP is omitted from the reaction. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions included 2 µM TrC template RNA, 50 µM cold GTP and 1 mM ATP, and CTP and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. Note that the 25 nt bands in panel D, lanes 2 and 3 could be due to kinase activity (either in the RSV RdRp or a contaminant of the preparation) phosphorylating the TrC template RNA. The long products detected with [α-32P]GTP in lanes 1 of panels C and D might be due to extensive 3′ nt addition, or repeated stuttering of the RdRp on the U tracts in the template.

Mentions: Experiments were performed to characterize the initiation and termination sites of the products of de novo RNA synthesis. During RSV infection, the TrC promoter directs synthesis of genome RNA, which is the full-length complement of the antigenome. Therefore, it would be expected that the RdRp would initiate RNA synthesis from the 3′ terminal nt of the TrC promoter, the +1 position, and continue RNA synthesis to the end of the template to generate a 25 nt product. The finding that the major de novo RNA synthesis product from the 25 nt TrC template was 21 nts in length indicated that the RSV RdRp either initiated internally and/or failed to extend to the end of the template RNA. To identify the initiation site(s), the RNA synthesis reaction was performed without UTP. As shown in Figure 1A, the first A residue in the template is at position +14, so omission of UTP should inhibit the RdRp from continuing RNA synthesis beyond nt 13. Reactions were performed with either [α-32P]ATP or [α-32P]GTP as a label (Figure 4, panels A and B, respectively). In both cases, omission of UTP resulted in a dominant band of 11 nts in length, and another band of 13 nts. However, products longer than 13 nts, including the 21 nt band, were still detectable, particularly in reactions containing [α-32P]ATP (Figure 4A and B, lane 2; note that there are more A than G residues in the Tr product which greatly increases the sensitivity of the [α-32P]ATP label). The presence of these bands suggested that either the NTP stocks were impure, or that the RdRp had poor fidelity in this assay, allowing it to insert an alternative NTP instead of UTP. Products less than 11 nts in length could also be detected, but their abundance was not affected by the presence or absence of UTP, indicating that these were premature termination products, rather than RNA initiated from downstream sites (Figure 4A and B, compare lanes 1 and 2). The fact that the 11 nt product was dominant specifically in reactions lacking UTP indicated that the RSV RdRp could initiate RNA synthesis opposite the position +3 of the TrC template. On the other hand, the 13 nt product could either be RNA that was initiated at +1 and terminated at the first A in the template at position +14, or RNA initiated at +3 and extended to the second A in the template at position +16, due to misincorporation of an NTP opposite position +14.


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

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

RNA products are generated from the +3 site on the TrC template.(A and B). Effect of omitting UTP from the RNA synthesis reaction. RNA synthesis reactions were performed with all four NTPs (lane 1) or with UTP omitted (lane 2). Reactions contained 2 µM TrC template RNA, wt RdRp, and 1 mM each NTP including either [α-32P]ATP (A), or [α-32P]GTP (B). (C) The 21 nt product is initiated with GTP. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions contained 2 µM TrC template RNA, 10 µM cold GTP and 1 mM ATP, CTP and UTP, and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. (D) [γ-32P]GTP is incorporated into 11 and 13 nt products if UTP is omitted from the reaction. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions included 2 µM TrC template RNA, 50 µM cold GTP and 1 mM ATP, and CTP and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. Note that the 25 nt bands in panel D, lanes 2 and 3 could be due to kinase activity (either in the RSV RdRp or a contaminant of the preparation) phosphorylating the TrC template RNA. The long products detected with [α-32P]GTP in lanes 1 of panels C and D might be due to extensive 3′ nt addition, or repeated stuttering of the RdRp on the U tracts in the template.
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ppat-1002980-g004: RNA products are generated from the +3 site on the TrC template.(A and B). Effect of omitting UTP from the RNA synthesis reaction. RNA synthesis reactions were performed with all four NTPs (lane 1) or with UTP omitted (lane 2). Reactions contained 2 µM TrC template RNA, wt RdRp, and 1 mM each NTP including either [α-32P]ATP (A), or [α-32P]GTP (B). (C) The 21 nt product is initiated with GTP. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions contained 2 µM TrC template RNA, 10 µM cold GTP and 1 mM ATP, CTP and UTP, and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. (D) [γ-32P]GTP is incorporated into 11 and 13 nt products if UTP is omitted from the reaction. RNA synthesis reactions were performed with either [α-32P]GTP (lane 1) or [γ-32P]GTP (lanes 2 and 3) as a label. The reactions included 2 µM TrC template RNA, 50 µM cold GTP and 1 mM ATP, and CTP and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. Note that the 25 nt bands in panel D, lanes 2 and 3 could be due to kinase activity (either in the RSV RdRp or a contaminant of the preparation) phosphorylating the TrC template RNA. The long products detected with [α-32P]GTP in lanes 1 of panels C and D might be due to extensive 3′ nt addition, or repeated stuttering of the RdRp on the U tracts in the template.
Mentions: Experiments were performed to characterize the initiation and termination sites of the products of de novo RNA synthesis. During RSV infection, the TrC promoter directs synthesis of genome RNA, which is the full-length complement of the antigenome. Therefore, it would be expected that the RdRp would initiate RNA synthesis from the 3′ terminal nt of the TrC promoter, the +1 position, and continue RNA synthesis to the end of the template to generate a 25 nt product. The finding that the major de novo RNA synthesis product from the 25 nt TrC template was 21 nts in length indicated that the RSV RdRp either initiated internally and/or failed to extend to the end of the template RNA. To identify the initiation site(s), the RNA synthesis reaction was performed without UTP. As shown in Figure 1A, the first A residue in the template is at position +14, so omission of UTP should inhibit the RdRp from continuing RNA synthesis beyond nt 13. Reactions were performed with either [α-32P]ATP or [α-32P]GTP as a label (Figure 4, panels A and B, respectively). In both cases, omission of UTP resulted in a dominant band of 11 nts in length, and another band of 13 nts. However, products longer than 13 nts, including the 21 nt band, were still detectable, particularly in reactions containing [α-32P]ATP (Figure 4A and B, lane 2; note that there are more A than G residues in the Tr product which greatly increases the sensitivity of the [α-32P]ATP label). The presence of these bands suggested that either the NTP stocks were impure, or that the RdRp had poor fidelity in this assay, allowing it to insert an alternative NTP instead of UTP. Products less than 11 nts in length could also be detected, but their abundance was not affected by the presence or absence of UTP, indicating that these were premature termination products, rather than RNA initiated from downstream sites (Figure 4A and B, compare lanes 1 and 2). The fact that the 11 nt product was dominant specifically in reactions lacking UTP indicated that the RSV RdRp could initiate RNA synthesis opposite the position +3 of the TrC template. On the other hand, the 13 nt product could either be RNA that was initiated at +1 and terminated at the first A in the template at position +14, or RNA initiated at +3 and extended to the second A in the template at position +16, due to misincorporation of an NTP opposite position +14.

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