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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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Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-32P]GTP, and lanes 4–6 show RNAs labeled with [α-32P]ATP.
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ppat-1002980-g007: Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-32P]GTP, and lanes 4–6 show RNAs labeled with [α-32P]ATP.

Mentions: The mechanism by which the nts were added to the TrC RNA was investigated. There were two potential mechanisms by which 3′ nt addition could occur: terminal transferase activity, or back-priming (also known as template dependent priming). In back-priming, the 3′ end of the RNA interacts with an internal sequence to form a hairpin structure, and the RdRp adds nts to the 3′ terminus using the folded RNA as a template [38], [45]. Visual inspection of the TrC RNA sequence showed there was possibility for two alternative hairpin loop structures to form in which nt 1U could base pair with either nts 14A or 16A, and nt 2G could base pair with either nts 13C or 15C. Pairing of nts 1 and 2 with 13 and 14 and extension by one to three nts would allow the RdRp to add a G, GU, or GUC, to the 3′ end of the TrC RNA by using nts 15C-17G as a template, whereas pairing of nts 1 and 2 with 15 and 16 would allow the RdRp to add a C (Figure 7A). This model was consistent with the results shown in Figure 6. To investigate this model, nts 1 or 14 and 16 in the 25 nt TrC RNA were substituted (Figure 7A) and NTP incorporation at the 3′ end of the RNA was examined using either a GTP or ATP label. Substitution of position 1U with an A caused a significant decrease in the levels of the 26–28 nt RNAs, suggesting that the identity of the 3′ terminal nt was significant for 3′ nt addition to occur (Figure 7B, compare lanes 1 and 2). Surprisingly, substitution of positions 14A and 16A with U residues did not block 3′ nt addition, but caused an alteration in the number and sequence of incorporated nts, with A being added, and G only being incorporated into longer products (Figure 7B, compare lanes 1 and 3, and 4 and 6). Thus, disruption of possible base-pairing between the 3′ terminus and nts 13 and 14, or 15 and 16 did not prevent 3′ addition, but altered the sequence of added nts. These results show that modification of the 3′ end of the TrC RNA involves an internal sequence, consistent with a back-priming mechanism, rather than terminal transferase activity.


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

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

Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-32P]GTP, and lanes 4–6 show RNAs labeled with [α-32P]ATP.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475672&req=5

ppat-1002980-g007: Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-32P]GTP, and lanes 4–6 show RNAs labeled with [α-32P]ATP.
Mentions: The mechanism by which the nts were added to the TrC RNA was investigated. There were two potential mechanisms by which 3′ nt addition could occur: terminal transferase activity, or back-priming (also known as template dependent priming). In back-priming, the 3′ end of the RNA interacts with an internal sequence to form a hairpin structure, and the RdRp adds nts to the 3′ terminus using the folded RNA as a template [38], [45]. Visual inspection of the TrC RNA sequence showed there was possibility for two alternative hairpin loop structures to form in which nt 1U could base pair with either nts 14A or 16A, and nt 2G could base pair with either nts 13C or 15C. Pairing of nts 1 and 2 with 13 and 14 and extension by one to three nts would allow the RdRp to add a G, GU, or GUC, to the 3′ end of the TrC RNA by using nts 15C-17G as a template, whereas pairing of nts 1 and 2 with 15 and 16 would allow the RdRp to add a C (Figure 7A). This model was consistent with the results shown in Figure 6. To investigate this model, nts 1 or 14 and 16 in the 25 nt TrC RNA were substituted (Figure 7A) and NTP incorporation at the 3′ end of the RNA was examined using either a GTP or ATP label. Substitution of position 1U with an A caused a significant decrease in the levels of the 26–28 nt RNAs, suggesting that the identity of the 3′ terminal nt was significant for 3′ nt addition to occur (Figure 7B, compare lanes 1 and 2). Surprisingly, substitution of positions 14A and 16A with U residues did not block 3′ nt addition, but caused an alteration in the number and sequence of incorporated nts, with A being added, and G only being incorporated into longer products (Figure 7B, compare lanes 1 and 3, and 4 and 6). Thus, disruption of possible base-pairing between the 3′ terminus and nts 13 and 14, or 15 and 16 did not prevent 3′ addition, but altered the sequence of added nts. These results show that modification of the 3′ end of the TrC RNA involves an internal sequence, consistent with a back-priming mechanism, rather than terminal transferase activity.

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