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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

Analysis of the effect of the 3′ extension on TrC promoter activity in vitro.RNA synthesis reactions were performed using 2 µM of TrC template RNA either lacking (lanes 2 and 4) or containing (lane 3) a 3′ CUG addition at the 3′ terminus, 1 mM of each NTP and [α-32P]ATP, and wt (lanes 2 and 3) or mutant (lane 4) RdRp. Both RNA oligonucleotide templates contained a PMN group at the 3′ end. Lane 1 shows the molecular weight ladder.
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ppat-1002980-g009: Analysis of the effect of the 3′ extension on TrC promoter activity in vitro.RNA synthesis reactions were performed using 2 µM of TrC template RNA either lacking (lanes 2 and 4) or containing (lane 3) a 3′ CUG addition at the 3′ terminus, 1 mM of each NTP and [α-32P]ATP, and wt (lanes 2 and 3) or mutant (lane 4) RdRp. Both RNA oligonucleotide templates contained a PMN group at the 3′ end. Lane 1 shows the molecular weight ladder.

Mentions: The presence of additional sequence at the 3′ end of almost half of the antigenome sequences that we examined indicated that the 3′ extension plays a role in RSV replication. The only known function of antigenome RNA is as a template for RNA synthesis. Therefore, we examined if the additional nts at the 3′ end of the TrC sequence affected promoter activity. The 1–25 TrC RNA template was compared to a “+CUG” RNA template, which contained 1–25 nts of TrC sequence and a 3′ CUG extension, using the in vitro RNA synthesis assay. Both RNA templates contained a 3′ terminal PMN group to ensure that neither was subject to further 3′ modification. Analysis of the RNA generated from these templates showed that the presence of a 3′ terminal CUG extension was highly deleterious to RNA synthesis, indicating that the 3′ extension inhibited access of the RdRp to the promoter (Figure 9, compare lanes 2 and 3). We considered the possibility that the extension might increase initiation from the +1 position, but there was no evidence of incorporation of a [γ-32P]ATP label into RNA synthesized from the +CUG template (data not shown). Thus, these data indicate that the 3′ terminal extension can inhibit antigenome promoter 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 effect of the 3′ extension on TrC promoter activity in vitro.RNA synthesis reactions were performed using 2 µM of TrC template RNA either lacking (lanes 2 and 4) or containing (lane 3) a 3′ CUG addition at the 3′ terminus, 1 mM of each NTP and [α-32P]ATP, and wt (lanes 2 and 3) or mutant (lane 4) RdRp. Both RNA oligonucleotide templates contained a PMN group at the 3′ end. Lane 1 shows the molecular weight ladder.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002980-g009: Analysis of the effect of the 3′ extension on TrC promoter activity in vitro.RNA synthesis reactions were performed using 2 µM of TrC template RNA either lacking (lanes 2 and 4) or containing (lane 3) a 3′ CUG addition at the 3′ terminus, 1 mM of each NTP and [α-32P]ATP, and wt (lanes 2 and 3) or mutant (lane 4) RdRp. Both RNA oligonucleotide templates contained a PMN group at the 3′ end. Lane 1 shows the molecular weight ladder.
Mentions: The presence of additional sequence at the 3′ end of almost half of the antigenome sequences that we examined indicated that the 3′ extension plays a role in RSV replication. The only known function of antigenome RNA is as a template for RNA synthesis. Therefore, we examined if the additional nts at the 3′ end of the TrC sequence affected promoter activity. The 1–25 TrC RNA template was compared to a “+CUG” RNA template, which contained 1–25 nts of TrC sequence and a 3′ CUG extension, using the in vitro RNA synthesis assay. Both RNA templates contained a 3′ terminal PMN group to ensure that neither was subject to further 3′ modification. Analysis of the RNA generated from these templates showed that the presence of a 3′ terminal CUG extension was highly deleterious to RNA synthesis, indicating that the 3′ extension inhibited access of the RdRp to the promoter (Figure 9, compare lanes 2 and 3). We considered the possibility that the extension might increase initiation from the +1 position, but there was no evidence of incorporation of a [γ-32P]ATP label into RNA synthesized from the +CUG template (data not shown). Thus, these data indicate that the 3′ terminal extension can inhibit antigenome promoter 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