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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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The +3 initiation site is utilized during RSV infection.(A) Primer extension analysis of Tr sense RNA generated during RSV infection. Two primers were utilized hybridizing to positions 13–35 or 32–55 relative to the 5′ terminus of RSV genome RNA (left and right panels, respectively). Lanes 3 and 4 show cDNAs generated from RNA isolated from mock or RSV infected cells, respectively. The sizes of the products were determined by co-migration of 32P end-labeled DNA oligonucleotides consisting of Tr sequence 3–35 or 1–35 (left panel, lanes 1 and 2, respectively), or 3–55 or 1–55 (right panel, lanes 1 and 2, respectively) to indicate the lengths of products initiated at +3 or +1. It should be noted that lanes 1–4 of the left panel are all from the same gel, but lanes 1 and 2 required a longer exposure to be detected. (B) Northern blot analysis of small genome sense RNA transcripts generated from the TrC promoter. Lanes 1 and 2 contain RNA isolated from mock or RSV infected cells, respectively. The blot was hybridized with a locked nucleic acid DNA oligonucleotide probe designed to anneal to nts 5–32 relative to the 5′ end of the RSV Tr sequence. (C) Alignment of the sequences from the 3′ terminus of the RSV TrC promoter and the ten nt L gene start (GS) signal. Identical nts are underlined and dashes indicate nts at the −1 and −2 positions relative to the L GS sequence, which are not part of the signal.
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ppat-1002980-g005: The +3 initiation site is utilized during RSV infection.(A) Primer extension analysis of Tr sense RNA generated during RSV infection. Two primers were utilized hybridizing to positions 13–35 or 32–55 relative to the 5′ terminus of RSV genome RNA (left and right panels, respectively). Lanes 3 and 4 show cDNAs generated from RNA isolated from mock or RSV infected cells, respectively. The sizes of the products were determined by co-migration of 32P end-labeled DNA oligonucleotides consisting of Tr sequence 3–35 or 1–35 (left panel, lanes 1 and 2, respectively), or 3–55 or 1–55 (right panel, lanes 1 and 2, respectively) to indicate the lengths of products initiated at +3 or +1. It should be noted that lanes 1–4 of the left panel are all from the same gel, but lanes 1 and 2 required a longer exposure to be detected. (B) Northern blot analysis of small genome sense RNA transcripts generated from the TrC promoter. Lanes 1 and 2 contain RNA isolated from mock or RSV infected cells, respectively. The blot was hybridized with a locked nucleic acid DNA oligonucleotide probe designed to anneal to nts 5–32 relative to the 5′ end of the RSV Tr sequence. (C) Alignment of the sequences from the 3′ terminus of the RSV TrC promoter and the ten nt L gene start (GS) signal. Identical nts are underlined and dashes indicate nts at the −1 and −2 positions relative to the L GS sequence, which are not part of the signal.

Mentions: Although initiation at the +3 site of the TrC promoter has been observed previously in experiments using the RSV minigenome system [44], it has never been described during RSV infection and the size of the RNA generated from this site has not been determined precisely. Examination of the TrC sequence showed that positions +3 to +12 are almost identical to the gene start signal sequence that lies at the beginning of the RSV L gene (Figure 5C), suggesting that initiation at +3 could occur by a mechanism analogous to transcription initiation at the gene start signals that lie internally on the RSV genome. To determine if the +3 initiation site is used during infection, RNA purified from wt RSV infected cells was analyzed by primer extension using TrC-sequence specific primers. Analysis using a primer that hybridized at positions 13–35 relative to the 5′ end of the Tr sequence clearly identified two bands, corresponding to initiation at positions +1 and +3 (Figure 5A, left panel, lane 4). This finding was consistent with the results obtained with the in vitro RNA synthesis assay, and indicates that nt +3 is a bona fide initiation site. Analysis with a primer that hybridized to positions 32–55 of Tr detected RNA initiated from +1 but not from +3, indicating that whereas the RNA initiated from +1 could be elongated, the RNA generated from the +3 initiation site was not extended far enough to hybridize to this primer (Figure 5A, right panel, lane 4). To determine the size of the RNA generated from the +3 site more precisely, RNA from RSV infected cells was also analyzed by Northern blotting with a probe specific to nts 5–32 of Tr, using conditions optimized for examination of RNA of 10–500 nts in length. This analysis identified an apparently abundant RNA transcript of ∼21–25 nts (Figure 5B, lane 2). This length is consistent with the primer extension analysis of the RNA generated from the +3 site, although the data do not exclude the possibility that some of the small RNA was initiated at +1. These data show that the RSV TrC promoter has the unusual property of having two closely positioned initiation sites, one at +1 that is required to generate genome RNA, and another at +3 that yields small RNA transcripts.


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

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

The +3 initiation site is utilized during RSV infection.(A) Primer extension analysis of Tr sense RNA generated during RSV infection. Two primers were utilized hybridizing to positions 13–35 or 32–55 relative to the 5′ terminus of RSV genome RNA (left and right panels, respectively). Lanes 3 and 4 show cDNAs generated from RNA isolated from mock or RSV infected cells, respectively. The sizes of the products were determined by co-migration of 32P end-labeled DNA oligonucleotides consisting of Tr sequence 3–35 or 1–35 (left panel, lanes 1 and 2, respectively), or 3–55 or 1–55 (right panel, lanes 1 and 2, respectively) to indicate the lengths of products initiated at +3 or +1. It should be noted that lanes 1–4 of the left panel are all from the same gel, but lanes 1 and 2 required a longer exposure to be detected. (B) Northern blot analysis of small genome sense RNA transcripts generated from the TrC promoter. Lanes 1 and 2 contain RNA isolated from mock or RSV infected cells, respectively. The blot was hybridized with a locked nucleic acid DNA oligonucleotide probe designed to anneal to nts 5–32 relative to the 5′ end of the RSV Tr sequence. (C) Alignment of the sequences from the 3′ terminus of the RSV TrC promoter and the ten nt L gene start (GS) signal. Identical nts are underlined and dashes indicate nts at the −1 and −2 positions relative to the L GS sequence, which are not part of the signal.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002980-g005: The +3 initiation site is utilized during RSV infection.(A) Primer extension analysis of Tr sense RNA generated during RSV infection. Two primers were utilized hybridizing to positions 13–35 or 32–55 relative to the 5′ terminus of RSV genome RNA (left and right panels, respectively). Lanes 3 and 4 show cDNAs generated from RNA isolated from mock or RSV infected cells, respectively. The sizes of the products were determined by co-migration of 32P end-labeled DNA oligonucleotides consisting of Tr sequence 3–35 or 1–35 (left panel, lanes 1 and 2, respectively), or 3–55 or 1–55 (right panel, lanes 1 and 2, respectively) to indicate the lengths of products initiated at +3 or +1. It should be noted that lanes 1–4 of the left panel are all from the same gel, but lanes 1 and 2 required a longer exposure to be detected. (B) Northern blot analysis of small genome sense RNA transcripts generated from the TrC promoter. Lanes 1 and 2 contain RNA isolated from mock or RSV infected cells, respectively. The blot was hybridized with a locked nucleic acid DNA oligonucleotide probe designed to anneal to nts 5–32 relative to the 5′ end of the RSV Tr sequence. (C) Alignment of the sequences from the 3′ terminus of the RSV TrC promoter and the ten nt L gene start (GS) signal. Identical nts are underlined and dashes indicate nts at the −1 and −2 positions relative to the L GS sequence, which are not part of the signal.
Mentions: Although initiation at the +3 site of the TrC promoter has been observed previously in experiments using the RSV minigenome system [44], it has never been described during RSV infection and the size of the RNA generated from this site has not been determined precisely. Examination of the TrC sequence showed that positions +3 to +12 are almost identical to the gene start signal sequence that lies at the beginning of the RSV L gene (Figure 5C), suggesting that initiation at +3 could occur by a mechanism analogous to transcription initiation at the gene start signals that lie internally on the RSV genome. To determine if the +3 initiation site is used during infection, RNA purified from wt RSV infected cells was analyzed by primer extension using TrC-sequence specific primers. Analysis using a primer that hybridized at positions 13–35 relative to the 5′ end of the Tr sequence clearly identified two bands, corresponding to initiation at positions +1 and +3 (Figure 5A, left panel, lane 4). This finding was consistent with the results obtained with the in vitro RNA synthesis assay, and indicates that nt +3 is a bona fide initiation site. Analysis with a primer that hybridized to positions 32–55 of Tr detected RNA initiated from +1 but not from +3, indicating that whereas the RNA initiated from +1 could be elongated, the RNA generated from the +3 initiation site was not extended far enough to hybridize to this primer (Figure 5A, right panel, lane 4). To determine the size of the RNA generated from the +3 site more precisely, RNA from RSV infected cells was also analyzed by Northern blotting with a probe specific to nts 5–32 of Tr, using conditions optimized for examination of RNA of 10–500 nts in length. This analysis identified an apparently abundant RNA transcript of ∼21–25 nts (Figure 5B, lane 2). This length is consistent with the primer extension analysis of the RNA generated from the +3 site, although the data do not exclude the possibility that some of the small RNA was initiated at +1. These data show that the RSV TrC promoter has the unusual property of having two closely positioned initiation sites, one at +1 that is required to generate genome RNA, and another at +3 that yields small RNA transcripts.

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