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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 isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-32P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-32P]GTP. Lane 2 is a control containing all four NTPs and [α-32P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-32P]GTP. In each panel, lane 1 shows the molecular weight ladder.
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ppat-1002980-g002: The isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-32P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-32P]GTP. Lane 2 is a control containing all four NTPs and [α-32P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-32P]GTP. In each panel, lane 1 shows the molecular weight ladder.

Mentions: To enable detailed analysis of the mechanisms involved in RSV RNA synthesis initiation, an assay was developed in which RSV RNA synthesis was reconstituted in vitro using isolated components. To date, the only recombinant NNS virus RdRps that have been expressed and purified in functional form are those of VSV, Chandipura and Sendai virus [18]–[23]; the purification of recombinant RdRp of RSV or any other human pathogens in the paramyxovirus family has not been described. Therefore, a strategy for purification of recombinant RSV RdRp from baculovirus infected insect cells was developed. Based on previous studies it was known that the catalytic domain for RSV RNA synthesis is located in domain III of the 250 kDa large (L) protein [24], [25], and that in infected cells, L forms a complex with the viral phosphoprotein (P), which is thought to act as a bridge between the L protein and the N protein of the nucleocapsid template [26]–[28]. Purification of the RSV L protein proved challenging for two major reasons. First, numerous attempts to express L without P were unsuccessful, indicating that whereas the VSV, Chandipura and Sendai virus L proteins can be expressed in isolation, in the case of RSV, the P protein might be necessary to stabilize L. Second, expression of L protein using the RSV gene sequence resulted in very poor expression of full-length L protein. This problem was overcome by using a codon-optimized version of the L open reading frame. By co-expressing codon-optimized L with P, it was possible to purify microgram quantities of L/P complex to near homogeneity. Figure 1B shows characteristic examples of isolated L/P complexes, with the bands corresponding to the correct migration pattern for full length L and P indicated. Note that the 27 kDa P protein has previously been shown to migrate anomalously [29], [30]. Analysis of these and other bands from a representative gel by excision, trypsin digestion and mass spectrometry, determined that the bands indicated with an asterisk or dots contained L and P specific polypeptides, respectively. The smaller L fragment may arise as a consequence of premature translation termination or proteolytic cleavage of the full length L protein. The relative abundance of this band compared to full-length L protein varied depending on the preparation. The P protein is known to be differentially phosphorylated and to exist as a highly stable oligomer [30], which could account for the multiple P bands present. The band migrating between 70 and 80 kDa was also consistently observed and identified as Hsp70 and/or HSC70 by Western blot analysis (Figure 1C). Hsp70 has previously been shown to affect RSV RdRp activity in an assay involving an infected cell extract [31], but its relevance to RSV RdRp function in the in vitro RNA synthesis assay described here is not yet known. Because the L/P preparations were not completely pure, and because there was variation in the relative levels of full-length and truncated L proteins, the experiments described in Figures 1 and 2 were performed with three independent preparations of wt and mutant L/P complexes and essentially identical results were obtained with each preparation.


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 isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-32P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-32P]GTP. Lane 2 is a control containing all four NTPs and [α-32P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-32P]GTP. In each panel, lane 1 shows the molecular weight ladder.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002980-g002: The isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (LN812A) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-32P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-32P]GTP. Lane 2 is a control containing all four NTPs and [α-32P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-32P]GTP. In each panel, lane 1 shows the molecular weight ladder.
Mentions: To enable detailed analysis of the mechanisms involved in RSV RNA synthesis initiation, an assay was developed in which RSV RNA synthesis was reconstituted in vitro using isolated components. To date, the only recombinant NNS virus RdRps that have been expressed and purified in functional form are those of VSV, Chandipura and Sendai virus [18]–[23]; the purification of recombinant RdRp of RSV or any other human pathogens in the paramyxovirus family has not been described. Therefore, a strategy for purification of recombinant RSV RdRp from baculovirus infected insect cells was developed. Based on previous studies it was known that the catalytic domain for RSV RNA synthesis is located in domain III of the 250 kDa large (L) protein [24], [25], and that in infected cells, L forms a complex with the viral phosphoprotein (P), which is thought to act as a bridge between the L protein and the N protein of the nucleocapsid template [26]–[28]. Purification of the RSV L protein proved challenging for two major reasons. First, numerous attempts to express L without P were unsuccessful, indicating that whereas the VSV, Chandipura and Sendai virus L proteins can be expressed in isolation, in the case of RSV, the P protein might be necessary to stabilize L. Second, expression of L protein using the RSV gene sequence resulted in very poor expression of full-length L protein. This problem was overcome by using a codon-optimized version of the L open reading frame. By co-expressing codon-optimized L with P, it was possible to purify microgram quantities of L/P complex to near homogeneity. Figure 1B shows characteristic examples of isolated L/P complexes, with the bands corresponding to the correct migration pattern for full length L and P indicated. Note that the 27 kDa P protein has previously been shown to migrate anomalously [29], [30]. Analysis of these and other bands from a representative gel by excision, trypsin digestion and mass spectrometry, determined that the bands indicated with an asterisk or dots contained L and P specific polypeptides, respectively. The smaller L fragment may arise as a consequence of premature translation termination or proteolytic cleavage of the full length L protein. The relative abundance of this band compared to full-length L protein varied depending on the preparation. The P protein is known to be differentially phosphorylated and to exist as a highly stable oligomer [30], which could account for the multiple P bands present. The band migrating between 70 and 80 kDa was also consistently observed and identified as Hsp70 and/or HSC70 by Western blot analysis (Figure 1C). Hsp70 has previously been shown to affect RSV RdRp activity in an assay involving an infected cell extract [31], but its relevance to RSV RdRp function in the in vitro RNA synthesis assay described here is not yet known. Because the L/P preparations were not completely pure, and because there was variation in the relative levels of full-length and truncated L proteins, the experiments described in Figures 1 and 2 were performed with three independent preparations of wt and mutant L/P complexes and essentially identical results were obtained with each preparation.

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