Limits...
Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo.

Karran RA, Hudak KA - Nucleic Acids Res. (2008)

Bottom Line: Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product.This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis.These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario, Canada.

ABSTRACT
Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

Show MeSH

Related in: MedlinePlus

Effect of depurination location on replicase activity in vitro. Regions of RNA3 were treated with PAP and then ligated to the remaining RNA to regenerate full-length RNA3 used as template in the replicase assay. (A) For the type I template, the 5′ region was PAP-treated or untreated and ligated to the remaining 3′ end, which was untreated. For the type II template, the 3′ region was PAP-treated or untreated and ligated to the 5′ UTR, which was untreated. For the type III template, the intergenic region was PAP-treated or untreated and ligated to the remaining 5′ and 3′ regions. (B) Synthesis of RNA3 following the replicase assay using the template RNAs described in (A). The radiolabeled RNA products were separated in a 7 M urea/12% acrylamide gel and visualized by autoradiography. Std represents radiolabeled, positive-strand RNA3 in vitro transcript to serve as a size marker. Values are means of intensities ± SE for three separate experiments quantified with a phosphorimager.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602774&req=5

Figure 4: Effect of depurination location on replicase activity in vitro. Regions of RNA3 were treated with PAP and then ligated to the remaining RNA to regenerate full-length RNA3 used as template in the replicase assay. (A) For the type I template, the 5′ region was PAP-treated or untreated and ligated to the remaining 3′ end, which was untreated. For the type II template, the 3′ region was PAP-treated or untreated and ligated to the 5′ UTR, which was untreated. For the type III template, the intergenic region was PAP-treated or untreated and ligated to the remaining 5′ and 3′ regions. (B) Synthesis of RNA3 following the replicase assay using the template RNAs described in (A). The radiolabeled RNA products were separated in a 7 M urea/12% acrylamide gel and visualized by autoradiography. Std represents radiolabeled, positive-strand RNA3 in vitro transcript to serve as a size marker. Values are means of intensities ± SE for three separate experiments quantified with a phosphorimager.

Mentions: Our observation that prior treatment of RNA3 with PAP reduced the accumulation of RNA product and also reduced the binding of the replicase complex suggested that depurination outside the defined replicase promoter and initiation sites within the 3′ terminus could affect the activity of the enzyme complex. To investigate this possibility, PAP-treated regions of RNA3 were ligated to remaining untreated portions, to regenerate the full-length RNA. For the type I template, the 3′-terminal 200 nucleotides were ligated to the PAP-treated 5′ region of 1913 nucleotides. In addition, two other ligated RNA3 molecules were made, one in which the 5′ UTR (92 nucleotides) was untreated and ligated to the 2021 nucleotides downstream that were first incubated with PAP (type II template), and the third template in which the intergenic region, comprised of nucleotides 1004–1246, was PAP-treated and ligated to the 5′ and 3′ regions (type III template), as illustrated in Figure 4A. Each fragment treated with PAP was assessed by primer extension analysis to confirm the expected depurination sites prior to ligation (data not shown). These ligated RNAs were then used as templates in the replicase assay, to determine whether depurination outside the 5′ and 3′ termini could influence replicase activity. Decreased RNA synthesis was observed with the type I template; therefore, depurination upstream of the characterized replicase binding site at the 3′ end inhibited replicase activity (Figure 4B). Results of the replicase assay using the type II template indicated that depurination downstream of the 5′ UTR also inhibited replicase activity. The decrease in RNA product seen from prior PAP treatment of the 5′ region in type I template and the 3′ region in type II template suggested that depurination within the intergenic region may affect replicase activity. To assess this possibility, type III template was designed to allow PAP treatment only within the intergenic region of RNA3, prior to its ligation to the 5′ and 3′ regions. The observed decrease in amount of RNA3 product indicated that depurination solely within the intergenic region reduced replicase activity (Figure 4C).Figure 4.


Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo.

Karran RA, Hudak KA - Nucleic Acids Res. (2008)

Effect of depurination location on replicase activity in vitro. Regions of RNA3 were treated with PAP and then ligated to the remaining RNA to regenerate full-length RNA3 used as template in the replicase assay. (A) For the type I template, the 5′ region was PAP-treated or untreated and ligated to the remaining 3′ end, which was untreated. For the type II template, the 3′ region was PAP-treated or untreated and ligated to the 5′ UTR, which was untreated. For the type III template, the intergenic region was PAP-treated or untreated and ligated to the remaining 5′ and 3′ regions. (B) Synthesis of RNA3 following the replicase assay using the template RNAs described in (A). The radiolabeled RNA products were separated in a 7 M urea/12% acrylamide gel and visualized by autoradiography. Std represents radiolabeled, positive-strand RNA3 in vitro transcript to serve as a size marker. Values are means of intensities ± SE for three separate experiments quantified with a phosphorimager.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602774&req=5

Figure 4: Effect of depurination location on replicase activity in vitro. Regions of RNA3 were treated with PAP and then ligated to the remaining RNA to regenerate full-length RNA3 used as template in the replicase assay. (A) For the type I template, the 5′ region was PAP-treated or untreated and ligated to the remaining 3′ end, which was untreated. For the type II template, the 3′ region was PAP-treated or untreated and ligated to the 5′ UTR, which was untreated. For the type III template, the intergenic region was PAP-treated or untreated and ligated to the remaining 5′ and 3′ regions. (B) Synthesis of RNA3 following the replicase assay using the template RNAs described in (A). The radiolabeled RNA products were separated in a 7 M urea/12% acrylamide gel and visualized by autoradiography. Std represents radiolabeled, positive-strand RNA3 in vitro transcript to serve as a size marker. Values are means of intensities ± SE for three separate experiments quantified with a phosphorimager.
Mentions: Our observation that prior treatment of RNA3 with PAP reduced the accumulation of RNA product and also reduced the binding of the replicase complex suggested that depurination outside the defined replicase promoter and initiation sites within the 3′ terminus could affect the activity of the enzyme complex. To investigate this possibility, PAP-treated regions of RNA3 were ligated to remaining untreated portions, to regenerate the full-length RNA. For the type I template, the 3′-terminal 200 nucleotides were ligated to the PAP-treated 5′ region of 1913 nucleotides. In addition, two other ligated RNA3 molecules were made, one in which the 5′ UTR (92 nucleotides) was untreated and ligated to the 2021 nucleotides downstream that were first incubated with PAP (type II template), and the third template in which the intergenic region, comprised of nucleotides 1004–1246, was PAP-treated and ligated to the 5′ and 3′ regions (type III template), as illustrated in Figure 4A. Each fragment treated with PAP was assessed by primer extension analysis to confirm the expected depurination sites prior to ligation (data not shown). These ligated RNAs were then used as templates in the replicase assay, to determine whether depurination outside the 5′ and 3′ termini could influence replicase activity. Decreased RNA synthesis was observed with the type I template; therefore, depurination upstream of the characterized replicase binding site at the 3′ end inhibited replicase activity (Figure 4B). Results of the replicase assay using the type II template indicated that depurination downstream of the 5′ UTR also inhibited replicase activity. The decrease in RNA product seen from prior PAP treatment of the 5′ region in type I template and the 3′ region in type II template suggested that depurination within the intergenic region may affect replicase activity. To assess this possibility, type III template was designed to allow PAP treatment only within the intergenic region of RNA3, prior to its ligation to the 5′ and 3′ regions. The observed decrease in amount of RNA3 product indicated that depurination solely within the intergenic region reduced replicase activity (Figure 4C).Figure 4.

Bottom Line: Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product.This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis.These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario, Canada.

ABSTRACT
Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

Show MeSH
Related in: MedlinePlus