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

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.

Figure 1.

Effect of PAP on the synthesis of RNA3 by the viral replicase. (A) PAP-treated and untreated BMV RNA3 in vitro transcripts were used as template for the BMV replicase assay in the presence of radiolabeled CTP. Product RNA3 was separated in a 7 M urea/12% acrylamide gel and visualized by autoradiography. Control is positive-strand, radiolabeled BMV RNA3 in vitro transcript to serve as a size marker. (B) PAP-treated and untreated BMV RNA3 in vitro transcripts were incubated with BMV replicase in the presence of radiolabeled CTP and aliquots were removed at the indicated times. The RNA products were precipitated and the amount synthesized over time was quantified by scintillation counting. Points are means ± SE for three separate experiments. Circles represent PAP-treated template RNA3 and squares represent untreated RNA3. (C) A time-course analysis of the status of PAP-treated BMV RNA3 in vitro transcripts used as template for the replicase assay and primer extension. PAP-treated and untreated BMV RNA3 transcripts were incubated with the replicase (RdRp) or in buffer alone, and aliquots were removed at the indicated times. RNA was analyzed by northern blot for positive-strand BMV RNA3.

Figure 2.

In vitro depurination of BMV RNA3 by PAP. (A) Schematic of BMV RNA3 indicating the sites of depurination by their nucleotide number. The 5′ untranslated region (5′ UTR; 1–91 nt), the open reading frame of RNA3 (ORF3; 92–1003 nt), the intergenic region (IGR; 1004–1246 nt), the open reading frame for RNA4 (ORF4; 1247–1813 nt) and the 3′ untranslated region (3′ UTR; 1814–2113 nt) are also indicated. (B) Representative depurination sites determined by primer extension. In vitro transcript of RNA3, either PAP-treated or untreated, was extended with reverse transcriptase using cDNA primers distributed over the length of the viral RNA. Radiolabeled cDNA products were separated in a 7M urea/6% acrylamide gel and visualized by autoradiography. The same primers were used to identify the depurination sites by deoxynucleotide sequencing of BMV DNA3.

Figure 3.

Replicase interaction with PAP-treated RNA3. (A) PAP-treated and untreated BMV RNA3 in vitro transcripts were used as template RNA with increasing concentration of the 3′ end RNA3 competitor RNA in the replicase assay. Radiolabeled products were separated in a 7 M urea/4.5% acrylamide gel and visualized by autoradiography. (B) PAP-treated and untreated BMV RNA3 in vitro transcripts were incubated with increasing amount of BMV replicase before passage through a nitrocellulose membrane underlaid by a nylon membrane. The retained RNA was visualized by probing the membranes for positive-strand BMV RNA3. (C) Filter-binding assay of PAP-treated and untreated radiolabeled BMV RNA3 transcripts incubated with increasing amount of BMV replicase. The amount of retained RNA was quantified by scintillation counting and corrected by subtracting background counts in the absence of replicase. Points are means ± SE for three separate experiments. Circles represent PAP-treated template RNA3 and squares represent untreated RNA3.

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.

Figure 5.

Effect of depurination location on BMV RNA replication in vivo. (A) Barley protoplasts were transfected with PAP-treated, ligated RNA3 templates described in Figure 4A, plus intact BMV RNA1 and 2 in vitro transcripts and allowed to replicate. Total protoplast RNA was analyzed by northern blot for the presence of positive-strand BMV RNAs. Std is in vitro transcript of positive-strand RNAs 1, 2 and 3 to serve as size markers. Values are means of intensities for RNAs 1, 2 and 3 ± SE for three separate experiments, quantified with a phosphorimager. Blots were also probed for 28S rRNA as a loading control for total RNA. (B) Stability of type I template in barley protoplasts. Type I template (ligated) or non-ligated RNA3 (control) was transfected into barley protoplasts and aliquots of cells were removed at the indicated time points. Total protoplast RNA was probed by northern blot for positive-strand BMV RNA3. The blot was also probed for 28S rRNA as a loading control for total RNA.

Figure 6.

Replicase binding to RNA3 depurinated within the intergenic region. The intergenic region of RNA3 was PAP-treated or untreated, prior to ligation to the remaining 5′ and 3′ regions of RNA3. A filter-binding assay was performed with the ligated, radiolabeled BMV RNA3 transcripts and increasing amount of BMV replicase. The amount of retained RNA was quantified by scintillation counting and corrected by subtracting background counts in the absence of replicase. Points are means ± SE for three separate experiments. Circles represent PAP-treated, ligated RNA3 and squares represent untreated, ligated RNA3.

Figure 7.

Effect of depurination 3′ of the intergenic region on replicase activity in vitro. (A) The 3′ end (terminal 400 nucleotides) of RNA3 was PAP-treated or untreated and ligated to the remaining 5′ region of RNA3. The resulting depurination site following PAP treatment is indicated by the nucleotide number (1971). (B) The RNA3 described in (A) was used as template in the replicase assay and radiolabeled products were separated in a 7 M urea/6% acrylamide gel and visualized by autoradiography. Marker indicates in vitro transcripts of known size.