Replicase-binding sites on plus- and minus-strand brome mosaic virus RNAs and their roles in RNA replication in plant cells

Abstract

The cis-acting elements for Brome mosaic virus (BMV) RNA synthesis have been characterized primarily for RNA3. To identify additional replicase-binding elements, nested fragments of all three of the BMV RNAs, both plus- and minus-sense fragments, were constructed and tested for binding enriched BMV replicase in a template competition assay. Ten RNA fragments containing replicase-binding sites were identified; eight were characterized further because they were more effective competitors. All eight mapped to noncoding regions of BMV RNAs, and the positions of seven localized to sequences containing previously characterized core promoter elements (C. C. Kao, Mol. Plant Pathol. 3:55-62, 2001), thus suggesting the identities of the replicase-binding sites. Three contained the tRNA-like structures that direct minus-strand RNA synthesis, three were within the 3' region of each minus-strand RNA that contained the core promoter for genomic plus-strand initiation, and one was in the core subgenomic promoter. Single-nucleotide mutations known previously to abolish RNA synthesis in vitro prevented replicase binding. When tested in the context of the respective full-length RNAs, the same mutations abolished BMV RNA synthesis in transfected barley protoplasts. The eighth site was within the intercistronic region (ICR) of plus-strand RNA3. Further mapping showed that a sequence of 22 consecutive adenylates was responsible for binding the replicase, with 16 being the minimal required length. Deletion of the poly(A) sequence was previously shown to severely debilitate BMV RNA replication in plants (E. Smirnyagina, Y. H. Hsu, N. Chua, and P. Ahlquist, Virology 198:427-436, 1994). Interestingly, the B box motif in the ICR of RNA3, which has previously been determined to bind the 1a protein, does not bind the replicase. These results identify the replicase-binding sites in all of the BMV RNAs and suggest that the recognition of RNA3 is different from that of RNA1 and RNA2.

FIG. 1.

Summary of previously reported cis-acting sequences in BMV RNAs. Locations of the cis-acting sequences are denoted by shaded rectangles. Near each rectangle is a brief description of the name of each motif and the nucleotide positions at which the motif resides. Plus- and minus-strand RNAs are denoted with “+” or “−. ” The first nucleotide at the 5′ end of the plus- and minus-strand RNAs are designated, respectively, with +1 and −1. The amount of area shaded denotes the approximate location within the RNA and is not to scale.

FIG. 2.

Regions in BMV RNAs that confer higher-affinity binding to the BMV replicase in vitro. (A) Schema used to generate desired competitor RNAs. Overlapping cDNA fragments flanked by T7 and SP6 promoters were produced by PCR using appropriate primers and BMV plasmids from the work of Janda and Ahlquist (23). (B) Results from a typical template competition assay used to determine the IC₅₀ for each competitor RNA. The gel image shows the effects of a competitor, B3(−)8, that binds the replicase and of B3(−)6, which does not. Template −20/13 at 2 nM served as the reference RNA in these assays. The amount of product from −20/13 is quantified against each concentration of competitor to derive the IC₅₀. % Syn., percent synthesized. (C) Summary of the 10 RNA fragments that we detected to bind the BMV replicase, their IC₅₀s, the location of the BMV that they encode, and the motif that may bind the replicase. Eight of the binders for which IC₅₀s were lower than 5 nM are considered effective and are characterized further in this work. The motifs within these eight were predicted based on previous characterizations detailed in the introduction. The weak replicase-binding motif within B2(+)5 was not previously identified, and its relevance in BMV replication remains to be determined.

FIG. 3.

Roles of SLC in replicase binding and BMV RNA accumulation in barley protoplasts. (A) Schematic representation of the BMV RNAs and the locations of the eight effective replicase-binding sequences. The shaded regions containing SLC are flanked with numbers denoting the nucleotide positions of the RNA fragment. The bars represent BMV RNAs, and the portion of SLC containing the clamped adenine (circled) is shown in the box. The term “SLC^m” designates an RNA with a mutation in the clamped adenine. (B) IC₅₀s of a number of RNAs that were used in a template competition assay. RNA −20/3 served as a positive control, and its replicase-binding properties were characterized by Sivakumaran et al. (45). The other RNAs contain the 3′ NCR of the three BMV RNAs (Table 1). SLC^m indicates that the clamped adenine was changed to a guanine. (C) Effects of SLC mutations in transfected barley protoplasts. The gel image shows the effect of SLC mutations on genomic minus-strand and genomic plus-strand accumulation. The identities of the most relevant RNAs used in the transfections are shown above the image. “All” indicates that the RNAs were from protoplasts transfected with all three BMV RNAs that had mutated SLCs. The gel image of the 18S rRNAs is intended to show an internal loading control. WT, wild type.

FIG. 4.

Effects of the subgenomic core promoter on replicase binding and BMV RNA accumulation in cells. (A) Schematic of plus- and minus-strand BMV RNA3s, with the eight identified replicase-binding sites (shaded) and the location of the subgenomic core promoter. The secondary structure of the subgenomic core promoter sequences was predicted by the MFOLD program (55) and validated by mutational analysis performed as described in the work of Sivakumaran et al. (45). The locations of the key residues in the core promoter are indicated by their positions relative to the initiation cytidylate (+1). The mutation at position −14A (mutated to a U) is shown by the arrow. (B) Quantitative results from competition assays in which the amounts of products synthesized from a 2 nM concentration of −20/13 are plotted against each concentration of B3(−)8 and B3(−)8-14A/U. The IC₅₀s of the two RNAs were from two independent assays that were highly similar. (C) Effects of the −14U mutation that abolished replicase binding on BMV RNA accumulation in barley protoplast. The identities of the most-relevant RNAs in the transfected mixture are indicated above the gel image. 18S rRNA serves as an internal loading control. WT, wild type.

FIG. 5.

Role of the cB box on replicase binding and on BMV RNA accumulation in barley protoplasts. (A) Schematic of the RNAs examined in this set of experiments. The shaded regions denote the positions of the eight replicase-binding sites. The three sites that were analyzed in this set of experiments have numbers that denote the nucleotide positions flanking the fragments. The intercistronic B site, denoted with an asterisk, is also examined in this figure. The RNA secondary structures are the predicted structures that contain a cB box in minus-strand RNA1 and RNA2 or the AAUU-box in RNA3 (47). Arrows identify single-nucleotide changes in the cB box motif in RNA1 and RNA2 and in the cB-like sequence of RNA3. For RNA1 and RNA2, the mutations are simply named cB/B box mutations. In minus-strand RNA3, two mutations were made, and the name of each is given in parentheses. Locations of the cB box and B box in the RNA3 ICR are denoted by asterisks. (B) Results from template competition assays. RNA fragments with mutations are denoted by an addition to the names of the RNA. (C) Northern blot analysis of mutated RNA accumulated in barley protoplasts. The most-relevant RNA in the transfected mixture is noted above the gel image. The identities of wild-type (WT) and mutant RNAs are shown to the side of the image. (D) Effects of mutations in the 3′ end of BMV RNA3 on RNA accumulation in barley protoplasts. (E) Results from competition assay using B3(−)1 or B3(+)1 with the SK1 or SK2 mutation. The reference template is −20/13. The results were tested twice, yielding consistent results. % Syn., percent synthesis.

FIG. 6.

The poly(A) sequence in the ICR can bind the BMV replicase. (A) Mapped sequences in B3(+)8 that contained at least one replicase-binding sequence. The IC₅₀s of RNAs tested in template competition assays are summarized to the right of the bars that denote the portion of the RNA derived from B3(+)8. The initial mapping led to the identification of the sequence between nt 1190 and 1240 as the one encoding a replicase-binding sequence. RNAs B3(+)8a, B3(+)8b, and B3(+)8c were made to further address the responsible sequence. In the sequences shown, lowercase g's represents nucleotides that were added to the BMV sequence (in uppercase letters) to allow transcription by the T7 RNA polymerase. (B) Preparation of poly(A) molecules of various lengths for use in template competition assays. The gel contains the purified RNAs separated in a 20% denaturing polyacrylamide gel and stained with toluidine blue. The nucleotide lengths of several marker RNAs are shown to the left side of the gel. (C) Summary of the results from template competition assays for poly(A) molecules of different lengths or a chemically synthesized molecule of 22 deoxyadenylates. The two gel images are from template competition assays performed with competitors of 22 or 15 adenylates. % Syn., percent synthesis.

FIG. 7.

Requirement for compatible interaction between 5′ and 3′ NCR in BMV RNA2. A Northern blot examining the accumulation of the minus-strand replication products from several chimeric BMV RNAs is shown. The names of the RNAs denote the origin of the 5′ NCR, the protein-coding region, and the 3′ NCR of a BMV RNA. For example, R2/1/2 indicates that the 5′ and 3′ NCR came from RNA2 but that the protein-coding region was from RNA1. Plus-strand RNA products are not shown but exhibited the same trends seen with the minus-strand RNAs. The combinations of the RNAs transfected into protoplasts are shown above the gel image. Total RNAs were purified from protoplasts at the times (in hours) indicated below the gel images. Asterisks identify the positions expected of the chimeric RNAs. All of the samples in this experiment were on one blot but were cropped to present the results in a more logical manner.