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. 2000 Nov;74(22):10323-31.
doi: 10.1128/jvi.74.22.10323-10331.2000.

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus

K Sivakumaran  1 , Y Bao, M J Roossinck, C C Kao
Affiliations

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus

K Sivakumaran et al. J Virol. 2000 Nov.

Abstract

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.

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Figures

FIG. 1
FIG. 1
Predicted secondary structure of the Fny-CMV tRNA-like region and minimal SLC constructs. (A) Secondary structure predicted for the Fny-CMV tRNA-like region as depicted in Rizzo and Paulakaitis (42). Stem-loops are named A to D and indicated in bold letters. (B) Schematic of the predicted structure for B-SLdel+8, C-SLC3del+6, and C-SLC5del+6 using the mfold program (24). The nucleotides in the triloop and the C-G closing base pair of B-SLdel+8 and C-SLC3del+6 as well as the nucleotides contributing to the pentaloop of C-SLC5del+6 are indicated in bold.
FIG. 2
FIG. 2
Initiation of genomic plus- and minus-strand and subgenomic RNA synthesis by Fny-CMV replicase. (A) Templates used in the assay. CF(+)208 indicates a 208-nt RNA corresponding to the 3′ end of Fny-CMV RNA3. CF(−)26G indicates a 27-nt RNA with a 3′ nontemplated nucleotide that is complementary to the 5′ end of Fny-CMV RNA3. CF−223/+15 indicates part of the region complementary to the intercistronic region and the initiation cytidylate for subgenomic RNA synthesis. An arrow denotes the +1 initiation cytidylate in the three RNAs. (B) Autoradiograms of genomic and subgenomic RNA products made by the enriched Fny-CMV replicase preparation. Arrows denote the 208-nt, 26-nt, and 15-nt genomic minus-strand, genomic plus-strand, and subgenomic RNA products, respectively. RNAs with a wild-type initiation site are indicated by +, while those with the initiation site mutated to guanylate(s) are indicated by −. The genomic minus-strand, genomic plus-strand, and subgenomic RNA products were separated on 5, 12, and 20% PAGE, respectively.
FIG. 3
FIG. 3
Recognition of the tRNA-like region by BMV and Fny-CMV replicases. Autoradiogram of the 206- and 208-nt RNA products synthesized by the BMV and CMV replicases from the tRNA-like regions of BMV and CMV. Initiation-competent RNAs are indicated by +. Initiation-incompetent RNAs are indicated by −.
FIG. 4
FIG. 4
Effect of nucleotide substitutions within C-SLC3 in the context of the tRNA-like structure. (A) Autoradiogram of the RNA products made by BMV replicase. (B) Autoradiogram of the RNA products made by Fny-CMV replicase. The arrow identifies the 208-nt RNA product. Initiation-competent RNAs (+) initiation-incompetent RNAs (−) are indicated. The wild-type and mutated sequences of C-SLC3 are shown near the top of the autoradiogram. The effect of nucleotide substitutions on RNA synthesis is denoted as a percentage relative to the amount of synthesis directed by the wild-type sequence, indicated as AAC. All results presented are from at least three independent trials with a standard deviation of ≤12%.
FIG. 5
FIG. 5
Effect of nucleotide substitutions within the minimal SLC of BMV and Fny-CMV on RNA synthesis. (A) Autoradiogram of RNA products made by BMV and Fny-CMV replicases. The arrow identifies a 38-nt RNA replicase product. The different nucleotide substitutions within the triloop and the closing base pair are shown near the top of the autoradiogram. I, inosine; P, purine lacking any exocyclic groups (26). The effect of nucleotide substitutions on RNA synthesis is shown as a percentage relative to the amount of synthesis directed by B-SLdel+8. (B) Autoradiogram of RNA products made by Fny-CMV and BMV replicases. The arrow identifies a 36-nt RNA replicase product. The effect of nucleotide substitutions on RNA synthesis is shown as a percentage relative to the amount of synthesis directed by C-SLC3del+6. All results presented are from at least three independent trials with a standard deviation of <12%.
FIG. 6
FIG. 6
Alignment of the sequences containing the SLC-like region of 30 CMV strains, predicted stem-loop structures of C-SLC3 and C-SLC5, and nondenaturing and denaturing gel analyses of C-SLC3 and C-SLC5. (A) SLC-like region of 1A and 1B isolates of 30 CMV isolates. Sequence data were obtained from the GenBank accessions in Table 1. The different isolates analyzed are indicated on the left side of the figure. Isolates having a stem region followed by a triloop are shown in the C-SLC3-like group. Isolates having a stem region followed by a pentaloop are shown in the C-SLC5-like group. The invariant CA dinucleotide is shown in bold. The sequences of the putative internal bulge are shown under the bracket. (B) Schematic depicting the lower portion of the predicted structure of C-SLC3 and C-SLC5. The invariant CA dinucleotide is indicated in bold. The predicted structure was generated using the mfold program (24). (C) Nondenaturing and denaturing gel analyses of C-SLC3 and C-SLC5 RNAs. The RNAs contained a 17-nt region corresponding to the lower stem-loop region of C-SLC3 and C-SLC5. The RNAs for nondenaturing gel analysis were heated to 90°C for 2 min, cooled on ice, and electrophoresed through a nondenaturing 20% polyacrylamide gel. The RNAs are SL13, a 13-nt stem with a triloop (lane 1), C-SLC3 (lane 2), and C-SLC5 (lane 3). For denaturing gel analysis, the RNAs were analyzed on a denaturing 20% polyacrylamide gel. The RNAs are C-SLC3 (lane 4) and C-SLC5 (lane 5).
FIG. 7
FIG. 7
Effect of nucleotide substitutions within C-SLC5 on RNA synthesis. (A) Autoradiogram showing the RNA products made by Fny-CMV and BMV replicases. CF(+)208 indicates a 208-nt RNA corresponding to the 3′ end of Fny-CMV RNA3. CI(+)208 indicates a 208-nt RNA corresponding to the 3′ end of Ix-CMV RNA3. CI-loop indicates a 208-nt RNA with the 5′CAAGA3′ sequence in the pentaloop mutated to 5′CGGGA3′. CI-SL indicates a 208-nt RNA with the 5′CAAGAUG3′ stem-loop sequence mutated to 5′AAAGUAU3′. Quantification of the effect of nucleotide substitutions on RNA synthesis is shown as the percentage of three independent assays relative to the amount of synthesis directed by CF(+)208 as well as CI(+)208. (B) Autoradiogram showing the effect of nucleotide substitutions within C-SLC5del+6 on RNA synthesis. The nucleotide substitutions are indicated at the top of the autoradiogram. The effect of nucleotide substitutions on RNA synthesis is shown as a percentage relative to the amount of synthesis directed by C-SLC5del+6. All results presented are from at least three independent trials with a standard deviation of <12%.
FIG. 8
FIG. 8
Schematic illustrating the common features present in SLC of BMV and CMV subgroup I and II RNAs. The RNAs have a short stem region with an internal bulge, followed by a rigid stem-loop region. The conserved CA dinucleotide in CMV subgroups I and II is indicated in bold. B-SLC, C-SLC3, C-SLC5, and Q-SLC were generated using the mfold program (24).
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References

    1. Adkins S, Kao C C. Subgenomic RNA promoters dictate the mode of recognition by bromoviral RNA-dependent RNA polymerase. Virology. 1998;252:1–8. - PubMed
    1. Adkins S, Siegel R, Sun J H, Kao C C. Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase. RNA. 1997;3:634–647. - PMC - PubMed
    1. Ahlquist P. Bromovirus RNA replication and transcription. Curr Opin Genet Dev. 1992;2:71–76. - PubMed
    1. Ahlquist P, Dasgupta R, Kaesberg P. Near identity of 3′ RNA secondary structure in bromo-viruses and cucumber mosaic virus. Cell. 1981;23:183–189. - PubMed
    1. Ahola T, Ahlquist P. Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a. J Virol. 1999;73:10061–10069. - PMC - PubMed

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