A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo.
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Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication. (+info)
The N-terminal half of the brome mosaic virus 1a protein has RNA capping-associated activities: specificity for GTP and S-adenosylmethionine.
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The N-terminal half of the brome mosaic virus (BMV) 1a replication-associated protein contains sequence motifs found in RNA methyltransferases. We demonstrate that recombinant BMV methyltransferase-like (MT) domain expressed in Escherichia coli forms an adduct with a guanine nucleotide in a reaction that requires S-adenosylmethionine (AdoMet) and divalent cations. Moieties in GTP and AdoMet required for adduct formation were determined using a competition assay and chemical analogues. In the guanine nucleotide the ribose 2' hydroxyl, the triphosphates, the base C6 keto group, and possibly the N1 imine are required. In AdoMet, the methyl group and the ability to transfer a methyl group to guanine nucleotide were demonstrated to be required for adduct formation. The effects of methyltransferase inhibitors on viral RNA synthesis was determined using an in vitro RNA synthesis assay. These results are consistent with the previously reported activities of alphaviral nsP1 methyltransferase protein and identify the chemical moieties required for the BMV methyltransferase activity. (+info)
Effect of C-terminal deletions in the movement protein of cowpea chlorotic mottle virus on cell-to-cell and long-distance movement.
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In order to elucidate the function of the C-terminal region of cowpea chlorotic mottle bromovirus (CCMV) movement protein (MP) in cell-to-cell movement, a set of deletions ranging from 10 to 80 amino acids (deltaMP10, deltaMP20, deltaMP33, deltaMP43, deltaMP60 and deltaMP80) was engineered into the MP gene encoded by the biologically active clone C3/deltaCP-EGFP, a variant of CCMV RNA3 that contained wild-type (wt) MP and the enhanced green fluorescent protein (EGFP) gene in place of the coat protein (CP). The effect of each MP deletion on cell-to-cell movement was examined in three susceptible host plants: Chenopodium quinoa, Nicotiana benthamiana and cowpea (Vigno sinensis cv. Black Eye). The results indicate that, except for mutant deltaMP43, infections resulting from the deletion mutants remained subliminal. Interestingly, infections resulting from inoculating mutant deltaMP43, which lacked the 43 most C-terminal amino acids, spread rapidly between cells and the number of infected cells expressing EGFP approached that of control inoculations made with C3/deltaCP-EGFP. To verify whether the presence of wt CP altered the movement behaviour of these mutants, each MP deletion was also incorporated into the genetic background of wt CCMV RNA3 (pCC3) and inoculated independently to all three hosts. The results suggest that the overall movement process exhibited by each MP mutant is influenced profoundly by the presence of CP and the particular host plant tested. (+info)
RNA recombination in brome mosaic virus, a model plus strand RNA virus.
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Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research. (+info)
Initiation of genomic plus-strand RNA synthesis from DNA and RNA templates by a viral RNA-dependent RNA polymerase.
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In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site. (+info)
Use of DNA, RNA, and chimeric templates by a viral RNA-dependent RNA polymerase: evolutionary implications for the transition from the RNA to the DNA world.
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All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position -11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes. (+info)
Mapping the molecular determinant of pathogenicity in a hammerhead viroid: a tetraloop within the in vivo branched RNA conformation.
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Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398-399 nt that can adopt hammerhead structures in both polarity strands. We have identified by Northern-blot hybridization a nonsymptomatic strain (CChMVd-NS) that protects against challenge inoculation with the symptomatic strain (CChMVd-S). Analysis of CChMVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the mutations observed in CChMVd-NS molecular variants were previously identified in CChMVd-S RNA, but others were never found in this RNA. When bioassayed in chrysanthemum, cDNA clones containing the CChMVd-NS specific mutations were infectious but nonsymptomatic. Site-directed mutagenesis showed that one of the CChMVd-NS-specific mutations, a UUUC --> GAAA substitution, was sufficient to change the symptomatic phenotype into the nonsymptomatic one without altering the final accumulation level of the viroid RNA. The pathogenicity determinant-to our knowledge, a determinant of this class has not been described previously in hammerhead viroids-is located in a tetraloop of the computer-predicted branched conformation for CChMVd RNA. Analysis of the sequence heterogeneity found in CChMVd-S and -NS variants strongly supports the existence of such a conformation in vivo, showing that the rod-like or quasi-rod-like secondary structure is not a universal paradigm for viroids. (+info)
Brome mosaic virus defective RNAs generated during infection of barley plants.
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Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV. (+info)