scholarly journals Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

1999 ◽  
Vol 73 (8) ◽  
pp. 6415-6423 ◽  
Author(s):  
K. Sivakumaran ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Initiation Site ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna

ABSTRACT 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.

scholarly journals Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

1999 ◽  
Vol 73 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Scott Stevenson Stawicki ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna ◽  
In The Wild ◽  
Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

scholarly journals Recognition of the Core RNA Promoter for Minus-Strand RNA Synthesis by the Replicases of Brome Mosaic Virus andCucumber Mosaic Virus

2000 ◽  
Vol 74 (22) ◽  
pp. 10323-10331 ◽  
Author(s):  
K. Sivakumaran ◽  
Y. Bao ◽  
M. J. Roossinck ◽  
C. C. Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Direct Synthesis ◽  
Specific Interaction ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Genomic Rnas ◽  
Promoter Recognition

ABSTRACT Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of theBromovirus 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. InBrome 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.

scholarly journals Enhancer-Like Activity of a Brome Mosaic Virus RNA Promoter

2003 ◽  
Vol 77 (3) ◽  
pp. 1830-1839 ◽  
Author(s):  
C. T. Ranjith-Kumar ◽  
Xin Zhang ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Initiation Site ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Minus Strand ◽  
Rna Sequences ◽  
Dna Templates ◽  
Virus Rna

ABSTRACT As with transcription from DNA templates, RNA synthesis from viral RNA templates must initiate accurately. RNA sequences named specificity and initiation determinants allow recognition of and coordinated interaction with the viral replication enzyme. Using enriched replicase from brome mosaic virus (BMV)-infected plants and variants of the promoter template for minus-strand and subgenomic RNA initiation, we found that a specificity determinant for minus-strand initiation could function at variable distances and positions from the 3′ initiation site in a manner similar to enhancers of transcription from DNA templates. This determinant's addition could convert a cellular tRNA into a template for RNA synthesis by the BMV replicase in vitro. Furthermore, the same specificity element could direct internal initiation, which occurred at a highly preferred site in a manner distinct from initiation at the 3′ terminus of the template. These results document two distinct modes of initiation site recognition by a viral RNA replicase.

scholarly journals Minimal Template Requirements for Initiation of Minus-Strand Synthesis In Vitro by the RNA-Dependent RNA Polymerase of Turnip Yellow Mosaic Virus

1998 ◽  
Vol 72 (5) ◽  
pp. 3965-3972 ◽  
Author(s):  
B. A. L. M. Deiman ◽  
A. K. Koenen ◽  
P. W. G. Verlaan ◽  
C. W. A. Pleij
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Mutational Analysis ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Minus Strand ◽  
Rna Dependent Rna Polymerase ◽  
Substitution Mutations ◽  
Internal Initiation

ABSTRACT From mutational analysis of the 3′-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3′ ACC(A) end. Base pairing of this 3′ end reduces the transcription efficiency drastically, and deletion of only the 3′-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3′ ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5′ A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3′ ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated ΔG value should be less than −10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.

scholarly journals Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

2000 ◽  
Vol 74 (24) ◽  
pp. 11671-11680 ◽  
Author(s):  
T. A. M. Osman ◽  
C. L. Hemenway ◽  
K. W. Buck
Keyword(s):  
Rna Polymerase ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Central Core ◽  
Minus Strand ◽  
Stem Loop ◽  
Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

scholarly journals 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

1999 ◽  
Vol 73 (8) ◽  
pp. 6424-6429 ◽  
Author(s):  
Robert W. Siegel ◽  
Laurent Bellon ◽  
Leonid Beigelman ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Competition Assay ◽  
Rna Dependent Rna Polymerase ◽  
Dna Molecule ◽  
Mechanism Of Catalysis ◽  
Insight Into

ABSTRACT 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.

scholarly journals Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts

2003 ◽  
Vol 77 (10) ◽  
pp. 5703-5711 ◽  
Author(s):  
K. Sivakumaran ◽  
M. Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Previous Analysis ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Stem Loop ◽  
Virus Rna ◽  
Different Levels

ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

scholarly journals Template Sequence near the Initiation Nucleotide Can Modulate Brome Mosaic Virus RNA Accumulation in Plant Protoplasts

2004 ◽  
Vol 78 (3) ◽  
pp. 1169-1180 ◽  
Author(s):  
M. Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Initiation Site ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Wild Type ◽  
Wild Type Level ◽  
The Third ◽  
Template Sequence ◽  
Rna Accumulation

ABSTRACT Bromoviral templates for plus-strand RNA synthesis are rich in A or U nucleotides in comparison to templates for minus-strand RNA synthesis. Previous studies demonstrated that plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase is more efficient if the template contains an A/U-rich template sequence near the initiation site (K. Sivakumaran and C. C. Kao, J. Virol. 73:6415-6423, 1999). These observations led us to examine the effects of nucleotide changes near the template's initiation site on the accumulation of BMV RNA3 genomic minus-strand, genomic plus-strand, and subgenomic RNAs in barley protoplasts transfected with wild-type and mutant BMV transcripts. Mutations in the template for minus-strand synthesis had only modest effects on BMV replication in barley protoplasts. Mutants with changes to the +3, +5, and +7 template nucleotides accumulated minus-strand RNA at levels similar to the the wild-type level. However, mutations at positions adjacent to the initiation cytidylate in the templates for genomic and subgenomic plus-strand RNA synthesis significantly decreased RNA accumulation. For example, changes at the third template nucleotide for plus-strand RNA3 synthesis resulted in RNA accumulation at between 18 and 24% of the wild-type level, and mutations in the third template nucleotide for subgenomic RNA4 resulted in accumulations at between 7 and 14% of the wild-type level. The effects of the mutations generally decreased as the mutations occurred further from the initiation nucleotide. These findings demonstrate that there are different requirements of the template sequence near the initiation nucleotide for BMV RNA accumulation in plant cells.

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