scholarly journals Replicase-Binding Sites on Plus- and Minus-Strand Brome Mosaic Virus RNAs and Their Roles in RNA Replication in Plant Cells

2004 ◽  
Vol 78 (24) ◽  
pp. 13420-13429 ◽  
Author(s):  
S.-K. Choi ◽  
M. Hema ◽  
K. Gopinath ◽  
J. Santos ◽  
C. Kao
Keyword(s):  
Mosaic Virus ◽  
Binding Sites ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
The Core ◽  
Strand Initiation

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.

scholarly journals Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

2004 ◽  
Vol 78 (12) ◽  
pp. 6091-6101 ◽  
Author(s):  
K. Sivakumaran ◽  
Seung-Kook Choi ◽  
Masarapu Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Core ◽  
Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

scholarly journals Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

1998 ◽  
Vol 72 (8) ◽  
pp. 6546-6553 ◽  
Author(s):  
Julie A. Lemm ◽  
Anders Bergqvist ◽  
Carol M. Read ◽  
Charles M. Rice
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Functional Assay ◽  
Minus Strand ◽  
Virus Rna ◽  
A Genome ◽  
Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

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 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 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 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 Efficient and Specific Initiation of Subgenomic RNA Synthesis by Cucumber Mosaic Virus Replicase In Vitro Requires an Upstream RNA Stem-Loop

2000 ◽  
Vol 74 (23) ◽  
pp. 11201-11209 ◽  
Author(s):  
M.-H. Chen ◽  
M. J. Roossinck ◽  
C. C. Kao
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Stem Loop ◽  
Promoter Sequences ◽  
Promoter Recognition

ABSTRACT We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions −28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions −28 to −5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.

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 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.

Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

Virology ◽  
1987 ◽  
Vol 158 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Mamoru Horikoshi ◽  
Masaharu Nakayama ◽  
Naoto Yamaoka ◽  
Iwao Furusawa ◽  
Jiko Shishiyama
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Virus Coat Protein ◽  
Virus Coat
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