scholarly journals Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination

2004 ◽  
Vol 78 (16) ◽  
pp. 8552-8564 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Aleksandra Dzianott ◽  
Jozef Bujarski
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
De Novo ◽  
Brome Mosaic Virus ◽  
Rna Recombination ◽  
Recombination Activity ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Subgenomic Promoter

ABSTRACT Previously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3 (A. Bruyere et al., J. Virol. 74:4214-4219, 2000; R. Wierzchoslawski et al., J. Virol. 77:6769-6776, 2003). In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the +1 or +2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.

scholarly journals Brome Mosaic Virus Protein 1a Recruits Viral RNA2 to RNA Replication through a 5′ Proximal RNA2 Signal

2001 ◽  
Vol 75 (7) ◽  
pp. 3207-3219 ◽  
Author(s):  
Jianbo Chen ◽  
Amine Noueiry ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Protein ◽  
Membrane Association ◽  
Stem Loop ◽  
Induced Membrane ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5′-proximal third of RNA2. The RNA2 5′ untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5′-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TΨC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

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 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 cis-Acting Elements Required for Efficient Packaging of Brome Mosaic Virus RNA3 in Barley Protoplasts

2003 ◽  
Vol 77 (18) ◽  
pp. 9979-9986 ◽  
Author(s):  
Tri Asmira Damayanti ◽  
Satoshi Tsukaguchi ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Brome Mosaic Virus ◽  
Proximal Region ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Reading Frame ◽  
Cis Acting ◽  
Infected Cells ◽  
Packaging Efficiency

ABSTRACT Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3′-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3′-proximal region of the CMV 3a ORF in B3Cmp with the 3′-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3′-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Ahlquist ◽  
M Janda
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
In Vitro Translation ◽  
In Vitro Production ◽  
Genomic Rnas

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

scholarly journals Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus

2006 ◽  
Vol 80 (12) ◽  
pp. 6182-6187 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Recombination Frequency ◽  
In Vivo Studies ◽  
Rna Recombination ◽  
In Vitro System ◽  
Brome Mosaic Bromovirus ◽  
Subgenomic Promoter

ABSTRACT Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (−)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.

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