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

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.

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Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

Virology ◽

10.1016/0042-6822(87)90232-7 ◽

1987 ◽

Vol 158 (1) ◽

pp. 15-19 ◽

Cited By ~ 23

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

Journal of Virology ◽

10.1128/jvi.74.22.10323-10331.2000 ◽

2000 ◽

Vol 74 (22) ◽

pp. 10323-10331 ◽

Cited By ~ 38

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.

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Brome mosaic virus defective RNAs generated during infection of barley plants

Journal of General Virology ◽

10.1099/0022-1317-80-9-2511 ◽

1999 ◽

Vol 80 (9) ◽

pp. 2511-2518 ◽

Cited By ~ 30

Author(s):

Tri Asmira Damayanti ◽

Hideaki Nagano ◽

Kazuyuki Mise ◽

Iwao Furusawa ◽

Tetsuro Okuno

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Helper Virus ◽

Brome Mosaic Virus ◽

Cdna Clones ◽

Post Inoculation ◽

Dependent Manner ◽

Genomic Rnas ◽

Defective Rnas

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.

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Enhancer-Like Activity of a Brome Mosaic Virus RNA Promoter

Journal of Virology ◽

10.1128/jvi.77.3.1830-1839.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 1830-1839 ◽

Cited By ~ 21

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.

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Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins

Phytopathology ◽

10.1094/phyto.2003.93.5.588 ◽

2003 ◽

Vol 93 (5) ◽

pp. 588-595 ◽

Cited By ~ 14

Author(s):

Jorge M. Vivanco ◽

Nilgun E. Tumer

Keyword(s):

Mosaic Virus ◽

Brome Mosaic Virus ◽

In Vitro Translation ◽

Type I ◽

Antiviral Protein ◽

Entry Site ◽

Viral Rnas ◽

Ribosome Inactivating Proteins ◽

Cell Extracts

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP.

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Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

Journal of Virology ◽

10.1128/jvi.00833-16 ◽

2016 ◽

Vol 90 (17) ◽

pp. 7748-7760 ◽

Cited By ~ 17

Author(s):

Haley S. Hoover ◽

Joseph Che-Yen Wang ◽

Stefani Middleton ◽

Peng Ni ◽

Adam Zlotnick ◽

...

Keyword(s):

Gene Expression ◽

Viral Infection ◽

Mosaic Virus ◽

Posttranslational Modifications ◽

Rna Replication ◽

Brome Mosaic Virus ◽

Viral Rna ◽

Genomic Rnas ◽

The Stability ◽

Rna Interaction

ABSTRACTThe four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.IMPORTANCEHow icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

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Further studies on inhibitory effect of brome mosaic virus coat protein on the viral RNA synthesis in vitro.

Japanese Journal of Phytopathology ◽

10.3186/jjphytopath.54.533 ◽

1988 ◽

Vol 54 (4) ◽

pp. 533-535

Author(s):

Mamoru HORIKOSHI ◽

Kazuyuki MISE ◽

Iwao FURUSAWA ◽

Jiko SHISHIYAMA

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Rna Synthesis ◽

Inhibitory Effect ◽

Brome Mosaic Virus ◽

Viral Rna ◽

Virus Coat Protein ◽

Virus Coat

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In Vitro Transcription by the Turnip Yellow Mosaic Virus RNA Polymerase: a Comparison with the Alfalfa Mosaic Virus and Brome Mosaic Virus Replicases

Journal of Virology ◽

10.1128/jvi.74.1.264-271.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 264-271 ◽

Cited By ~ 22

Author(s):

Birgit A. L. M. Deiman ◽

Paul W. G. Verlaan ◽

Cornelis W. A. Pleij

Keyword(s):

Rna Polymerase ◽

Mosaic Virus ◽

Transcription Initiation ◽

Alfalfa Mosaic Virus ◽

Brome Mosaic Virus ◽

Yellow Mosaic Virus ◽

Turnip Yellow Mosaic Virus ◽

Virus Rna ◽

Internal Initiation

ABSTRACT Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3′ ACCA end. By mutational analysis of the 3′-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3′-proximal hairpin is the most favorable, in that it has the lowest ΔG value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.

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