scholarly journals Interactions between Brome Mosaic Virus RNAs and Cytoplasmic Processing Bodies

2007 ◽  
Vol 81 (18) ◽  
pp. 9759-9768 ◽  
Author(s):  
Carla J. Beckham ◽  
Heather R. Light ◽  
T. Amar Nissan ◽  
Paul Ahlquist ◽  
Roy Parker ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Yeast Cells ◽  
Processing Bodies ◽  
Genomic Rnas ◽  
Replication Complex ◽  
P Bodies ◽  
Body Component ◽  
Positive Strand Rna

ABSTRACT Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.

scholarly journals Brome Mosaic Virus Polymerase-Like Protein 2a Is Directed to the Endoplasmic Reticulum by Helicase-Like Viral Protein 1a

2000 ◽  
Vol 74 (9) ◽  
pp. 4310-4318 ◽  
Author(s):  
Jianbo Chen ◽  
Paul Ahlquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Yeast Cells ◽  
Cell Fractionation ◽  
Cytoplasmic Organelle ◽  
Replication Complex ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.

scholarly journals Brome Mosaic Virus RNA Replication Proteins 1a and 2a Colocalize and 1a Independently Localizes on the Yeast Endoplasmic Reticulum

1999 ◽  
Vol 73 (12) ◽  
pp. 10303-10309 ◽  
Author(s):  
María Restrepo-Hartwig ◽  
Paul Ahlquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Mosaic Virus ◽  
Plant Cells ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Replication Proteins ◽  
Replication Complex ◽  
Virus Rna ◽  
Positive Strand Rna

ABSTRACT The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.

scholarly journals Identification of a Ribonucleoprotein Intermediate of Tomato Mosaic Virus RNA Replication Complex Formation

2006 ◽  
Vol 81 (6) ◽  
pp. 2584-2591 ◽  
Author(s):  
Keisuke Komoda ◽  
Natsuki Mawatari ◽  
Yuka Hagiwara-Komoda ◽  
Satoshi Naito ◽  
Masayuki Ishikawa
Keyword(s):  
Complex Formation ◽  
Mosaic Virus ◽  
Rna Replication ◽  
Tomato Mosaic Virus ◽  
Membrane Targeting ◽  
Genomic Rnas ◽  
Positive Strand ◽  
Replication Complex ◽  
Ribonucleoprotein Complex ◽  
Positive Strand Rna

ABSTRACT The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the “pre-membrane-targeting complex.” Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.

scholarly journals Yeast Lsm1p-7p/Pat1p Deadenylation-Dependent mRNA-Decapping Factors Are Required for Brome Mosaic Virus Genomic RNA Translation

2003 ◽  
Vol 23 (12) ◽  
pp. 4094-4106 ◽  
Author(s):  
Amine O. Noueiry ◽  
Juana Diez ◽  
Shaun P. Falk ◽  
Jianbo Chen ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Genomic Rna ◽  
Reading Frame ◽  
Mrna Decapping ◽  
Rna Translation ◽  
Positive Strand Rna

ABSTRACT Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5′ and 3′ noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.

scholarly journals 5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

2009 ◽  
Vol 83 (7) ◽  
pp. 2976-2988 ◽  
Author(s):  
Priscilla M. Van Wynsberghe ◽  
Paul Ahlquist
Keyword(s):  
Complex Formation ◽  
Mutational Analysis ◽  
Protein A ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Positive Strand ◽  
Replication Complex ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

scholarly journals Mutation of Host dnaJ Homolog Inhibits Brome Mosaic Virus Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (5) ◽  
pp. 2990-2997 ◽  
Author(s):  
Yuriko Tomita ◽  
Tomomitsu Mizuno ◽  
Juana Díez ◽  
Satoshi Naito ◽  
Paul Ahlquist ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Positive Strand ◽  
Replication Complex ◽  
Negative Strand ◽  
Rna Accumulation ◽  
Positive Strand Rna ◽  
Mutant Cells

ABSTRACT The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.

scholarly journals Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

2017 ◽  
Vol 114 (7) ◽  
pp. E1282-E1290 ◽  
Author(s):  
Kiwamu Hyodo ◽  
Kenji Hashimoto ◽  
Kazuyuki Kuchitsu ◽  
Nobuhiro Suzuki ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Plant Stress ◽  
Rna Replication ◽  
Plant Viruses ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Genome Replication ◽  
Dependent Manner ◽  
Positive Strand Rna

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant–virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

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 Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

2005 ◽  
Vol 79 (3) ◽  
pp. 1438-1451 ◽  
Author(s):  
Valery Z. Grdzelishvili ◽  
Hernan Garcia-Ruiz ◽  
Tokiko Watanabe ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Brome Mosaic Virus ◽  
Genomic Rna ◽  
Mrna Transcription ◽  
Rna Amplification ◽  
Genomic Rnas ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna ◽  
Replication Factors

ABSTRACT Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle. Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication. Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis. We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positive-strand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components. Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5′-untranslated region. Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors. In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2.

scholarly journals The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 1045-fold amplification

2000 ◽  
Vol 81 (9) ◽  
pp. 2307-2311 ◽  
Author(s):  
Masayuki Ishikawa ◽  
Michael Janda ◽  
Paul Ahlquist
Keyword(s):  
Cell Division ◽  
Mosaic Virus ◽  
Rna Virus ◽  
Cell Movement ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Rna ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Rna Replicon

In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85–95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura+ colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

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