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 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 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 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 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 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.

scholarly journals An Alternate Pathway for Recruiting Template RNA to the Brome Mosaic Virus RNA Replication Complex

2003 ◽  
Vol 77 (4) ◽  
pp. 2568-2577 ◽  
Author(s):  
Jianbo Chen ◽  
Amine Noueiry ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Terminal Region ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Stem Loop ◽  
Reading Frame ◽  
Replication Complex ◽  
Alternate Pathway

ABSTRACT The multidomain RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, plays key roles in assembly and function of the viral RNA replication complex. 1a, which encodes RNA capping and helicase-like domains, localizes to endoplasmic reticulum membranes, recruits BMV 2a polymerase and viral RNA templates, and forms membrane-bound, capsid-like spherules in which RNA replication occurs. cis-acting signals necessary and sufficient for RNA recruitment by 1a have been mapped in BMV genomic RNA2 and RNA3. Both signals comprise an extended stem-loop whose apex matches the conserved sequence and structure of the TΨC stem-loop in tRNAs (box B). Mutations show that this box B motif is crucial to 1a responsiveness of wild-type RNA2 and RNA3. We report here that, unexpectedly, some chimeric mRNAs expressing the 2a polymerase open reading frame from RNA2 were recruited by 1a to the replication complex and served as templates for negative-strand RNA synthesis, despite lacking the normally essential, box B-containing 5′ signal. Further studies showed that this template recruitment required high-efficiency translation of the RNA templates. Moreover, multiple small frameshifting insertion or deletion mutations throughout the N-terminal region of the open reading frame inhibited this template recruitment, while an in-frame insertion did not. Providing 2a in trans did not restore template recruitment of RNAs with frameshift mutations. Only those deletions in the N-terminal region of 2a that abolished 2a interaction with 1a abolished template recruitment of the RNA. These and other results indicate that this alternate pathway for 1a-dependent RNA recruitment involves 1a interaction with the translating mRNA via the 1a-interactive N-terminal region of the nascent 2a polypeptide. Interaction with nascent 2a also may be involved in 1a recruitment of 2a polymerase to membranes.

scholarly journals Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates

2005 ◽  
Vol 79 (21) ◽  
pp. 13747-13758 ◽  
Author(s):  
Xiaofeng Wang ◽  
Wai-Ming Lee ◽  
Tokiko Watanabe ◽  
Michael Schwartz ◽  
Michael Janda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mosaic Virus ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Helicase Domain ◽  
Positive Strand Rna ◽  
Resistant State ◽  
Nucleoside Triphosphatase

ABSTRACT Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2apol has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2apol, and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2apol, 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2apol. Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.

scholarly journals Host Deadenylation-Dependent mRNA Decapping Factors Are Required for a Key Step in Brome Mosaic Virus RNA Replication

2006 ◽  
Vol 80 (1) ◽  
pp. 246-251 ◽  
Author(s):  
Antonio Mas ◽  
Isabel Alves-Rodrigues ◽  
Amine Noueiry ◽  
Paul Ahlquist ◽  
Juana Díez
Keyword(s):  
Viral Replication ◽  
Mosaic Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Rna ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Positive Strand Rna

ABSTRACT The genomes of positive-strand RNA [(+)RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of (+)RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic (+)RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3′ noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.

scholarly journals Interactions between the Structural Domains of the RNA Replication Proteins of Plant-Infecting RNA Viruses

1998 ◽  
Vol 72 (9) ◽  
pp. 7160-7169 ◽  
Author(s):  
Erin K. O’Reilly ◽  
Zhaohui Wang ◽  
Roy French ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Replication Proteins ◽  
Mutant Proteins ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

scholarly journals Mutation of Host Δ9 Fatty Acid Desaturase Inhibits Brome Mosaic Virus RNA Replication between Template Recognition and RNA Synthesis

2001 ◽  
Vol 75 (5) ◽  
pp. 2097-2106 ◽  
Author(s):  
Wai-Ming Lee ◽  
Masayuki Ishikawa ◽  
Paul Ahlquist
Keyword(s):  
Fatty Acid ◽  
Mosaic Virus ◽  
Unsaturated Fatty Acid ◽  
Rna Synthesis ◽  
Fatty Acid Desaturase ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Positive Strand Rna ◽  
Er Membrane

ABSTRACT All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding Δ9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis.OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.

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