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 A Brome Mosaic Virus Intergenic RNA3 Replication Signal Functions with Viral Replication Protein 1a To Dramatically Stabilize RNA In Vivo

1999 ◽  
Vol 73 (4) ◽  
pp. 2622-2632 ◽  
Author(s):  
Michael L. Sullivan ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Rna Polymerase Iii ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Positive Strand ◽  
Signal Functions ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified thecis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous β-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV 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 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 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 Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

2001 ◽  
Vol 75 (23) ◽  
pp. 11664-11676 ◽  
Author(s):  
David J. Miller ◽  
Michael D. Schwartz ◽  
Paul Ahlquist
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Protein A ◽  
Rna Replication ◽  
Yeast Cells ◽  
Flock House Virus ◽  
Mitochondrial Membranes ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection ofDrosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.

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 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 cis- and trans-Acting Functions of Brome Mosaic Virus Protein 1a in Genomic RNA1 Replication

2007 ◽  
Vol 82 (6) ◽  
pp. 3045-3053 ◽  
Author(s):  
Guanghui Yi ◽  
Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Brome Mosaic Virus ◽  
Virus Protein ◽  
Helicase Domain ◽  
Replication Factory ◽  
Positive Strand Rna Virus ◽  
Rna Motif ◽  
Rna Accumulation ◽  
Positive Strand Rna

ABSTRACT RNA viruses employ a combination of mechanisms to regulate their gene expression and replication. Brome mosaic virus (BMV) is a tripartite positive-strand RNA virus used to study the requirements for virus infection. BMV genomic RNA1 encodes protein 1a, which contains a methyltransferase (MT) domain and a helicase domain that are required for replication. 1a forms a complex with the 2a RNA-dependent RNA polymerase for the replication and transcription of all BMV RNAs. RNA1 expressed with 2a from Agrobacterium-based vectors can result in RNA1 replication in Nicotiana benthamiana. A mutation in the 1a translation initiation codon significantly decreased RNA1 accumulation even when wild-type (WT) 1a and 2a were provided in trans. Therefore, efficient RNA1 replication requires 1a translation from RNA1 in cis, indicating a linkage between replication and translation. Mutation analyses showed that the full-length 1a protein was required for efficient RNA1 replication, not just the process of translation. Three RNA1s with mutations in the 1a MT domain could be partially rescued by WT 1a expressed in trans, indicating that the cis-acting function of 1a was retained. Furthermore, an RNA motif in the 5′-untranslated region of RNA1, named the B box, was required for 1a to function in cis and in trans for BMV RNA accumulation. The B box is required for the formation of the replication factory (M. Schwartz, J. Chen, M. Janda, M. Sullivan, J. den Boon, and P. Ahlquist, Mol. Cell 9:505-514, 2002). Results in this work demonstrate a linkage between BMV RNA1 translation and replication.

scholarly journals Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity

2007 ◽  
Vol 81 (20) ◽  
pp. 11402-11412 ◽  
Author(s):  
Anna Jakubiec ◽  
Gabrièle Drugeon ◽  
Laurent Camborde ◽  
Isabelle Jupin
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Proteolytic Processing ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Replication Proteins ◽  
Sequence Comparisons ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA.

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