The Experts below are selected from a list of 66000 Experts worldwide ranked by ideXlab platform
Santiago F Elena - One of the best experts on this subject based on the ideXlab platform.
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Going, going, gone: predicting the fate of genomic insertions in Plant RNA viruses
Heredity, 2018Co-Authors: Anouk Willemsen, Santiago F Elena, José L. Carrasco, Mark P ZwartAbstract:Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a Plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate—the rate at which deletions removing the insertion occur—and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.
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predicting the stability of homologous gene duplications in a Plant RNA virus
Genome Biology and Evolution, 2016Co-Authors: Mark P Zwart, Santiago F Elena, Anouk Willemsen, Pablo Higueras, Josep SardanyesAbstract:One of the striking features of many eukaryotes is the apparent amount of redundancy in coding and non-coding elements of their genomes. Despite the possible evolutionary advantages, there are fewer examples of redundant sequences in viral genomes, particularly those with RNA genomes. The factors constraining the maintenance of redundant sequences in present-day RNA virus genomes are not well known. Here, we use Tobacco etch virus, a Plant RNA virus, to investigate the stability of genetically redundant sequences by generating viruses with potentially beneficial gene duplications. Subsequently, we tested the viability of these viruses and performed experimental evolution. We found that all gene duplication events resulted in a loss of viability or in a significant reduction in viral fitness. Moreover, upon analyzing the genomes of the evolved viruses, we always observed the deletion of the duplicated gene copy and maintenance of the ancestral copy. Interestingly, there were clear differences in the deletion dynamics of the duplicated gene associated with the passage duration and the size and position of the duplicated copy. Based on the experimental data, we developed a mathematical model to characterize the stability of genetically redundant sequences, and showed that fitness effects are not enough to predict genomic stability. A context-dependent recombination rate is also required, with the context being the duplicated gene and its position. Our results therefore demonstrate experimentally the deleterious nature of gene duplications in RNA viruses. Beside previously described constraints on genome size, we identified additional factors that reduce the likelihood of the maintenance of duplicated genes.
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predicting the stability of homologous gene duplications in a Plant RNA virus
bioRxiv, 2016Co-Authors: Anouk Willemsen, Mark P Zwart, Pablo Higueras, Josep Sardanyes, Santiago F ElenaAbstract:One of the striking features of many eukaryotes is the apparent amount of redundancy in coding and non-coding elements of their genomes. Despite the possible evolutionary advantages, there are fewer examples of redundant sequences in viral genomes, particularly those with RNA genomes. The low prevalence of gene duplication in RNA viruses most likely reflects the strong selective constraints against increasing genome size. Here we investigated the stability of genetically redundant sequences and how adaptive evolution proceeds to remove them. We generated Plant RNA viruses with potentially beneficial gene duplications, measured their fitness and performed experimental evolution, hereby exploring their genomic stability and evolutionary potential. We found that all gene duplication events resulted in a loss of viability or significant reductions in fitness. Moreover, upon evolving the viable viruses and analyzing their genomes, we always observed the deletion of the duplicated gene copy and maintenance of the ancestral copy. Interestingly, there were clear differences in the deletion dynamics of the duplicated gene associated with the passage duration, the size of the gene and the position for duplication. Based on the experimental data, we developed a mathematical model to characterize the stability of genetically redundant sequences, and showed that the fitness of viruses with duplications is not enough information to predict genomic stability as a recombination rate dependent on the genetic context - the duplicated gene and its position - is also required. Our results therefore demonstrate experimentally the deleterious nature of gene duplications in RNA viruses, and we identify factors that constrain the maintenance of duplicated genes.
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estimation of the in vivo recombination rate for a Plant RNA virus
Journal of General Virology, 2014Co-Authors: Nicolas Tromas, Mark P Zwart, Maite Poulain, Santiago F ElenaAbstract:Phylogenomic evidence suggested that recombination is an important evolutionary force for potyviruses, one of the larger families of Plant RNA viruses. However, mixed-genotype potyvirus infections are marked by low levels of cellular coinfection, precluding template switching and recombination events between virus genotypes during genomic RNA replication. To reconcile these conflicting observations, we evaluated the in vivo recombination rate (r g) of Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) by coinfecting Plants with pairs of genotypes marked with engineered restriction sites as neutral markers. The recombination rate was then estimated using two different approaches: (i) a classical approach that assumed recombination between marked genotypes can occur in the whole virus population, rendering an estimate of r g = 7.762×10−8 recombination events per nucleotide site per generation, and (ii) an alteRNAtive method that assumed recombination between marked genotypes can occur only in coinfected cells, rendering a much higher estimate of r g = 3.427×10−5 recombination events per nucleotide site per generation. This last estimate is similar to the TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. Finally, we compared our mutation and recombination rate estimates to those reported for animal RNA viruses. Our analysis suggested that high recombination rates may be an unavoidable consequence of selection for fast replication at the cost of low fidelity.
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effects of the number of genome segments on primary and systemic infections with a multipartite Plant RNA virus
Journal of Virology, 2013Co-Authors: J A Sancheznavarro, Mark P Zwart, Santiago F ElenaAbstract:Multipartite Plant viruses were discovered because of discrepancies between the observed dose response and predictions of the independent-action hypothesis (IAH) model. Theory suggests that the number of genome segments predicts the shape of the dose-response curve, but a rigorous test of this hypothesis has not been reported. Here, Alfalfa mosaic virus (AMV), a tripartite Alfamovirus, and transgenic Nicotianatabacum Plants expressing no (wild type), one (P2), or two (P12) viral genome segments were used to test whether the number of genome segments necessary for infection predicts the dose response. The dose-response curve of wild-type Plants was steep and congruent with the predicted kinetics of a multipartite virus, confirming previous results. Moreover, for P12 Plants, the data support the IAH model, showing that the expression of virus genome segments by the host Plant can modulate the infection kinetics of a tripartite virus to those of a monopartite virus. However, the different types of virus particles occurred at different frequencies, with a ratio of 116:45:1 (RNA1 to RNA2 to RNA3), which will affect infection kinetics and required analysis with a more comprehensive infection model. This analysis showed that each type of virus particle has a different probability of invading the host Plant, at both the primary- and systemic-infection levels. While the number of genome segments affects the dose response, taking into consideration differences in the infection kinetics of the three types of AMV particles results in a better understanding of the infection process.
Tetsuro Okuno - One of the best experts on this subject based on the ideXlab platform.
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harnessing host ros generating machinery for the robust genome replication of a Plant RNA virus
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Kiwamu Hyodo, Tetsuro Okuno, Kenji Hashimoto, Kazuyuki Kuchitsu, Nobuhiro SuzukiAbstract: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.
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host and viral RNA binding proteins involved in membrane targeting replication and intercellular movement of Plant RNA virus genomes
Frontiers in Plant Science, 2014Co-Authors: Kiwamu Hyodo, Masanori Kaido, Tetsuro OkunoAbstract:Many Plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host Plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to several viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of Plant RNA virus genomes.
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traffic jam on the cellular secretory pathway generated by a replication protein from a Plant RNA virus
Plant Signaling & Behavior, 2014Co-Authors: Kiwamu Hyodo, Masanori Kaido, Tetsuro OkunoAbstract:Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a Plant RNA virus, Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on Plant–virus interaction are discussed.
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adp ribosylation factor 1 plays an essential role in the replication of a Plant RNA virus
Journal of Virology, 2013Co-Authors: Kiwamu Hyodo, Masanori Kaido, Kazuyuki Mise, Akira Mine, Takako Taniguchi, Hisaaki Taniguchi, Tetsuro OkunoAbstract:ABSTRACT Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA Plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.
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a Plant RNA virus suppresses RNA silencing through viral RNA replication
The EMBO Journal, 2005Co-Authors: Atsushi Takeda, Masanori Kaido, Kazuyuki Mise, Misato Tsukuda, Hiroyuki Mizumoto, Kimiyuki Okamoto, Tetsuro OkunoAbstract:RNA interference (RNAi) is a post-transcriptional gene-regulatory mechanism that operates in many eukaryotes. RNAi is induced by double-stranded RNA (dsRNA) and is mainly involved in defence against transposons and viruses. To counteract RNAi, viruses have RNAi suppressors. Here we show a novel mechanism of RNAi suppression by a Plant virus Red clover necrotic mosaic virus (RCNMV). To suppress RNAi, RCNMV needs multiple viral components, which include viral RNAs and putative RNA replicase proteins. A close relationship between the RNA elements required for negative-strand RNA synthesis and RNAi suppression suggests a strong link between the viral RNA replication machinery and the RNAi machinery. In a transient assay, RCNMV interferes with the accumulation of small-interfering RNA (siRNAs) in RNAi induced by a hairpin dsRNA and it also interferes with microRNA (miRNA) biogenesis. An Arabidopsis dcl1 mutant showed reduced susceptibility to RCNMV infection. Based on these results, we propose a model in which, to replicate, RCNMV deprives the RNAi machinery of Dicer-like enzymes that are involved in both siRNA and miRNA biogenesis.
Mark P Zwart - One of the best experts on this subject based on the ideXlab platform.
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Going, going, gone: predicting the fate of genomic insertions in Plant RNA viruses
Heredity, 2018Co-Authors: Anouk Willemsen, Santiago F Elena, José L. Carrasco, Mark P ZwartAbstract:Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a Plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate—the rate at which deletions removing the insertion occur—and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.
-
predicting the stability of homologous gene duplications in a Plant RNA virus
Genome Biology and Evolution, 2016Co-Authors: Mark P Zwart, Santiago F Elena, Anouk Willemsen, Pablo Higueras, Josep SardanyesAbstract:One of the striking features of many eukaryotes is the apparent amount of redundancy in coding and non-coding elements of their genomes. Despite the possible evolutionary advantages, there are fewer examples of redundant sequences in viral genomes, particularly those with RNA genomes. The factors constraining the maintenance of redundant sequences in present-day RNA virus genomes are not well known. Here, we use Tobacco etch virus, a Plant RNA virus, to investigate the stability of genetically redundant sequences by generating viruses with potentially beneficial gene duplications. Subsequently, we tested the viability of these viruses and performed experimental evolution. We found that all gene duplication events resulted in a loss of viability or in a significant reduction in viral fitness. Moreover, upon analyzing the genomes of the evolved viruses, we always observed the deletion of the duplicated gene copy and maintenance of the ancestral copy. Interestingly, there were clear differences in the deletion dynamics of the duplicated gene associated with the passage duration and the size and position of the duplicated copy. Based on the experimental data, we developed a mathematical model to characterize the stability of genetically redundant sequences, and showed that fitness effects are not enough to predict genomic stability. A context-dependent recombination rate is also required, with the context being the duplicated gene and its position. Our results therefore demonstrate experimentally the deleterious nature of gene duplications in RNA viruses. Beside previously described constraints on genome size, we identified additional factors that reduce the likelihood of the maintenance of duplicated genes.
-
predicting the stability of homologous gene duplications in a Plant RNA virus
bioRxiv, 2016Co-Authors: Anouk Willemsen, Mark P Zwart, Pablo Higueras, Josep Sardanyes, Santiago F ElenaAbstract:One of the striking features of many eukaryotes is the apparent amount of redundancy in coding and non-coding elements of their genomes. Despite the possible evolutionary advantages, there are fewer examples of redundant sequences in viral genomes, particularly those with RNA genomes. The low prevalence of gene duplication in RNA viruses most likely reflects the strong selective constraints against increasing genome size. Here we investigated the stability of genetically redundant sequences and how adaptive evolution proceeds to remove them. We generated Plant RNA viruses with potentially beneficial gene duplications, measured their fitness and performed experimental evolution, hereby exploring their genomic stability and evolutionary potential. We found that all gene duplication events resulted in a loss of viability or significant reductions in fitness. Moreover, upon evolving the viable viruses and analyzing their genomes, we always observed the deletion of the duplicated gene copy and maintenance of the ancestral copy. Interestingly, there were clear differences in the deletion dynamics of the duplicated gene associated with the passage duration, the size of the gene and the position for duplication. Based on the experimental data, we developed a mathematical model to characterize the stability of genetically redundant sequences, and showed that the fitness of viruses with duplications is not enough information to predict genomic stability as a recombination rate dependent on the genetic context - the duplicated gene and its position - is also required. Our results therefore demonstrate experimentally the deleterious nature of gene duplications in RNA viruses, and we identify factors that constrain the maintenance of duplicated genes.
-
estimation of the in vivo recombination rate for a Plant RNA virus
Journal of General Virology, 2014Co-Authors: Nicolas Tromas, Mark P Zwart, Maite Poulain, Santiago F ElenaAbstract:Phylogenomic evidence suggested that recombination is an important evolutionary force for potyviruses, one of the larger families of Plant RNA viruses. However, mixed-genotype potyvirus infections are marked by low levels of cellular coinfection, precluding template switching and recombination events between virus genotypes during genomic RNA replication. To reconcile these conflicting observations, we evaluated the in vivo recombination rate (r g) of Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) by coinfecting Plants with pairs of genotypes marked with engineered restriction sites as neutral markers. The recombination rate was then estimated using two different approaches: (i) a classical approach that assumed recombination between marked genotypes can occur in the whole virus population, rendering an estimate of r g = 7.762×10−8 recombination events per nucleotide site per generation, and (ii) an alteRNAtive method that assumed recombination between marked genotypes can occur only in coinfected cells, rendering a much higher estimate of r g = 3.427×10−5 recombination events per nucleotide site per generation. This last estimate is similar to the TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. Finally, we compared our mutation and recombination rate estimates to those reported for animal RNA viruses. Our analysis suggested that high recombination rates may be an unavoidable consequence of selection for fast replication at the cost of low fidelity.
-
effects of the number of genome segments on primary and systemic infections with a multipartite Plant RNA virus
Journal of Virology, 2013Co-Authors: J A Sancheznavarro, Mark P Zwart, Santiago F ElenaAbstract:Multipartite Plant viruses were discovered because of discrepancies between the observed dose response and predictions of the independent-action hypothesis (IAH) model. Theory suggests that the number of genome segments predicts the shape of the dose-response curve, but a rigorous test of this hypothesis has not been reported. Here, Alfalfa mosaic virus (AMV), a tripartite Alfamovirus, and transgenic Nicotianatabacum Plants expressing no (wild type), one (P2), or two (P12) viral genome segments were used to test whether the number of genome segments necessary for infection predicts the dose response. The dose-response curve of wild-type Plants was steep and congruent with the predicted kinetics of a multipartite virus, confirming previous results. Moreover, for P12 Plants, the data support the IAH model, showing that the expression of virus genome segments by the host Plant can modulate the infection kinetics of a tripartite virus to those of a monopartite virus. However, the different types of virus particles occurred at different frequencies, with a ratio of 116:45:1 (RNA1 to RNA2 to RNA3), which will affect infection kinetics and required analysis with a more comprehensive infection model. This analysis showed that each type of virus particle has a different probability of invading the host Plant, at both the primary- and systemic-infection levels. While the number of genome segments affects the dose response, taking into consideration differences in the infection kinetics of the three types of AMV particles results in a better understanding of the infection process.
Kiwamu Hyodo - One of the best experts on this subject based on the ideXlab platform.
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harnessing host ros generating machinery for the robust genome replication of a Plant RNA virus
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Kiwamu Hyodo, Tetsuro Okuno, Kenji Hashimoto, Kazuyuki Kuchitsu, Nobuhiro SuzukiAbstract: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.
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host and viral RNA binding proteins involved in membrane targeting replication and intercellular movement of Plant RNA virus genomes
Frontiers in Plant Science, 2014Co-Authors: Kiwamu Hyodo, Masanori Kaido, Tetsuro OkunoAbstract:Many Plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host Plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to several viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of Plant RNA virus genomes.
-
traffic jam on the cellular secretory pathway generated by a replication protein from a Plant RNA virus
Plant Signaling & Behavior, 2014Co-Authors: Kiwamu Hyodo, Masanori Kaido, Tetsuro OkunoAbstract:Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a Plant RNA virus, Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on Plant–virus interaction are discussed.
-
adp ribosylation factor 1 plays an essential role in the replication of a Plant RNA virus
Journal of Virology, 2013Co-Authors: Kiwamu Hyodo, Masanori Kaido, Kazuyuki Mise, Akira Mine, Takako Taniguchi, Hisaaki Taniguchi, Tetsuro OkunoAbstract:ABSTRACT Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA Plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.
Masanori Kaido - One of the best experts on this subject based on the ideXlab platform.
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host and viral RNA binding proteins involved in membrane targeting replication and intercellular movement of Plant RNA virus genomes
Frontiers in Plant Science, 2014Co-Authors: Kiwamu Hyodo, Masanori Kaido, Tetsuro OkunoAbstract:Many Plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host Plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to several viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of Plant RNA virus genomes.
-
traffic jam on the cellular secretory pathway generated by a replication protein from a Plant RNA virus
Plant Signaling & Behavior, 2014Co-Authors: Kiwamu Hyodo, Masanori Kaido, Tetsuro OkunoAbstract:Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a Plant RNA virus, Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on Plant–virus interaction are discussed.
-
adp ribosylation factor 1 plays an essential role in the replication of a Plant RNA virus
Journal of Virology, 2013Co-Authors: Kiwamu Hyodo, Masanori Kaido, Kazuyuki Mise, Akira Mine, Takako Taniguchi, Hisaaki Taniguchi, Tetsuro OkunoAbstract:ABSTRACT Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA Plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.
-
a Plant RNA virus suppresses RNA silencing through viral RNA replication
The EMBO Journal, 2005Co-Authors: Atsushi Takeda, Masanori Kaido, Kazuyuki Mise, Misato Tsukuda, Hiroyuki Mizumoto, Kimiyuki Okamoto, Tetsuro OkunoAbstract:RNA interference (RNAi) is a post-transcriptional gene-regulatory mechanism that operates in many eukaryotes. RNAi is induced by double-stranded RNA (dsRNA) and is mainly involved in defence against transposons and viruses. To counteract RNAi, viruses have RNAi suppressors. Here we show a novel mechanism of RNAi suppression by a Plant virus Red clover necrotic mosaic virus (RCNMV). To suppress RNAi, RCNMV needs multiple viral components, which include viral RNAs and putative RNA replicase proteins. A close relationship between the RNA elements required for negative-strand RNA synthesis and RNAi suppression suggests a strong link between the viral RNA replication machinery and the RNAi machinery. In a transient assay, RCNMV interferes with the accumulation of small-interfering RNA (siRNAs) in RNAi induced by a hairpin dsRNA and it also interferes with microRNA (miRNA) biogenesis. An Arabidopsis dcl1 mutant showed reduced susceptibility to RCNMV infection. Based on these results, we propose a model in which, to replicate, RCNMV deprives the RNAi machinery of Dicer-like enzymes that are involved in both siRNA and miRNA biogenesis.