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Jozef J Bujarski - One of the best experts on this subject based on the ideXlab platform.
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characterization of a novel 5 subgenomic rna3a derived from rna3 of brome mosaic bromovirus
Journal of Virology, 2006Co-Authors: Rafal Wierzchoslawski, Jozef J Bujarski, Marek Figlerowicz, Aleksandra Dzianott, Anna UrbanowiczAbstract:The synthesis of 3′ subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5′ sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3′ oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5′ sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.
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Characterization of a Novel 5′ Subgenomic RNA3a Derived from RNA3 of Brome Mosaic Bromovirus
Journal of Virology, 2006Co-Authors: Rafal Wierzchoslawski, Marek Figlerowicz, Aleksandra Dzianott, Anna Urbanowicz, Jozef J BujarskiAbstract:The synthesis of 3′ subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5′ sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3′ oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5′ sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.
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Homologous crossovers among molecules of brome mosaic bromovirus RNA1 or RNA2 segments in vivo.
Journal of Virology, 2005Co-Authors: Anna Urbanowicz, Marek Figlerowicz, Magdalena Alejska, Piotr Formanowicz, Jacek Blazewicz, Jozef J BujarskiAbstract:Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3' and 5' noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.
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RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts
Journal of Virology, 2002Co-Authors: René C. L. Olsthoorn, Aleksandra Dzianott, A. Bruyere, Jozef J BujarskiAbstract:A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.
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Engineering of homologous recombination hotspots with AU-rich sequences in brome mosaic virus.
Journal of virology, 1997Co-Authors: Peter D. Nagy, Jozef J BujarskiAbstract:Previously, we observed that crossovers sites of RNA recombinants clustered within or close to AU-rich regions during genetic recombination in brome mosaic bromovirus (BMV) (P. D. Nagy and J. J. Bujarski. J. Virol. 70:415-426, 1996). To test whether AU-rich sequences can facilitate homologous recombination, AU-rich sequences were introduced into parental BMV RNAs (RNA2 and RNA3). These insertions created a homologous RNA2-RNA3 recombination hotspot. Two other AU-rich sequences also supported high-frequency homologous recombination if a common sequence with high or average G/C content was present immediately upstream of the AU-rich element. Homologous RNA recombination did not require any additional sequence motifs or RNA structures and was position nonspecific within the 3' noncoding region. These results suggest that nucleotide content (i.e., the presence of common 5' GC-rich or moderately AU-rich and 3' AU-rich regions) is the important factor that determines the sites of homologous recombination. A mechanism that involves replicase switching during synthesis of positive-sense RNA strands is presented to explain the observed results.
Anette Schneemann - One of the best experts on this subject based on the ideXlab platform.
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capsid protein synthesis from replicating rna directs specific packaging of the genome of a multipartite positive strand rna virus
Journal of Virology, 2005Co-Authors: Arno P Venter, Neel K Krishna, Anette SchneemannAbstract:Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.
Gregor Bucher - One of the best experts on this subject based on the ideXlab platform.
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exploring systemic rna interference in insects a genome wide survey for RNAI genes in tribolium
Genome Biology, 2008Co-Authors: Yoshinori Tomoyasu, Sherry Miller, Shuichiro Tomita, Michael Schoppmeier, Daniela Grossmann, Gregor BucherAbstract:Background: RNA interference (RNAI) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAI response can be transmitted systemically. Some insects also exhibit a systemic RNAI response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAI response, necessitating another model system to study the molecular mechanism of systemic RNAI in insects. Results: We used Tribolium, which exhibits robust systemic RNAI, as an alternative model system. We have identified the core RNAI genes, as well as genes potentially involved in systemic RNAI, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAI in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAI. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAI in C. elegans. Conclusion: Although both Tribolium and C. elegans show a robust systemic RNAI response, our genome-wide survey reveals significant differences between the RNAI mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAI response. Understanding this process would assist with rendering other insects amenable to systemic RNAI, and may influence pest control approaches.
René C. L. Olsthoorn - One of the best experts on this subject based on the ideXlab platform.
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RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts
Journal of Virology, 2002Co-Authors: René C. L. Olsthoorn, Aleksandra Dzianott, A. Bruyere, Jozef J BujarskiAbstract:A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.
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role of an essential triloop hairpin and flanking structures in the 3 untranslated region of alfalfa mosaic virus rna in in vitro transcription
Journal of Virology, 2002Co-Authors: René C. L. OlsthoornAbstract:The family Bromoviridae consists of five genera of plant viruses with a tripartite RNA genome. RNA1 and RNA2 encode the viral subunits of the replicase. RNA3 is dicistronic and codes for a movement protein that is required for cell-to-cell movement and a coat protein (CP) needed for cell-to-cell and long-distance transport. CP is translated from a subgenomic messenger, RNA4, that is coterminal with the 3′ 800 to 1,000 nucleotides (nt) of RNA3. Some members of the Bromoviridae family produce a second subgenomic RNA (sgRNA) which is derived from RNA2. sgRNA synthesis is thought to occur by internal transcription on the minus strand of RNA3 (or RNA2). It has been suggested that a hairpin structure is required for subgenomic promoter (sgp) activity in all Bromoviridae (10). This hypothesis was experimentally verified for three genera within this family (4, 8, 9). Within this family, bromo- and cucumoviruses possess a tRNA-like structure (TLS) that can be charged with tyrosine whereas the RNAs of Alfalfa mosaic virus (AMV) and ilarviruses cannot be charged with an amino acid, although their 3′ ends were recently shown to adopt a putative TLS (13). The 3′ untranslated region (UTR) of the genomic RNAs of Olive latent virus type 2, the type species of the fifth genus, Oleavirus, can be folded into a TLS similar to that of the bromovirus RNAs (13). A unique property of the 3′ UTR of the Alfamovirus and Ilarvirus RNAs is their ability to adopt two mutually exclusive conformations: one recognized by the CP and one, a pseudoknotted conformation, recognized by the viral replicase. The latter conformer resembles the TLS of Brome mosaic virus (BMV) RNA and, for AMV, was shown to be essential for viral replication. The binding sites for CP in AMV RNAs have been characterized extensively in the past (reviewed in reference 2). The requirements for binding of the polymerase are less well defined: a minimal promoter element was delimited to the 3′-terminal 145 nt of RNA3 (18). In the present study, we performed an extensive mutation analysis of the 3′ UTR of RNA3 to identify a putative polymerase binding site. We have identified a triloop hairpin, hairpin E (hpE), as the most crucial element in minus-strand synthesis in vitro. The TLS domain, although harboring the initiation site for replication, by itself showed no template activity, nor did it compete with the full 3′ UTR for the replicase. Interestingly, we observed that severe deletions in the TLS caused transcription to initiate at a position upstream of hpE. This mode of transcription is similar to the action of the sgp. This promoter region was recently characterized in our laboratory and shown to require the formation of a similar triloop hairpin (8). We propose that (i) the AMV subgenomic and minus-strand promoters are basically the same and that (ii) the role of the TLS is to ensure that transcription initiates at the very 3′ terminus.
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Role of an Essential Triloop Hairpin and Flanking Structures in the 3′ Untranslated Region of Alfalfa Mosaic Virus RNA in In Vitro Transcription
Journal of Virology, 2002Co-Authors: René C. L. OlsthoornAbstract:The family Bromoviridae consists of five genera of plant viruses with a tripartite RNA genome. RNA1 and RNA2 encode the viral subunits of the replicase. RNA3 is dicistronic and codes for a movement protein that is required for cell-to-cell movement and a coat protein (CP) needed for cell-to-cell and long-distance transport. CP is translated from a subgenomic messenger, RNA4, that is coterminal with the 3′ 800 to 1,000 nucleotides (nt) of RNA3. Some members of the Bromoviridae family produce a second subgenomic RNA (sgRNA) which is derived from RNA2. sgRNA synthesis is thought to occur by internal transcription on the minus strand of RNA3 (or RNA2). It has been suggested that a hairpin structure is required for subgenomic promoter (sgp) activity in all Bromoviridae (10). This hypothesis was experimentally verified for three genera within this family (4, 8, 9). Within this family, bromo- and cucumoviruses possess a tRNA-like structure (TLS) that can be charged with tyrosine whereas the RNAs of Alfalfa mosaic virus (AMV) and ilarviruses cannot be charged with an amino acid, although their 3′ ends were recently shown to adopt a putative TLS (13). The 3′ untranslated region (UTR) of the genomic RNAs of Olive latent virus type 2, the type species of the fifth genus, Oleavirus, can be folded into a TLS similar to that of the bromovirus RNAs (13). A unique property of the 3′ UTR of the Alfamovirus and Ilarvirus RNAs is their ability to adopt two mutually exclusive conformations: one recognized by the CP and one, a pseudoknotted conformation, recognized by the viral replicase. The latter conformer resembles the TLS of Brome mosaic virus (BMV) RNA and, for AMV, was shown to be essential for viral replication. The binding sites for CP in AMV RNAs have been characterized extensively in the past (reviewed in reference 2). The requirements for binding of the polymerase are less well defined: a minimal promoter element was delimited to the 3′-terminal 145 nt of RNA3 (18). In the present study, we performed an extensive mutation analysis of the 3′ UTR of RNA3 to identify a putative polymerase binding site. We have identified a triloop hairpin, hairpin E (hpE), as the most crucial element in minus-strand synthesis in vitro. The TLS domain, although harboring the initiation site for replication, by itself showed no template activity, nor did it compete with the full 3′ UTR for the replicase. Interestingly, we observed that severe deletions in the TLS caused transcription to initiate at a position upstream of hpE. This mode of transcription is similar to the action of the sgp. This promoter region was recently characterized in our laboratory and shown to require the formation of a similar triloop hairpin (8). We propose that (i) the AMV subgenomic and minus-strand promoters are basically the same and that (ii) the role of the TLS is to ensure that transcription initiates at the very 3′ terminus.
Arno P Venter - One of the best experts on this subject based on the ideXlab platform.
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capsid protein synthesis from replicating rna directs specific packaging of the genome of a multipartite positive strand rna virus
Journal of Virology, 2005Co-Authors: Arno P Venter, Neel K Krishna, Anette SchneemannAbstract:Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.
