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José R Castón - One of the best experts on this subject based on the ideXlab platform.
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The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.
Journal of virology, 2018Co-Authors: Carlos P. Mata, Johann Mertens, Juan Fontana, Daniel Luque, Carolina Allende-ballestero, David Reguera, Benes L. Trus, Alasdair C. Steven, José L. Carrascosa, José R CastónAbstract:Infectious bursal disease Virus (IBDV), a nonenveloped, Double-Stranded RNA (dsRNA) Virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an inteRNAl scaffold shell similar to that of tailed Double-Stranded DNA (dsDNA) Viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-A-thick inteRNAl shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA Viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) Viruses.IMPORTANCE Structural analyses highlight the constraint of Virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease Virus (IBDV), a Double-Stranded RNA Virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an inteRNAl scaffold during IBDV assembly. The use of an inteRNAl scaffold is reminiscent of tailed dsDNA Viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA Viruses; we suggest that IBDV is a modern descendant of ancestral Viruses and comprises different features of current viral lineages.
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The biological attributes, genome architecture and packaging of diverse multi-component fungal Viruses.
Current opinion in virology, 2018Co-Authors: Yukiyo Sato, José R Castón, Nobuhiro SuzukiAbstract:Many fungal Viruses or mycoViruses have multi-segmented, rather than single-segmented, genomes. This multi-segment nature is frequently possessed by Double-Stranded RNA Viruses, which include members of the Chrysoviridae, Quadriviridae, MegabiRNAviridae, Partitiviridae, and Reoviridae families, and unassigned groups. Their genome segments are often packaged separately with the exception of mycoreoViruses, which are multi-segmented but mono-particulate Viruses. These multi-segmented fungal dsRNA Viruses, as exemplified by reoViruses, have been extensively studied among structural biologists, and contributed to discoveries of novel virion structures. Multi-component systems, interactions of Viruses with subviral agents such as satellite and defective RNAs as typified by the yeast killer, and the rule-breaking neo-Virus lifestyle exhibited by a capsidless single-stranded RNA Virus hosted in an unrelated Double-Stranded RNA Virus are also discussed. Fungal multi-segmented Viruses and multicomponent Virus systems would continue to provide virologists with interesting future challenges.
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infectious bursal disease Virus ribonucleoprotein complexes of a double stranded RNA Virus
Journal of Molecular Biology, 2009Co-Authors: Daniel Luque, José L. Carrascosa, Jose F Rodriguez, Irene Saugar, Maria Teresa Rejas, José R CastónAbstract:Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some Double-Stranded DNA (dsDNA) Viruses, and the nucleocapsid proteins of single-stranded RNA Viruses. dsRNA Viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T=2 core involved in the metabolism and insulation of the dsRNA genome. The biRNAViruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T=13 capsid only. Moreover, as in many negative single-stranded RNA Viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease Virus, the best characterized member of the BiRNAviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at approximately 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNAse III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA Viruses.
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three dimensional structure of penicillium chrysogenum Virus a double stranded RNA Virus with a genuine t 1 capsid
Journal of Molecular Biology, 2003Co-Authors: José R Castón, Daniel Luque, Daohong Jiang, German Rivas, Carlos Alfonso, Ramon Roca, José L. CarrascosaAbstract:Abstract Although Double-Stranded (ds) RNA Viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA Viruses, from the mammalian reoViruses to the bacteriophage φ6, including fungal Viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA Viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26 A resolution of the Penicillium chrysogenum Virus (PcV) capsid. In contrast to all previous studies of dsRNA Viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA Virus capsids.
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c terminus of infectious bursal disease Virus major capsid protein vp2 is involved in definition of the t number for capsid assembly
Journal of Virology, 2001Co-Authors: José R Castón, Jorge L Martineztorrecuadrada, Antonio Maraver, Eleuterio Lombardo, Jose F Rodriguez, J I Casal, José L. CarrascosaAbstract:Infectious bursal disease Virus (IBDV), a member of the BiRNAviridae family, is a Double-Stranded RNA Virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and Virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.
Juan Carlos Ribas - One of the best experts on this subject based on the ideXlab platform.
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a new wine torulaspora delbrueckii killer strain with broad antifungal activity and its toxin encoding double stranded RNA Virus
Frontiers in Microbiology, 2015Co-Authors: Manuel Ramirez, Rocio Velazquez, Matilde Maqueda, Antonio Lopezpineiro, Juan Carlos RibasAbstract:Wine Torulaspora delbrueckii strains producing a new killer toxin (Kbarr-1) were isolated and selected for wine making. They killed all the previously known S. cerevisiae killer strains, in addition to other non-Saccharomyces yeasts. The Kbarr-1 phenotype is encoded by a medium-size 1.7 kb dsRNA, TdV-Mbarr-1, which seems to depend on a large-size 4.6 kb dsRNA Virus (TdV-LAbarr) for stable maintenance and replication. The TdV-Mbarr-1 dsRNA was sequenced by new generation sequencing techniques. Its genome structure is similar to those of S. cerevisiae killer M dsRNAs, with a 5´-end coding region followed by an inteRNAl A-rich sequence and a 3´-end non-coding region. Mbarr-1 RNA positive strand carries cis acting signals at its 5´ and 3´ termini for transcription and replication respectively, similar to those RNAs of yeast killer Viruses. The ORF at the 5´ region codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No relevant sequence identity was found either between the full sequence of Mbarr-1 dsRNA and other yeast M dsRNAs, or between their respective toxin-encoded proteins. However, a relevant identity of TdV-Mbarr-1 RNA regions to the putative replication and packaging signals of most of the M-Virus RNAs suggests that they are all evolutionarily related.
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the gag domain of the gag pol fusion protein directs incorporation into the l a double stranded RNA viral particles insaccharomyces cerevisiae
Journal of Biological Chemistry, 1998Co-Authors: Juan Carlos Ribas, Reed B. WicknerAbstract:Abstract The L-A Double-Stranded RNA Virus of yeast encodes its major coat protein, Gag, and a Gag-Pol fusion protein made by a −1 ribosomal frameshift, a coding strategy used by many retroViruses. We find that cells expressing only Gag from one plasmid and only Gag-Pol (in frame) from a separate plasmid can support the propagation of M1 Double-Stranded RNA, encoding the killer toxin. We use this system to separately investigate the functions of Gag and the Gag part of Gag-Pol. L-A contains two fusion protein molecules per particle, and although N-terminal acetylation of Gag is essential for viral assembly, it is completely dispensable for function of Gag-Pol. In general, the requirements on Gag for viral assembly and propagation are more stringent than on the Gag part of Gag-Pol. Finally, we directly show that it is Gag that instructs the incorporation of Gag-Pol into the viral particles.
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essential RNA binding and packaging domains of the gag pol fusion protein of the l a double stranded RNA Virus of saccharomyces cerevisiae
Journal of Biological Chemistry, 1994Co-Authors: Juan Carlos Ribas, Tsutomu FujimuraAbstract:Abstract The crucial process in the assembly of the L-A Double-Stranded RNA Virus is the recognition of its (+) single-stranded RNA by the Gag-Pol protein. The Pol region of this protein has RNA binding activity and is necessary for RNA packaging. Here we show that there are actually two in vitro RNA-binding domains of Pol (residues 172-190 and 770-819), and both are necessary for viral propagation, (but not for particle assembly). Furthermore, the N-terminal RNA-binding domain is necessary for in vivo packaging of viral (+) single-stranded RNA. We precisely define the extent of the Pol packaging domain (residues 67-213), which includes the N-terminal RNA-binding domain. This suggests that the N-terminal RNA-binding domain is responsible for binding the genomic RNA in the process of packaging and that additional surrounding residues are responsible for the specificity of binding.
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pol of gag pol fusion protein required for encapsidation of viral RNA of yeast l a Virus
Nature, 1992Co-Authors: Tsutomu Fujimura, Juan Carlos Ribas, Alexandr M MakhovAbstract:Double-Stranded RNA Viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A Double-Stranded RNA Virus we have investigated the mechanism by which the Virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag–pol fusion protein which is formed by a −1 ribosomal frameshift1–3. Here we show that Gag alone is sufficient for Virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A Virus encapsidation model1: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.
José L. Carrascosa - One of the best experts on this subject based on the ideXlab platform.
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The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.
Journal of virology, 2018Co-Authors: Carlos P. Mata, Johann Mertens, Juan Fontana, Daniel Luque, Carolina Allende-ballestero, David Reguera, Benes L. Trus, Alasdair C. Steven, José L. Carrascosa, José R CastónAbstract:Infectious bursal disease Virus (IBDV), a nonenveloped, Double-Stranded RNA (dsRNA) Virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an inteRNAl scaffold shell similar to that of tailed Double-Stranded DNA (dsDNA) Viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-A-thick inteRNAl shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA Viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) Viruses.IMPORTANCE Structural analyses highlight the constraint of Virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease Virus (IBDV), a Double-Stranded RNA Virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an inteRNAl scaffold during IBDV assembly. The use of an inteRNAl scaffold is reminiscent of tailed dsDNA Viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA Viruses; we suggest that IBDV is a modern descendant of ancestral Viruses and comprises different features of current viral lineages.
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infectious bursal disease Virus ribonucleoprotein complexes of a double stranded RNA Virus
Journal of Molecular Biology, 2009Co-Authors: Daniel Luque, José L. Carrascosa, Jose F Rodriguez, Irene Saugar, Maria Teresa Rejas, José R CastónAbstract:Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some Double-Stranded DNA (dsDNA) Viruses, and the nucleocapsid proteins of single-stranded RNA Viruses. dsRNA Viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T=2 core involved in the metabolism and insulation of the dsRNA genome. The biRNAViruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T=13 capsid only. Moreover, as in many negative single-stranded RNA Viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease Virus, the best characterized member of the BiRNAviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at approximately 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNAse III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA Viruses.
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three dimensional structure of penicillium chrysogenum Virus a double stranded RNA Virus with a genuine t 1 capsid
Journal of Molecular Biology, 2003Co-Authors: José R Castón, Daniel Luque, Daohong Jiang, German Rivas, Carlos Alfonso, Ramon Roca, José L. CarrascosaAbstract:Abstract Although Double-Stranded (ds) RNA Viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA Viruses, from the mammalian reoViruses to the bacteriophage φ6, including fungal Viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA Viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26 A resolution of the Penicillium chrysogenum Virus (PcV) capsid. In contrast to all previous studies of dsRNA Viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA Virus capsids.
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c terminus of infectious bursal disease Virus major capsid protein vp2 is involved in definition of the t number for capsid assembly
Journal of Virology, 2001Co-Authors: José R Castón, Jorge L Martineztorrecuadrada, Antonio Maraver, Eleuterio Lombardo, Jose F Rodriguez, J I Casal, José L. CarrascosaAbstract:Infectious bursal disease Virus (IBDV), a member of the BiRNAviridae family, is a Double-Stranded RNA Virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and Virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.
Tsutomu Fujimura - One of the best experts on this subject based on the ideXlab platform.
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5 ’ diphosphate terminus of L-A transcript 1 YEAST Double-Stranded RNA Virus L-A DELIBERATELY SYNTHESIZES RNA TRANSCRIPTS WITH 5 ’ DIPHOSPHATE*
2016Co-Authors: Tsutomu Fujimura, Rosa EstebanAbstract:L-A is a persistent Double-Stranded RNA Virus commonly found in the yeast Saccharomyces cerevisiae. Isolated L-A Virus synthesizes positive strand transcripts in vitro. We found that the 5 ’ termini of the transcripts are diphosphorylated. The 5 ’ terminal nucleotide is G and GDP was the best substrate among those examined to prime the reaction. When GTP was used, the triphosphate of GTP incorporated into the 5 ’ end was converted to diphosphate. This activity was not dependent on host CTL1 RNA triphosphatase. The 5 ’ end of GMP-primed transcript was also converted to diphosphate, the β phosphate of which was derived from the γ phosphate of ATP present i
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20s RNA naRNAVirus defies the antiviral activity of ski1 xrn1 in saccharomyces cerevisiae
Journal of Biological Chemistry, 2008Co-Authors: Rosa Esteban, Lorena Vega, Tsutomu FujimuraAbstract:Abstract 20S RNA Virus is a persistent positive strand RNA Virus found in Saccharomyces cerevisiae. We previously observed that the Virus generated in vivo from a launching vector possessed the correct RNA termini without extra sequences. Here we present evidence that the SKI1/XRN1 5′-exonuclease plays a major role in the elimination of the non-viral upstream sequences from the primary transcripts. The Virus, once generated, however, is fairly unaffected by overexpression or deletion of SKI1/XRN1. By contrast, the copy number of the L-A Double-Stranded RNA Virus in the same host is greatly increased by the deletion of SKI1/XRN1, and overexpression of the gene cured L-A Virus from the cells at a high frequency. 20S RNA Virus, unlike L-A Virus, has a strong secondary structure at its 5′-end: the first four nucleotides are G, and they are buried at the bottom of a long stem structure, features known to inhibit the SKI1/XRN1 5′-exonuclease progression. Mutations that weakened the 5′-stem structure made 20S RNA Virus vulnerable to SKI1/XRN1. These results, together with the data on L-A Virus, indicate a strong anti-RNA Virus activity of SKI1/XRN1. Given that 20S RNA Virus resides and replicates in the cytoplasm without a protective capsid, our results suggest that the strong secondary structure at the 5′-end is crucial for the 20S RNA Virus to evade the host SKI1/XRN1 defense.
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essential RNA binding and packaging domains of the gag pol fusion protein of the l a double stranded RNA Virus of saccharomyces cerevisiae
Journal of Biological Chemistry, 1994Co-Authors: Juan Carlos Ribas, Tsutomu FujimuraAbstract:Abstract The crucial process in the assembly of the L-A Double-Stranded RNA Virus is the recognition of its (+) single-stranded RNA by the Gag-Pol protein. The Pol region of this protein has RNA binding activity and is necessary for RNA packaging. Here we show that there are actually two in vitro RNA-binding domains of Pol (residues 172-190 and 770-819), and both are necessary for viral propagation, (but not for particle assembly). Furthermore, the N-terminal RNA-binding domain is necessary for in vivo packaging of viral (+) single-stranded RNA. We precisely define the extent of the Pol packaging domain (residues 67-213), which includes the N-terminal RNA-binding domain. This suggests that the N-terminal RNA-binding domain is responsible for binding the genomic RNA in the process of packaging and that additional surrounding residues are responsible for the specificity of binding.
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pol of gag pol fusion protein required for encapsidation of viral RNA of yeast l a Virus
Nature, 1992Co-Authors: Tsutomu Fujimura, Juan Carlos Ribas, Alexandr M MakhovAbstract:Double-Stranded RNA Viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A Double-Stranded RNA Virus we have investigated the mechanism by which the Virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag–pol fusion protein which is formed by a −1 ribosomal frameshift1–3. Here we show that Gag alone is sufficient for Virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A Virus encapsidation model1: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.
Daniel Luque - One of the best experts on this subject based on the ideXlab platform.
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The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.
Journal of virology, 2018Co-Authors: Carlos P. Mata, Johann Mertens, Juan Fontana, Daniel Luque, Carolina Allende-ballestero, David Reguera, Benes L. Trus, Alasdair C. Steven, José L. Carrascosa, José R CastónAbstract:Infectious bursal disease Virus (IBDV), a nonenveloped, Double-Stranded RNA (dsRNA) Virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an inteRNAl scaffold shell similar to that of tailed Double-Stranded DNA (dsDNA) Viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-A-thick inteRNAl shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA Viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) Viruses.IMPORTANCE Structural analyses highlight the constraint of Virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease Virus (IBDV), a Double-Stranded RNA Virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an inteRNAl scaffold during IBDV assembly. The use of an inteRNAl scaffold is reminiscent of tailed dsDNA Viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA Viruses; we suggest that IBDV is a modern descendant of ancestral Viruses and comprises different features of current viral lineages.
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infectious bursal disease Virus ribonucleoprotein complexes of a double stranded RNA Virus
Journal of Molecular Biology, 2009Co-Authors: Daniel Luque, José L. Carrascosa, Jose F Rodriguez, Irene Saugar, Maria Teresa Rejas, José R CastónAbstract:Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some Double-Stranded DNA (dsDNA) Viruses, and the nucleocapsid proteins of single-stranded RNA Viruses. dsRNA Viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T=2 core involved in the metabolism and insulation of the dsRNA genome. The biRNAViruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T=13 capsid only. Moreover, as in many negative single-stranded RNA Viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease Virus, the best characterized member of the BiRNAviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at approximately 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNAse III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA Viruses.
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three dimensional structure of penicillium chrysogenum Virus a double stranded RNA Virus with a genuine t 1 capsid
Journal of Molecular Biology, 2003Co-Authors: José R Castón, Daniel Luque, Daohong Jiang, German Rivas, Carlos Alfonso, Ramon Roca, José L. CarrascosaAbstract:Abstract Although Double-Stranded (ds) RNA Viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA Viruses, from the mammalian reoViruses to the bacteriophage φ6, including fungal Viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA Viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26 A resolution of the Penicillium chrysogenum Virus (PcV) capsid. In contrast to all previous studies of dsRNA Viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA Virus capsids.