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Said A Ghabrial - One of the best experts on this subject based on the ideXlab platform.
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Chrysovirus structure: repeated helical core as evidence of gene duplication.
Advances in Virus Research, 2020Co-Authors: Jose R Caston, Daniel Luque, Josué Gómez-blanco, Said A GhabrialAbstract:Abstract Chrysoviruses are double-stranded RNA viruses with a multipartite genome. Structure of two fungal chrysoviruses, Penicillium chrysogenum virus and Cryphonectria nitschkei chrysovirus 1, has been determined by three-dimensional cryo-electron microscopy analysis and in hydrodynamic studies. The capsids of both viruses are based on a T = 1 lattice containing 60 subunits, remain structurally undisturbed throughout the viral cycle, and participate in genome metabolism. The capsid protein is formed by a repeated α-helical core, indicative of gene duplication. Whereas the chrysovirus capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The arrangement of the chrysovirus α-helical core is conserved in the Totivirus L-A capsid protein, suggesting a shared basic fold. The encapsidated genome is organized in concentric shells; whereas inner dsRNA shells are diffuse, the outermost layer is organized into a dodecahedral cage beneath the protein capsid. This genome ordering could constitute a framework for dsRNA transcription in the capsid interior and/or have a structural role for capsid stability.
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Three-dimensional Structure of Victorivirus HvV190S Suggests Coat Proteins in Most Totiviruses Share a Conserved Core
PLOS Pathogens, 2013Co-Authors: Sarah E. Dunn, Said A Ghabrial, Max L Nibert, Hua Li, Giovanni Cardone, Timothy S BakerAbstract:Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300–450 A in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination–reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 A, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a “T = 2” icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other Totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all Totiviruses including, Leishmania RNA virus 1, to be similar as well.
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cryphonectria nitschkei virus 1 structure shows that the capsid protein of chrysoviruses is a duplicated helix rich fold conserved in fungal double stranded rna viruses
Journal of Virology, 2012Co-Authors: Josue Gomezblanco, Daniel Luque, Jose M Gonzalez, Jose L Carrascosa, Carlos Alfonso, Benes L Trus, Wendy M Havens, Said A Ghabrial, Jose R CastonAbstract:Cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This unusual organization is common to chrysoviruses. The arrangement of many of these putative α-helices is conserved in the Totivirus L-A capsid protein, suggesting a shared motif. Our results indicate that a 120-subunit T=1 capsid is a conserved architecture that optimizes dsRNA replication and organization.
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The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication
Journal of Virology, 2010Co-Authors: Daniel Luque, Jose M Gonzalez, Jose L Carrascosa, Benes L Trus, Wendy M Havens, Said A Ghabrial, Damià Garriga, Núria Verdaguer, Jose R CastonAbstract:Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 A resolution. The capsid protein has a high content of rod-like densities characteristic of α-helices, forming a repeated α-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the α-helical core resembles that found in the capsid protein of the L-A virus, a fungal Totivirus with an undivided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells; whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability.
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infectious myonecrosis virus has a Totivirus like 120 subunit capsid but with fiber complexes at the fivefold axes
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Jinghua Tang, Said A Ghabrial, Wendy F Ochoa, Robert S Sinkovits, Bonnie T Poulos, Donald V Lightner, Timothy S Baker, Max L NibertAbstract:Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-A resolution. Results reveal a Totivirus-like, 120-subunit T = 1 capsid, 450 A in diameter, but with fiber complexes protruding a further 80 A at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other Totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.
Nicholas Benjamin Daniel Phelps - One of the best experts on this subject based on the ideXlab platform.
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Molecular detection of a novel Totivirus from golden shiner (Notemigonus crysoleucas) baitfish in the USA
Archives of Virology, 2016Co-Authors: Nicholas Benjamin Daniel PhelpsAbstract:During regulatory and routine surveillance sampling of apparently healthy baitfish from the state of Minnesota, a novel Totivirus (tentatively named “golden shiner Totivirus”, GSTV) was detected in a homogenate of kidney and spleen of golden shiner ( Notemigonus crysoleucas ). The nearly complete genome is 7788 nt long with a complete 5’ untranslated region (UTR) of 135 nt (1-135 nt position), complete open reading frames (ORFs) and a partial 3’ UTR of 54 nt (7734-7788). The sequence is comprised of two ORFs (ORF1 and ORF2). The larger ORF1 encodes a 1659-aa polypeptide in frame +1 from nt position 136 to 5115 (4980 nt) with a start codon at position 136-138 and a stop codon at position 5113-5115. The ORF1 is 54 aa longer than the 1605-aa ORF1-encoded protein of a reference strain of infectious myonecrosis virus (IMNV), ID-EJ-12-1(AIC34743.1). The predicted ORF1 and ORF2 fusion protein sequence was NFQDGG. Hence, an overlapping region of 99 nt was observed, which is shorter than the 172-nt and 199-nt overlapping regions in Armigeres subalbatus Totivirus (AsTV) and IMNV, respectively. GSTV formed a separate lineage based on phylogenetic analysis of ORF1-encoded major capsid protein (MCP) and ORF2-encoded RNA-dependent RNA polymerase (RdRp) sequences. Based on ORF1 MCP sequence analysis, GSTV was most closely related to IMNV, with maximum aa sequence identity of 26.42-27.86 %, followed by 26.59, 22.94 and 21.75 % for Drosophila Totivirus (DTV), AsTV and Omono River virus (OMRV), respectively. Similar to ORF1, the ORF2 (RdRp) of GSTV formed a separate clade with maximum identity of 38.10 % and 38.50 % to IMNV and DTV, respectively. The virus identified here differs enough from its closest relative that it may represent a new genus in the family Totiviridae . The disease-causing potential and management impact of this novel virus is unknown at this time.
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molecular detection of a novel Totivirus from golden shiner notemigonus crysoleucas baitfish in the usa
Archives of Virology, 2016Co-Authors: Nicholas Benjamin Daniel PhelpsAbstract:During regulatory and routine surveillance sampling of apparently healthy baitfish from the state of Minnesota, a novel Totivirus (tentatively named “golden shiner Totivirus”, GSTV) was detected in a homogenate of kidney and spleen of golden shiner (Notemigonus crysoleucas). The nearly complete genome is 7788 nt long with a complete 5’ untranslated region (UTR) of 135 nt (1-135 nt position), complete open reading frames (ORFs) and a partial 3’ UTR of 54 nt (7734-7788). The sequence is comprised of two ORFs (ORF1 and ORF2). The larger ORF1 encodes a 1659-aa polypeptide in frame +1 from nt position 136 to 5115 (4980 nt) with a start codon at position 136-138 and a stop codon at position 5113-5115. The ORF1 is 54 aa longer than the 1605-aa ORF1-encoded protein of a reference strain of infectious myonecrosis virus (IMNV), ID-EJ-12-1(AIC34743.1). The predicted ORF1 and ORF2 fusion protein sequence was NFQDGG. Hence, an overlapping region of 99 nt was observed, which is shorter than the 172-nt and 199-nt overlapping regions in Armigeres subalbatus Totivirus (AsTV) and IMNV, respectively. GSTV formed a separate lineage based on phylogenetic analysis of ORF1-encoded major capsid protein (MCP) and ORF2-encoded RNA-dependent RNA polymerase (RdRp) sequences. Based on ORF1 MCP sequence analysis, GSTV was most closely related to IMNV, with maximum aa sequence identity of 26.42-27.86 %, followed by 26.59, 22.94 and 21.75 % for Drosophila Totivirus (DTV), AsTV and Omono River virus (OMRV), respectively. Similar to ORF1, the ORF2 (RdRp) of GSTV formed a separate clade with maximum identity of 38.10 % and 38.50 % to IMNV and DTV, respectively. The virus identified here differs enough from its closest relative that it may represent a new genus in the family Totiviridae. The disease-causing potential and management impact of this novel virus is unknown at this time.
Nobuhiro Suzuki - One of the best experts on this subject based on the ideXlab platform.
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Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature
Viruses, 2019Co-Authors: Atif Jamal, Hideki Kondo, Yukiyo Sato, Sabitree Shahi, Wajeeha Shamsi, Nobuhiro SuzukiAbstract:The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the Totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.
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Reprint of "Sequence and phylogenetic analyses of novel Totivirus-like double-stranded RNAs from field-collected powdery mildew fungi".
Virus research, 2016Co-Authors: Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro SuzukiAbstract:The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten Totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated Totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (
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sequence and phylogenetic analyses of novel Totivirus like double stranded rnas from field collected powdery mildew fungi
Virus Research, 2016Co-Authors: Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro SuzukiAbstract:The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten Totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated Totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known Totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2–8) resembled the genome of the prototype Totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another Totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast Totiviruses, each ScV-L-A-like RPaTV contains a –1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel Totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.
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Sequence and phylogenetic analyses of novel Totivirus-like double-stranded RNAs from field-collected powdery mildew fungi.
Virus Research, 2015Co-Authors: Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro SuzukiAbstract:The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten Totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated Totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (
Hideki Kondo - One of the best experts on this subject based on the ideXlab platform.
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Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature
Viruses, 2019Co-Authors: Atif Jamal, Hideki Kondo, Yukiyo Sato, Sabitree Shahi, Wajeeha Shamsi, Nobuhiro SuzukiAbstract:The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the Totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.
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Reprint of "Sequence and phylogenetic analyses of novel Totivirus-like double-stranded RNAs from field-collected powdery mildew fungi".
Virus research, 2016Co-Authors: Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro SuzukiAbstract:The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten Totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated Totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (
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sequence and phylogenetic analyses of novel Totivirus like double stranded rnas from field collected powdery mildew fungi
Virus Research, 2016Co-Authors: Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro SuzukiAbstract:The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten Totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated Totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known Totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2–8) resembled the genome of the prototype Totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another Totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast Totiviruses, each ScV-L-A-like RPaTV contains a –1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel Totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.
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Sequence and phylogenetic analyses of novel Totivirus-like double-stranded RNAs from field-collected powdery mildew fungi.
Virus Research, 2015Co-Authors: Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro SuzukiAbstract:The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten Totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated Totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (
Rosa Esteban - One of the best experts on this subject based on the ideXlab platform.
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cap snatching in yeast l bc double stranded rna Totivirus
Journal of Biological Chemistry, 2013Co-Authors: Tsutomu Fujimura, Rosa EstebanAbstract:Yeast L-A double-stranded RNA virus furnishes its transcript with a 5′ cap structure by a novel cap-snatching mechanism in which m7Gp from a host mRNA cap structure is transferred to the 5′-diphosphate terminus of the viral transcript. His-154 of the coat protein Gag forms an m7Gp adduct, and the H154R mutation abolishes both m7Gp adduct formation and cap snatching. Here we show that L-BC, another Totivirus closely related to L-A, also synthesizes 5′-diphosphorylated transcripts and transfers m7Gp from mRNA to the 5′ termini of the transcripts. L-BC Gag also covalently binds to the cap structure and the mutation H156R, which corresponds to H154R of L-A Gag, abolishes cap adduct formation. Cap snatching of the L-BC virus is very similar to that of L-A; N7 methylation of the mRNA cap is essential for cap donor activity, and only 5′-diphosphorylated RNA is used as cap acceptor. L-BC cap snatching is also activated by viral transcription. Furthermore, both viruses require Mg2+ and Mn2+ for cap snatching. These cations are not only required for transcription activation but also directly involved in the cap transfer process. These findings support our previous proposal that the cap-snatching mechanism of the L-A virus is shared by fungal Totiviruses closely related to L-A. Interestingly, L-A and L-BC viruses accept either viral transcript as cap acceptor in vitro. Because L-A and L-BC viruses cohabit in many yeast strains, it raises the possibility that their cohabitation in the same host may be beneficial for their mutual cap acquisition.
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l a lus a new variant of the l a Totivirus found in wine yeasts with klus killer toxin encoding mlus double stranded rna possible role of killer toxin encoding satellite rnas in the evolution of their helper viruses
Applied and Environmental Microbiology, 2013Co-Authors: Nieves Rodriguezcousino, Pilar Gomez, Rosa EstebanAbstract:This work has been supported by grant BFU2010-15768 from the Spanish Ministry of Education and Science.
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Yeast RNA viruses as indicators of exosome activity: human exosome hCsl4p participates in RNA degradation in Saccharomyces cerevisiae'
Yeast, 2011Co-Authors: Manuel Ramírez‐garrastacho, Rosa EstebanAbstract:The exosome is an evolutionarily conserved 10-mer complex involved in RNA metabolism, located in both the nucleus and the cytoplasm. The cytoplasmic exosome plays an important role in mRNA turnover through its 3′5′ exonucleolytic activity. The superkiller (SKI) phenotype of yeast was originally identified as an increase of killer toxin production due to elevated levels of the L-A double-stranded RNA (dsRNA) Totivirus and its satellite toxin-encoding M dsRNA. Most SKI genes were later shown to be either components of the exosome or modulators of its activity. Variations in the amount of Totivirus are, thus, good indicators of yeast exosome activity, and can be used to analyse its components. Furthermore, if exosome proteins of higher eukaryotes were functional in S. cerevisiae, these viruses would provide a simple tool to analyse their function. In this work, we have found that hCSL4, the human orthologue of SKI4 in the yeast exosome, rescues the null phenotype of the deletion mutant. hCsl4p shares with Ski4p conserved S1 RNA-binding domains, but lacks the N-terminal third of Ski4p. Nevertheless, it interacts with the Dis3p exonuclease of yeast exosome, and partially complements the superkiller phenotype of ski4-1 mutation. The elimination of the N-terminal third of Ski4p does not affect its activity, indicating that it is dispensable for RNA degradation. We have also identified the point mutation G152E in hCSL4, equivalent to the ski4-1 mutation G253E, which impairs the activity of the protein, thus validating our approach of using yeast RNA virus to analyse the exosome of higher eukaryotes. Copyright © 2011 John Wiley & Sons, Ltd.
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Cap-snatching mechanism in yeast L-A double-stranded RNA virus
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Tsutomu Fujimura, Rosa EstebanAbstract:The 5′ cap structure (m7GpppX-) is an essential feature of eukaryotic mRNA required for mRNA stability and efficient translation. Influenza virus furnishes its mRNA with this structure by a cap-snatching mechanism, in which the viral polymerase cleaves host mRNA endonucleolytically 10–13 nucleotides from the 5′ end and utilizes the capped fragment as a primer to synthesize viral transcripts. Here we report a unique cap-snatching mechanism by which the yeast double-stranded RNA Totivirus L-A furnishes its transcript with a cap structure derived from mRNA. Unlike influenza virus, L-A transfers only m7Gp from the cap donor to the 5′ end of the viral transcript, thus preserving the 5′ α- and β-phosphates of the transcript in the triphosphate linkage of the final product. This in vitro capping reaction requires His154 of the coat protein Gag, a residue essential for decapping of host mRNA and known to form m7Gp-His adduct. Furthermore, the synthesis of capped viral transcripts in vivo and their expression were greatly compromised by the Arg154 mutation, indicating the involvement of Gag in the cap-snatching reaction. The overall reaction and the structure around the catalytic site in Gag resemble those of guanylyltransferase, a key enzyme of cellular mRNA capping, suggesting convergent evolution. Given that Pol of L-A is confined inside the virion and unable to access host mRNA in the cytoplasm, the structural protein Gag rather than Pol catalyzing this unique cap-snatching reaction exemplifies the versatility as well as the adaptability of eukaryotic RNA viruses.
