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Wilmer J Cuellar - One of the best experts on this subject based on the ideXlab platform.
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characterization of distinct strains of an aphid transmitted ilarvirus fam Bromoviridae infecting different hosts from south america
Virus Research, 2020Co-Authors: Rocio Silvestre, Segundo Fuentes, Roger Risco, Alfredo Berrocal, Ian P Adams, Wilmer J CuellarAbstract:Abstract Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range and RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA3 sequences from potato and partial sequences of RNA1, 2 and 3 of isolates infecting potato and yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently described isolate from Solanum muricatum (pepino dulce), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.
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Characterization of distinct strains of an aphid-transmitted ilarvirus (Fam. Bromoviridae) infecting different hosts from South America.
Virus research, 2020Co-Authors: Rocio Silvestre, Segundo Fuentes, Roger Risco, Alfredo Berrocal, Wilmer J Cuellar, Ian Adams, Jan KreuzeAbstract:Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range, RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA 3 sequences from potato and partial sequences of RNA 1, 2 and 3 of isolates infecting papa y yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently describe isolate from Solanum muricatum (Pepino), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.
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first report of potato yellowing virus genus ilarvirus in solanum phureja from ecuador
Plant Disease, 2011Co-Authors: Rocio Silvestre, Milton Untiveros, Wilmer J CuellarAbstract:A bacilliform virus, named Potato yellowing virus (PYV), causing chlorosis of leaves was reported in 1992 in potato (Solanum tuberosum) fields in Peru (1) and symptomless wild potatoes (S. fernandezianum) in Chile (4). PYV is reported as an alfamo-like virus (1) (family Bromoviridae) but no sequence information is available for this virus, making its taxonomic position inside the Bromoviridae uncertain (currently this family is organized into five genera: Alfamovirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus). Herein we report the presence of PYV in native potatoes (Solanum phureja) collected from Ecuador where the crop constitutes an important source of income in rural communities. Forty accessions of S. phureja collected in Ecuador in June 1986 and maintained in vitro at the International Potato Center (CIP) germplasm bank were analyzed by double-antibody sandwich (DAS)-ELISA with antiserum raised against a Peruvian isolate of PYV (1). PYV was detected in six accessions (15% of the material) co...
S. W. Scott - One of the best experts on this subject based on the ideXlab platform.
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eLS - Bromoviridae and Allies
Encyclopedia of Life Sciences, 2001Co-Authors: S. W. ScottAbstract:The family Bromoviridae consists of six genera: Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus and Oleavirus. All viruses in these genera possess tripartite genomes of single-stranded, linear, positive-sense ribonucleic acid (RNA), in which expression of the downstream codon of the RNA3 is via a subgenomic messenger RNA (mRNA) (RNA4). Raspberry bushy dwarf virus (RBDV) was the sole member of the genus Idaeovirus for many years and shares features in common with members of the genus Ilarvirus and Alfamovirus, but possesses a bipartite genome and is not currently assigned to a family. Members of the genera Alfamovirus, Bromovirus and Cucumovirus have been used extensively as model systems for research on molecular aspects of plant virology. Key Concepts: Although all members of the Bromoviridae have similar particle morphologies (quasi-spherical and bacilliform) and possess tripartite genomes, a range of different strategies is used to express the products of the different genomes. The phenomenon of genome activation by the coat protein was unique to the ilarviruses and Alfalfa mosaic virus but has recently been demonstrated for RBDV. The natural host ranges of Cucumber mosaic virus and Alfalfa mosaic virus are extensive whereas many other viruses described in this article have an extremely limited natural host range. Complete genomic nucleotide sequences are available from GenBank for the majority of viruses referred to in this article. Keywords: Alfamovirus; Anulavirus; Bromovirus; Cucumovirus; Ilarvirus; Oleavirus; Idaeovirus
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Bromoviridae and allies
eLS, 2001Co-Authors: S. W. ScottAbstract:The family Bromoviridae consists of six genera: Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus and Oleavirus. All viruses in these genera possess tripartite genomes of single-stranded, linear, positive-sense ribonucleic acid (RNA), in which expression of the downstream codon of the RNA3 is via a subgenomic messenger RNA (mRNA) (RNA4). Raspberry bushy dwarf virus (RBDV) was the sole member of the genus Idaeovirus for many years and shares features in common with members of the genus Ilarvirus and Alfamovirus, but possesses a bipartite genome and is not currently assigned to a family. Members of the genera Alfamovirus, Bromovirus and Cucumovirus have been used extensively as model systems for research on molecular aspects of plant virology. Key Concepts: Although all members of the Bromoviridae have similar particle morphologies (quasi-spherical and bacilliform) and possess tripartite genomes, a range of different strategies is used to express the products of the different genomes. The phenomenon of genome activation by the coat protein was unique to the ilarviruses and Alfalfa mosaic virus but has recently been demonstrated for RBDV. The natural host ranges of Cucumber mosaic virus and Alfalfa mosaic virus are extensive whereas many other viruses described in this article have an extremely limited natural host range. Complete genomic nucleotide sequences are available from GenBank for the majority of viruses referred to in this article. Keywords: Alfamovirus; Anulavirus; Bromovirus; Cucumovirus; Ilarvirus; Oleavirus; Idaeovirus
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ilarviruses encode a cucumovirus like 2b gene that is absent in other genera within the Bromoviridae
Journal of Virology, 1998Co-Authors: Lianghui Ji, S. W. Scott, Robert H. Symons, Shou-wei DingAbstract:We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3′ end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.
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The complete sequence of the genomic RNAs of spinach latent virus
Archives of Virology, 1997Co-Authors: X Ge, S. W. Scott, M T ZimmermanAbstract:We describe the sequence for the complete genome of spinach latent virus (SpLV). Comparisons of this genome with that of the only other complete genome described for a species within the genus Ilarvirus (citrus leaf rugose virus – CiLRV) indicate that while there are marked differences between the RNA 3 of the two viruses, their respective RNAs 1 and 2 share many similarities. However, the putative 2a protein of SpLV contains a C_2H_2 type “zinc finger”-like motif located towards the carboxy terminal of the protein which is absent in CiLRV and has not been reported for other members of the family Bromoviridae. A second open reading frame (2b), located at a similar position to that described for the cucumoviruses, occurs in the RNA 2 of both SpLV and CiLRV. The putative coat protein of SpLV is similar to that of citrus variegation virus (CVV) and asparagus virus 2 (AV-2), both members of subgroup 2 of the ilarviruses. We have subsequently demonstrated a serological relationship between SpLV and other viruses in subgroup 2 and suggest that SpLV should be included in this subgroup rather than remain in a separate group (subgroup 6). However, while the putative movement protein of SpLV is remarkably similar to that of AV-2, it shows little relationship with the corresponding protein of CVV and the lack of similarity suggests that a recombination event may have occurred in the past. The relationship between the genera Alfamovirus and Ilarvirus is discussed in the light of the data for the genome of SpLV and recently published information for other members of the genus Ilarvirus .
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The nucleotide sequence of citrus leaf rugose virus RNA 1.
The Journal of general virology, 1995Co-Authors: S. W. Scott, X GeAbstract:The nucleotide sequence of citrus leaf rugose virus (CiLRV) RNA 1 consists of 3404 nucleotides and contains one open reading frame (ORF) which encodes a putative translation product of 1051 amino acids with a calculated M(r) of 118339. Both the nucleotide sequence of CiLRV RNA 1 and its translated polypeptide share similarities with those of the RNA 1 of alfalfa mosaic virus. However, the relationship is not as close as that which exists between the polymerase signatures of the two viruses, which are found on RNA 2. This is the first report of the full-length sequence for the RNA 1 of an ilarvirus and completes the first sequence for an entire ilarvirus genome. If it is typical of members of the genus then, as has long been speculated, the genomic organization of ilarviruses is identical to that of other genera in the family Bromoviridae.
Vicente Pallas - One of the best experts on this subject based on the ideXlab platform.
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Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus.
The Journal of general virology, 2020Co-Authors: Frederic Aparicio, José Ángel Sánchez-navarro, René C. L. Olsthoorn, Vicente PallasAbstract:Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family Bromoviridae: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.
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RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus.
The Journal of general virology, 2020Co-Authors: M Carmen Herranz, Vicente PallasAbstract:The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.
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ictv virus taxonomy profile Bromoviridae
Journal of General Virology, 2019Co-Authors: Joseph Bujarski, Vicente Pallas, Donato Gallitelli, Fernando Garciaarenal, Peter Palukaitis, Krishna M Reddy, Aiming WangAbstract:Bromoviridae is a family of plant viruses with tri-segmented, positive-sense, single-stranded RNA genomes of about 8 kb in total. Genomic RNAs are packaged in separate virions that may also contain subgenomic, defective or satellite RNAs. Virions are variable in morphology (spherical or bacilliform) and are transmitted between hosts mechanically, in/on the pollen and non-persistently by insect vectors. Members of the family are responsible for major disease epidemics in fruit, vegetable and fodder crops such as tomato, cucurbits, bananas, fruit trees and alfalfa. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bromoviridae, which is available at www.ictv.global/report/Bromoviridae.
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Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Mireya Martínez-pérez, Frederic Aparicio, Jesús A. Sánchez-navarro, María Pilar López-gresa, José M. Bellés, Vicente PallasAbstract:N6-methyladenosine (m6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. m6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.
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Implication of the C terminus of the Prunus necrotic ringspot virus movement protein in cell-to-cell transport and in its interaction with the coat protein
Journal of General Virology, 2010Co-Authors: Frederic Aparicio, Vicente Pallas, José Ángel Sánchez-navarroAbstract:The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.
Rocio Silvestre - One of the best experts on this subject based on the ideXlab platform.
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characterization of distinct strains of an aphid transmitted ilarvirus fam Bromoviridae infecting different hosts from south america
Virus Research, 2020Co-Authors: Rocio Silvestre, Segundo Fuentes, Roger Risco, Alfredo Berrocal, Ian P Adams, Wilmer J CuellarAbstract:Abstract Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range and RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA3 sequences from potato and partial sequences of RNA1, 2 and 3 of isolates infecting potato and yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently described isolate from Solanum muricatum (pepino dulce), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.
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Characterization of distinct strains of an aphid-transmitted ilarvirus (Fam. Bromoviridae) infecting different hosts from South America.
Virus research, 2020Co-Authors: Rocio Silvestre, Segundo Fuentes, Roger Risco, Alfredo Berrocal, Wilmer J Cuellar, Ian Adams, Jan KreuzeAbstract:Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range, RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA 3 sequences from potato and partial sequences of RNA 1, 2 and 3 of isolates infecting papa y yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently describe isolate from Solanum muricatum (Pepino), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.
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first report of potato yellowing virus genus ilarvirus in solanum phureja from ecuador
Plant Disease, 2011Co-Authors: Rocio Silvestre, Milton Untiveros, Wilmer J CuellarAbstract:A bacilliform virus, named Potato yellowing virus (PYV), causing chlorosis of leaves was reported in 1992 in potato (Solanum tuberosum) fields in Peru (1) and symptomless wild potatoes (S. fernandezianum) in Chile (4). PYV is reported as an alfamo-like virus (1) (family Bromoviridae) but no sequence information is available for this virus, making its taxonomic position inside the Bromoviridae uncertain (currently this family is organized into five genera: Alfamovirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus). Herein we report the presence of PYV in native potatoes (Solanum phureja) collected from Ecuador where the crop constitutes an important source of income in rural communities. Forty accessions of S. phureja collected in Ecuador in June 1986 and maintained in vitro at the International Potato Center (CIP) germplasm bank were analyzed by double-antibody sandwich (DAS)-ELISA with antiserum raised against a Peruvian isolate of PYV (1). PYV was detected in six accessions (15% of the material) co...
René C. L. Olsthoorn - One of the best experts on this subject based on the ideXlab platform.
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Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus.
The Journal of general virology, 2020Co-Authors: Frederic Aparicio, José Ángel Sánchez-navarro, René C. L. Olsthoorn, Vicente PallasAbstract:Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family Bromoviridae: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.
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Similarities and differences between the subgenomic and minus-strand promoters of an RNA plant virus.
Journal of Virology, 2004Co-Authors: René C. L. Olsthoorn, P. C. HaasnootAbstract:Promoter regions required for minus-strand and subgenomic RNA synthesis have been mapped for several plus-strand RNA viruses. In general, the two types of promoters do not share structural features even though they are recognized by the same viral polymerase. The minus-strand promoter of Alfalfa mosaic virus (AMV), a plant virus of the family Bromoviridae, consists of a triloop hairpin (hpE) which is attached to a 3′ tRNA-like structure (TLS). In contrast, the AMV subgenomic promoter consists of a single triloop hairpin that bears no sequence homology with hpE. Here we show that hpE, when detached from its TLS, can function as a subgenomic promoter in vitro and can replace the authentic subgenomic promoter in the live virus. Thus, the AMV subgenomic and minus-strand promoters are basically the same, but the minus-strand promoter is linked to a 3′ TLS to force the polymerase to initiate at the very 3′end.
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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.
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Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus
Journal of General Virology, 2001Co-Authors: Frederic Aparicio, José Ángel Sánchez-navarro, René C. L. Olsthoorn, Vicente PallasAbstract:Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera Alfamovirus and Ilarvirus, respectively, of the family Bromoviridae. Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3′ termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. Cis-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV–PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.
