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Stuart A. Macfarlane - One of the best experts on this subject based on the ideXlab platform.
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Immunogold localization of Tobravirus 2b nematode transmission helper protein associated with virus particles.
Virology, 2002Co-Authors: Evangelos Vellios, Derek J. F. Brown, George H. Duncan, Stuart A. MacfarlaneAbstract:Transmission of the Tobraviruses Tobacco rattle virus (TRV) and Pea early-browning virus (PEBV) by trichodorid vector nematodes requires the viral coat protein (CP) and the 2b protein, a nonstructural protein encoded by RNA2, the smaller of the two viral genomic RNAs. It is hypothesized that the 2b protein functions by interacting with a small, flexible domain located at the C-terminus of the CP, forming a bridge between the virus particle and the internal surface of the vector nematode feeding apparatus. Antibodies specific for the 2b protein of PEBV or TRV did not bind to virus particles that were adsorbed to electron microscope grids and were not able to trap virus particles from extracts of infected plants. However, electron microscopy of thin sections of plants infected with PEBV probed with 2b-specific antibodies which were further gold-labeled showed that the 2b protein localizes exclusively to virus particles. Similarly, immunogold localization studies showed that the 2b protein of TRV isolate PaY4 is associated only with TRV PaY4 virus particles. When a recombinant TRV encoding the PaY4 2b protein and the CP from TRV isolate PpK20 was examined, the 2b protein could not be detected by Western blotting and in IGL experiments was not associated with virus particles. These results suggest that in the absence of a specific interaction between compatible CP and 2b proteins, the 2b protein does not accumulate.
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Tobravirus 2b protein acts in trans to facilitate transmission by nematodes.
Virology, 2001Co-Authors: N. Vassilakos, Derek J. F. Brown, Evangelos Vellios, E.c. Brown, Stuart A. MacfarlaneAbstract:Abstract Analysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3′-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the Tobravirus 2b transmission protein is trans -acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20.
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Efficient Expression of Foreign Proteins in Roots from Tobravirus Vectors
Virology, 2000Co-Authors: Stuart A. Macfarlane, Alexandra H. PopovichAbstract:Viral vectors were constructed from infectious cDNA clones of each of the three Tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP) when inoculated to plants. The Tobravirus expression vectors have a wide host range and were able to express GFP in, for example, Nicotiana species, tomato, pea, arabidopsis, and sugar beet. The TRV vector was able to invade and express GFP very efficiently in roots, whereas the widely used PVX vector was not.
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gene silencing without dna rna mediated cross protection between viruses
The Plant Cell, 1999Co-Authors: Frank Ratcliff, Stuart A. Macfarlane, David C BaulcombeAbstract:Previously, it was shown that the upper leaves of plants infected with nepoviruses and caulimoviruses are symptom free and contain reduced levels of virus. These leaves are said to be recovered. Recovery is associated with RNA-mediated cross-protection against secondary virus infection. Here, by analyzing plants infected with viruses that are quite distinct from the nepovirus or caulimovirus groups, we demonstrate that this RNA-mediated defense is a general response to virus infection. Upon infection with a Tobravirus, plants exhibited RNA-mediated cross-protection and recovery, as occurs in nepovirus-infected plants. However, upon infection with a potexvirus, plants exhibited RNA-mediated cross-protection without recovery. In both instances, a transient gene expression assay showed that RNA-mediated cross-protection was functionally equivalent to post-transcriptional gene silencing. Combined, these data provide direct evidence that post-transcriptional gene silencing of nuclear genes is a manifestation of a natural defense mechanism that is induced by a wide range of viruses.
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Molecular biology of the Tobraviruses
1999Co-Authors: Stuart A. MacfarlaneAbstract:The genus Tobravirus comprises of three different viruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV) and pepper ringspot virus (PepRSV) (Harrison, 1973; Robinson & Harrison, 1989a, b). The Tobravirus genome is divided int
Joh F Atkins - One of the best experts on this subject based on the ideXlab platform.
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stimulation of stop codon readthrough frequent presence of an extended 3 rna structural element
Nucleic Acids Research, 2011Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.
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Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element
Nucleic acids research, 2011Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.
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Stimulation of stop codon readthrough: frequent presence of an extended 39 RNA structural element
2011Co-Authors: Andrew E Firth, Norma M Wills, Raymond F. Gestel, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 30-adjacent to the UGA. However, analysis of vari-ability at synonymous sites revealed strikingly enhanced conservation within the 150nt 30-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computa-tional analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 30 RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis
Andrew E Firth - One of the best experts on this subject based on the ideXlab platform.
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stimulation of stop codon readthrough frequent presence of an extended 3 rna structural element
Nucleic Acids Research, 2011Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.
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Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element
Nucleic acids research, 2011Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.
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Stimulation of stop codon readthrough: frequent presence of an extended 39 RNA structural element
2011Co-Authors: Andrew E Firth, Norma M Wills, Raymond F. Gestel, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 30-adjacent to the UGA. However, analysis of vari-ability at synonymous sites revealed strikingly enhanced conservation within the 150nt 30-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computa-tional analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 30 RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis
John F. Bol - One of the best experts on this subject based on the ideXlab platform.
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Nematode transmission of tobacco rattle virus serves as a bottleneck to clear the virus population from defective interfering RNAs.
Virology, 1999Co-Authors: Peter B. Visser, Derek J. F. Brown, Frans Th. Brederode, John F. BolAbstract:DI7 is a defective interfering RNA derived from RNA 2 of tobacco rattle Tobravirus (TRV) isolate PpK20. Tobacco was transformed with DI7 cDNA fused to the CaMV 35S promoter. Upon infection of the transgenic plants with TRV isolate PpK20 or the serologically unrelated isolate PaY4, the transgenic DI7 RNA started to accumulate at high levels and strongly interfered with accumulation of wild-type (wt) RNA 2. When DI7 transgenic plants infected with isolate PpK20 were used as source plants in nematode-transmission experiments, the vector Paratrichodorus pachydermus efficiently transmitted virus to healthy bait plants. However, the nematodes transmitted only the wt virus present in the transgenic source plants, whereas virus particles containing the abundant, accumulated DI7 RNA were excluded from transmission. Evidence is presented that wt RNA 2 and DI7 RNA are encapsidated in cis by their encoded CPs, which are known to be functional and nonfunctional in transmission, respectively. This mechanism would result in defective interfering RNAs, which rapidly arise after mechanical transmission of the virus in the laboratory, being eliminated from Tobraviruses under natural field conditions. Also this mechanism which acts with nematode transmitted virus isolates contrasts with that of vector-transmission of defective potyviruses and luteoviruses by wt helper viruses.
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Heterologous encapsidation of recombinant pea early browning virus
Journal of General Virology, 1994Co-Authors: Stuart A. Macfarlane, A Mathis, John F. BolAbstract:The coat protein gene of pea early browning virus (PEBV) was replaced with that of another Tobravirus, tobacco rattle virus (TRV strain PPK20). The recombinant virus multiplied efficiently in the systemic host Nicotiana benthamiana and, on the local lesion host Phaseolus vulgaris, produced symptoms typical of PEBV rather than TRV showing that viral coat protein is not a determinant for lesion morphology. Both viral RNAs were encapsidated by TRV coat protein although the shorter particles (encapsidated RNA-2) did not form a discrete population. Evidence is presented to suggest involvement of nucleotide sequences upstream of the coat protein gene in virus particle assembly.
Norma M Wills - One of the best experts on this subject based on the ideXlab platform.
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stimulation of stop codon readthrough frequent presence of an extended 3 rna structural element
Nucleic Acids Research, 2011Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.
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Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element
Nucleic acids research, 2011Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.
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Stimulation of stop codon readthrough: frequent presence of an extended 39 RNA structural element
2011Co-Authors: Andrew E Firth, Norma M Wills, Raymond F. Gestel, Joh F AtkinsAbstract:In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 30-adjacent to the UGA. However, analysis of vari-ability at synonymous sites revealed strikingly enhanced conservation within the 150nt 30-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computa-tional analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 30 RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis
