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Lee Gehrke - One of the best experts on this subject based on the ideXlab platform.
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Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent
Journal of virology, 2005Co-Authors: Laura M. Guogas, Siana M. Laforest, Lee GehrkeAbstract:Alfalfa Mosaic Virus (AMV) and ilarVirus RNAs are infectious only in the presence of the viral coat protein; therefore, an understanding of coat protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV coat protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that coat protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining coat protein's function in the earliest stages of AMV replication. To detect coat-protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by coat protein is concentration dependent; that is, replication was strongly stimulated at low coat protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing coat protein mRNA translation or disrupting coat protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of coat protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that coat protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local coat protein concentrations and low coat protein occupancy on the multiple binding sites present in the 3' untranslated regions of the viral RNAs.
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rna determinants of a specific rna coat protein peptide interaction in Alfalfa Mosaic Virus conservation of homologous features in ilarVirus rnas
Journal of Molecular Biology, 1998Co-Authors: Patricia Anselmckinney, Lee GehrkeAbstract:Abstract Alfalfa Mosaic Virus (AMV) coat protein and tobacco streak Virus (TSV) coat protein bind specifically to the 3′ untranslated regions of the viral RNAs and are required with the genomic RNAs to initiate Virus replication. A combination of nucleotide substitutions, hydroxyl radical footprinting, and ethylation and chemical modification interference analysis has been used to define the RNA determinants important for the specific binding of the 3′-terminal 39 nucleotides of AMV RNA 3/4 (AMV 843–881 ) to an amino-terminal coat protein peptide (CP26). The results demonstrate that potential phosphate and base-specific contacts as well as ribose moieties protected upon peptide binding cluster in lower hairpin stems and flanking AUGC sequences of the viral RNA, without direct involvement of loop nucleotides. Nucleotides identified in the modification-interference analyses as important for RNA-protein interactions are highly conserved among AMV and the ilarVirus RNAs. This RNA sequence homology, coupled with the recent identification of an RNA binding consensus sequence for AMV and ilarVirus coat proteins, provides a framework for understanding the functional equivalence of AMV and TSV coat proteins in binding RNA and activating Virus replication and may explain why heterologous AMV and ilarVirus coat protein-RNA mixtures are infectious.
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viral coat protein peptides with limited sequence homology bind similar domains of Alfalfa Mosaic Virus and tobacco streak Virus rnas
Journal of Virology, 1998Co-Authors: M M Swanson, Patricia Anselmckinney, Lee Gehrke, Felicia Houserscott, Vidadi Yusibov, Sue L LoeschfriesAbstract:An unusual and distinguishing feature of Alfalfa Mosaic Virus (AMV) and ilarViruses such as tobacco streak Virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating Virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarVirus coat proteins.
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Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of Alfalfa Mosaic Virus RNA 4.
Journal of virology, 1994Co-Authors: F Houser-scott, M L Baer, K F Liem, J M Cai, Lee GehrkeAbstract:The specific binding of Alfalfa Mosaic Virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of Alfalfa Mosaic Virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of Alfalfa Mosaic Virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of Alfalfa Mosaic RNA sequences with those from several closely related ilarViruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that Alfalfa Mosaic Virus coat protein and ilaVirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements.
John F Bol - One of the best experts on this subject based on the ideXlab platform.
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RNA 3
2015Co-Authors: Vera Thole, Lyda Neeleman, Maria-laura Garcia, Clemens M. A. Van Rossum, Frans T. Brederode, Huub J. M. Linthorst, John F BolAbstract:RNAs 1 and 2 of the tripartite genome of Alfalfa Mosaic Virus (AMV) encode the two viral replicase subunits. Full-length DNA copies of RNAs 1 and 2 were used to transform tobacco plants (R12 lines). None of the transgenic lines showed resistance to AMV infection. In healthy R12 plants, the transcripts of the viral cDNAs were copied by the transgenic viral replicase into minus-strand RNAs but sub-sequent steps in replication were blocked. When the R12 plants were inoculated with AMV RNA 3, this block was lifted and the transgenic RNAs 1 and 2 were amplified by the transgenic replicase together with RNA 3. The transgenic expression of RNAs 1 and 2 largely circumvented the role of coat protein (CP) in the inoculum that is required fo
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formation of unusually long Virus particles
2013Co-Authors: Vera Thole, Roberto Miglino, John F BolAbstract:Amino acids of Alfalfa Mosaic Virus coat protein that direc
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coat protein enhances translational efficiency of Alfalfa Mosaic Virus rnas and interacts with the eif4g component of initiation factor eif4f
Journal of General Virology, 2005Co-Authors: Ivo M Krab, Daniel R Gallie, Christian Caldwell, John F BolAbstract:The three plus-strand genomic RNAs of Alfalfa Mosaic Virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc-AMV), a poly(A) tail of 50 residues [Luc-poly(A)] or a short vector-derived sequence (Luc-control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc-poly(A) or Luc-control, but strongly stimulated Luc expression from Luc-AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc-AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc-AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.
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ability of tobacco streak Virus coat protein to substitute for late functions of Alfalfa Mosaic Virus coat protein
Journal of Virology, 1995Co-Authors: Chantal B E M Reusken, Lyda Neeleman, John F BolAbstract:The coat protein (CP) of tobacco streak Virus (TSV) can substitute for the early function of Alfalfa Mosaic Virus (AIMV) CP in genome activation. Replacement of the CP gene in AIMV RNA 3 with the TSV CP gene and analysis of the replication of the chimeric RNA indicated that the TSV CP could not substitute for the function of AIMV CP in asymmetric plus-strand RNA accumulation but could encapsidate the chimeric RNA and permitted a low level of cell-to-cell transport.
Patricia Anselmckinney - One of the best experts on this subject based on the ideXlab platform.
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rna determinants of a specific rna coat protein peptide interaction in Alfalfa Mosaic Virus conservation of homologous features in ilarVirus rnas
Journal of Molecular Biology, 1998Co-Authors: Patricia Anselmckinney, Lee GehrkeAbstract:Abstract Alfalfa Mosaic Virus (AMV) coat protein and tobacco streak Virus (TSV) coat protein bind specifically to the 3′ untranslated regions of the viral RNAs and are required with the genomic RNAs to initiate Virus replication. A combination of nucleotide substitutions, hydroxyl radical footprinting, and ethylation and chemical modification interference analysis has been used to define the RNA determinants important for the specific binding of the 3′-terminal 39 nucleotides of AMV RNA 3/4 (AMV 843–881 ) to an amino-terminal coat protein peptide (CP26). The results demonstrate that potential phosphate and base-specific contacts as well as ribose moieties protected upon peptide binding cluster in lower hairpin stems and flanking AUGC sequences of the viral RNA, without direct involvement of loop nucleotides. Nucleotides identified in the modification-interference analyses as important for RNA-protein interactions are highly conserved among AMV and the ilarVirus RNAs. This RNA sequence homology, coupled with the recent identification of an RNA binding consensus sequence for AMV and ilarVirus coat proteins, provides a framework for understanding the functional equivalence of AMV and TSV coat proteins in binding RNA and activating Virus replication and may explain why heterologous AMV and ilarVirus coat protein-RNA mixtures are infectious.
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viral coat protein peptides with limited sequence homology bind similar domains of Alfalfa Mosaic Virus and tobacco streak Virus rnas
Journal of Virology, 1998Co-Authors: M M Swanson, Patricia Anselmckinney, Lee Gehrke, Felicia Houserscott, Vidadi Yusibov, Sue L LoeschfriesAbstract:An unusual and distinguishing feature of Alfalfa Mosaic Virus (AMV) and ilarViruses such as tobacco streak Virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating Virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarVirus coat proteins.
K F Smith - One of the best experts on this subject based on the ideXlab platform.
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Assessment of nutritional characteristics of Virus-resistant transgenic white clover (Trifolium repens L.) grown under field and glasshouse conditions
Molecular Breeding, 2015Co-Authors: A. Lucas, Simone Rochfort, K F Smith, S. Panter, A. Mouradov, G. SpangenbergAbstract:White clover ( Trifolium repens L.) is an important pasture legume in temperate areas throughout the world, providing fodder for grazing animals and improving soil fertility via symbiotic nitrogen fixation. However, the persistence and stress tolerance of white clover are affected by a number of Viruses including Alfalfa Mosaic Virus. Transgenic white clover plants with ectopic expression of the Alfalfa Mosaic Virus coat protein were resistant to the Virus under field and greenhouse conditions. With all genetic modifications of major consequence, there is the possibility of unintended effects on forage quality and natural toxicant levels. In this paper, we describe the evaluation of a range of parameters related to the nutritive value of white clover herbage to grazing animals and a suite of naturally occurring secondary metabolites that have the potential to be natural toxicants in transgenic white clover plants and wild-type control plants with a similar genetic background. Samples were collected from plants grown under both field and glasshouse conditions. Several commercial cultivars were included for comparison. Although there was plant-to-plant variation, as expected from an obligate outcrossing species, there were no significant differences in the range of this variation between transgenic and wild-type plants. Furthermore, no consistent significant differences were found between groups of transgenic and wild-type plants from the same generation, when mean nutritional parameters (crude protein, in vitro dry matter digestibility, neutral detergent fibre and water-soluble carbohydrates) and natural toxicants (cyanogenic glucosides, phytoestrogens and saponins) were compared.
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molecular breeding of transgenic white clover trifolium repens l with field resistance to Alfalfa Mosaic Virus through the expression of its coat protein gene
Transgenic Research, 2012Co-Authors: S. Panter, Simone Rochfort, A. Mouradov, P W G Chu, Emma Ludlow, R Garrett, R Kalla, M Z Z Jahufer, A De Lucas Arbiza, K F SmithAbstract:Viral diseases, such as Alfalfa Mosaic Virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T0 and T1 generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T4 generation in an agronomically elite ‘Grasslands Sustain’ genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T4 generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.
C R Grau - One of the best experts on this subject based on the ideXlab platform.
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seasonal progression symptom development and yield effects of Alfalfa Mosaic Virus epidemics on soybean in wisconsin
Plant Disease, 2007Co-Authors: E E Mueller, C R GrauAbstract:The occurrence of Alfalfa Mosaic Virus (AMV) has increased in Wisconsin soybean fields in conjunction with the establishment of the soybean aphid (Aphis glycines). Field studies were conducted to determine the seasonal incidence of AMV-infected plants, progression of symptom severity caused by AMV, and the effect of AMV on soybean yield and seed quality. An isolate of AMV, collected from field-grown soybean, was introduced into plots by mechanical inoculation of plants at the V1 growth stage. The goal of the experiment was to achieve an incidence of AMV-infected plants of 0, 50, and 100% in 2002, and 0, 10, 25, 50, 75, and 100% in 2003. Severity of foliar symptoms was rated, and leaf samples were collected for serological assay (enzyme-linked immunosorbent assay [ELISA]) to estimate the incidence of AMV-infected plants from growth stages VC to R5. The maximum incidence of AMV-infected plants was 49% at growth stage R5, yet detection of the Virus by ELISA dropped dramatically thereafter in both years. Incidence of AMV-infected plants accounted for 53 to 67% of the variability observed for severity of foliar symptoms in 2002 and 2003, respectively. Maximum yield loss ranged from 32% in 2002 to 48% in 2003 based on the difference in yield between noninoculated plots and plots with the highest incidence of AMV-infected plants. Incidence of AMV-infected plants explained 31% of the variation in yield in 2002 and 26% in 2003. An AMV incidence of 30% or greater was required for yield loss caused by AMV. Results of this study suggest that AMV has the potential to lower soybean yield and warrants further study.
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first report of transmission of soybean Mosaic Virus and Alfalfa Mosaic Virus by aphis glycines in the new world
Plant Disease, 2001Co-Authors: John H Hill, R Alleman, D. B. Hogg, C R GrauAbstract:The recent discovery of the soybean aphid, Aphis glycines Matsamura, in the North Central region of the United States is significant because it is the first time that a soybean-colonizing aphid has been detected in the New World. Although the aphid has the potential to cause physiological loss of up to 52% on soybeans (4), it can also transmit Soybean Mosaic Virus (SMV). Transmission of Alfalfa Mosaic Virus (AMV) has not been reported. SMV, and less commonly AMV, are found in soybeans in the North Central states and are transmitted by numerous aphids in a nonpersistent manner (2; Grau, unpublished). For SMV, potential exists for specificity of transmission between Virus strain and aphid species (3). For these reasons, it was important to determine if an endemic isolate of these Viruses could be transmitted by this introduced species of aphid in the North Central region. Transmission experiments were conducted as described (3), using 3, 5, and 10 aphids per plant. Ten plants of the soybean cultivar William...
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first report of transmission of soybean Mosaic Virus and Alfalfa Mosaic Virus by aphis glycines in the new world
Plant Disease, 2001Co-Authors: John H Hill, R Alleman, D. B. Hogg, C R GrauAbstract:The recent discovery of the soybean aphid, Aphis glycines Matsamura, in the North Central region of the United States is significant because it is the first time that a soybean-colonizing aphid has been detected in the New World. Although the aphid has the potential to cause physiological loss of up to 52% on soybeans (4), it can also transmit Soybean Mosaic Virus (SMV). Transmission of Alfalfa Mosaic Virus (AMV) has not been reported. SMV, and less commonly AMV, are found in soybeans in the North Central states and are transmitted by numerous aphids in a nonpersistent manner (2; Grau, unpublished). For SMV, potential exists for specificity of transmission between Virus strain and aphid species (3). For these reasons, it was important to determine if an endemic isolate of these Viruses could be transmitted by this introduced species of aphid in the North Central region. Transmission experiments were conducted as described (3), using 3, 5, and 10 aphids per plant. Ten plants of the soybean cultivar Williams 82 were used for each treatment. To preclude confounding results by possible seed transmission, plants used in all tests were grown from seeds harvested from Virus-indexed plants grown in the greenhouse. For experiments involving SMV, the aphid-transmissible field isolate Al5 (GeneBank Accession no. AF242844) and, as a negative control, the non-aphid transmissible isolate N (GeneBank Accession no. D500507) were used. For experiments involving AMV, a field isolate of AMV, confirmed by ELISA and host range, was used. The aphid species Myzus persicae was maintained on broad bean and A. glycines was maintained on Virus-free soybean. The protocol for transmission studies of AMV was identical to that used in the SMV study, except only A. glycines was tested. For experiments, plants were periodically observed for symptom development and tested by ELISA 4 to 5 weeks after inoculation access. No transmission of SMV-N occurred in any tests, which together involved 180 aphids each of M. persicae or A. glycines. For the Al5 isolate, transmission efficiencies of 30, 50, and 50% were obtained with 3, 5, and 10 individuals, respectively, of M. persicae per plant. Efficiencies for A. glycines were 30, 40, and 40%. Transmission levels by the two aphid species did not differ significantly (t-test, P = 0.01). For AMV, corresponding transmission efficiencies were 0, 0, and 20%. The data suggest that the introduced A. glycines can be an efficient vector of SMV, but a less efficient vector of AMV, in the North Central region. Transmission of AMV by M. persicae has been documented (1) but was not examined in this study. Transmission of SMV and AMV by A. glycines is of concern because it may increase SMV and AMV incidence. With the recent outbreak of Bean pod mottle Virus (BPMV) in the region, the potential for synergism of SMV and BPMV is increased (2). References: (1) M. B. Castillo and G. G. Orlob. Phytopathology 56:1028, 1966. (2) G. L. Hartman et al., eds. 1999. Compendium of Soybean Diseases, 4th Ed. American Phytopathological Society, St. Paul, MN. (3) B. S. Lucas and J. H. Hill. Phytopathol. Z. 99:47, 1980. (4) C. L. Wang et al. Plant Prot. 20:12, 1994.