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1.
Transgenic Res ; 19(6): 987-99, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20191320

ABSTRACT

Soybean was used as a model for studies of chemical induction of gene expression in seeds. A chimeric transcriptional activator, VGE, driven by the soybean seed glycinin G1 promoter, was used to induce the expression of an ER-targeted GFP(KDEL) reporter protein upon addition of the chemical ligand, methoxyfenozide. The chemical gene switch activated gene expression under in vitro conditions in somatic cotyledonary embryos and zygotic seed embryos cultured from transgenic soybean plants, as well as in seeds in planta under greenhouse conditions. The efficiency of induction of GFP expression under different growth conditions was strongly influenced by the developmental stage of the seed and availability of the inducer. The formation of ER-derived GFP-containing protein bodies in seed storage parenchyma cells was correlated with the level of induced expression.


Subject(s)
Ecdysone/metabolism , Glycine max/genetics , Glycine max/metabolism , Base Sequence , DNA Primers/genetics , Endoplasmic Reticulum/metabolism , Gene Expression/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hydrazines/pharmacology , Juvenile Hormones/pharmacology , Plants, Genetically Modified , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seeds/genetics , Glycine max/drug effects , Glycine max/embryology , Trans-Activators/genetics , Trans-Activators/metabolism
2.
Science ; 327(5967): 833-4, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-20150494

ABSTRACT

Population growth, arable land and fresh water limits, and climate change have profound implications for the ability of agriculture to meet this century's demands for food, feed, fiber, and fuel while reducing the environmental impact of their production. Success depends on the acceptance and use of contemporary molecular techniques, as well as the increasing development of farming systems that use saline water and integrate nutrient flows.


Subject(s)
Agriculture/trends , Crops, Agricultural , Agriculture/legislation & jurisprudence , Agriculture/methods , Aquaculture/methods , Aquaculture/trends , Biotechnology , Climate Change , Food, Genetically Modified , Government Regulation , Population Growth , Private Sector , Public Sector , United States , United States Department of Agriculture
3.
Proc Natl Acad Sci U S A ; 104(29): 12157-62, 2007 Jul 17.
Article in English | MEDLINE | ID: mdl-17615233

ABSTRACT

Infections by plant virus generally cause disease symptoms by interfering with cellular processes. Here we demonstrated that infection of Nicotiana tabacum (N.t) by plant viruses representative of the Tobamoviridae, Potyviridae, and Potexviridae families altered accumulation of certain microRNAs (miRNAs). A correlation was observed between symptom severity and alteration in levels of miRNAs 156, 160, 164,166, 169, and 171 that is independent of viral posttranscriptional gene silencing suppressor activity. Hybrid transgenic plants that produced tobacco mosaic virus (TMV) movement protein (MP) plus coat protein (CP)(T42W) (a variant of CP) exhibited disease-like phenotypes, including abnormal plant development. Grafting studies with a plant line in which both transgenes are silenced confirmed that the disease-like phenotypes are due to the coexpression of CP and MP. In hybrid MPxCP(T42W) plants and TMV-infected plants, miRNAs 156, 164, 165, and 167 accumulated to higher levels compared with nontransgenic and noninfected tissues. Bimolecular fluorescence complementation assays revealed that MP interacts with CP(T42W) in vivo and leads to the hypothesis that complexes formed between MP and CP caused increases in miRNAs that result in disease symptoms. This work presents evidence that virus infection and viral proteins influence miRNA balance without affecting posttranscriptional gene silencing and contributes to the hypothesis that viruses exploit miRNA pathways during pathogenesis.


Subject(s)
Capsid Proteins/metabolism , MicroRNAs/metabolism , Nicotiana/growth & development , Nicotiana/virology , Plant Diseases/virology , Plant Viral Movement Proteins/metabolism , Tobacco Mosaic Virus/physiology , Gene Silencing , Phenotype , Plant Leaves/cytology , Plant Leaves/virology , Plants, Genetically Modified , Protein Binding , Nicotiana/genetics
4.
Virology ; 366(1): 98-106, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17493658

ABSTRACT

Tobacco mosaic virus (TMV) coat protein (CP) in absence of RNA self-assembles into several different structures depending on pH and ionic strength. Transgenic plants that produce self-assembling CP are resistant to TMV infection, a phenomenon referred to as coat-protein-mediated resistance (CP-MR). The mutant CP Thr42Trp (CP(T42W)) produces enhanced CP-MR compared to wild-type CP. To establish the relationship between the formation of 20S CP aggregates and CP-MR, virus-like particles (VLPs) produced by TMV variants that yield high levels of CP-MR were characterized. We demonstrate that non-helical structures are found in VLPs formed in vivo by CP(T42W) but not by wild-type CP and suggest that the mutation shifts the intracellular equilibrium of aggregates from low to higher proportions of non-helical 20S aggregates. A similar shift in equilibrium of aggregates was observed with CP(D77R), another mutant that confers high level of CP-MR. The mutant CP(D50R) confers a level of CP-MR similar to wild-type CP and aggregates in a manner similar to wild-type CP. We conclude that increased CP-MR is correlated with a shift in intracellular equilibrium of CP aggregates, including aggregates that interfere with virus replication.


Subject(s)
Capsid Proteins/physiology , Tobacco Mosaic Virus/physiology , Amino Acid Substitution , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cloning, Molecular , Cryoelectron Microscopy , DNA, Viral/genetics , Drug Resistance, Viral , Genetic Variation , Microscopy, Electron , Models, Molecular , Protein Conformation , Tobacco Mosaic Virus/ultrastructure , Transcription, Genetic
5.
Arch Virol ; 151(11): 2111-22, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16773235

ABSTRACT

The disease caused by rice yellow mottle virus (RYMV) is a major, economically important constraint to rice production in Africa. RYMV is mechanically transmitted by a variety of agents, including insect vectors. The production of resistant rice varieties would be an important advance in the control of the disease and increase rice production in Africa. We produced transgenic plants of the Oryza sativa japonica variety, TP309, to express a RYMV coat protein gene (CP) and mutants of the CP under the control of a ubiquitin promoter. Transgenic plants expressing genes that encode wild-type CP (wt.CP), deleted CP (DeltaNLS.CP), mRNA of the CP, or antisense CP sequences of the CP gene were characterised. Eighty per cent (80%) of independent transgenic lines analysed contained CP gene sequences. Transgenic plants were challenged with RYMV and produced two types of reactions. Most of the plants expressing antisense sequences of the CP and untranslatable CP mRNA exhibited a delay in virus accumulation of up to a week, and the level of virus accumulation was reduced compared with non-transgenic TP309 plants. Transgenic plants expressing RYMV wild-type CP (wt.CP) and deleted CP (DeltaNLS.CP) accumulated the highest levels of virus particles. These results suggest that antisense CP and untranslatable CP mRNA induced moderate resistance, whereas transgenic CP enhanced virus infection.


Subject(s)
Capsid Proteins/metabolism , Oryza/virology , Plant Diseases/virology , RNA Viruses/pathogenicity , Capsid Proteins/genetics , Gene Deletion , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/virology , RNA Viruses/genetics , RNA Viruses/metabolism , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
6.
J Gen Virol ; 87(Pt 4): 1005-1012, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16528051

ABSTRACT

Replication of Potato virus X (PVX) was reduced in transgenic protoplasts that accumulated wild-type coat protein (CPWT) of Tobacco mosaic virus (TMV) or a mutant CP, CP(T42W), that produced highly ordered states of aggregation, including pseudovirions. This reaction is referred to as heterologous CP-mediated resistance. However, protoplasts expressing a CP mutant that abolished aggregation and did not produce pseudovirions, CPT28W, did not reduce PVX replication. Similarly, in transgenic tobacco plants producing TMV CPWT or CP(T42W), there was a delay in local cell-to-cell spread of PVX infection that was not observed in CP(T28W) plants or in non-transgenic plants. The results suggest that the quaternary structure of the TMV CP regulates the mechanism(s) of heterologous CP-mediated resistance. Similarly, transgenic protoplasts that produced PVX CP conferred transient protection against infection by TMV RNA. Transgenic plants that accumulated PVX CP reduced the cell-to-cell spread of infection and resulted in a delay in systemic infection following inoculation with TMV or TMV RNA. Heterologous CP-mediated resistance was characterized by a brief delay in systemic infection, whilst homologous CP-mediated resistance conferred reduced or no systemic infection.


Subject(s)
Capsid Proteins/metabolism , Nicotiana/virology , Plant Diseases/virology , Plants, Genetically Modified , Potexvirus/pathogenicity , Tobacco Mosaic Virus/pathogenicity , Capsid Proteins/genetics , Cell Line , Potexvirus/genetics , Potexvirus/metabolism , Nicotiana/genetics , Tobacco Mosaic Virus/genetics , Tobacco Mosaic Virus/metabolism , Virus Replication
7.
Phytopathology ; 96(11): 1175-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-18943953

ABSTRACT

ABSTRACT The expression of a gene that encodes coat protein (CP) of Potato virus X (PVX) in transgenic tobacco plants confers a high level of CP-mediated rresistance (CP-MR) against PVX infection. To determine if posttranscriptional gene silencing (PTGS) plays a role in resistance, transgenic plants expressing PVX CP were challenged against PVX under conditions in which PTGS was suppressed by low temperatures or using viruses carrying PTGS suppressors. The data demonstrate that PTGS does not play a significant role in PVX CP-MR.

8.
Arch Virol ; 150(12): 2567-82, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16086100

ABSTRACT

The resistance of transgenic Nicotiana benthamiana plants encoding wild type, truncated and point mutants of the tobacco etch virus (TEV) coat protein (CP) was analyzed. After R1 plants from 45 transgenic lines were challenged with TEV, six percent of the lines exhibited high resistance, 38% exhibited low resistance, and the remainder were susceptible. The phenomenon of recovery and delay in symptom development was observed in 65% and 56% of the resistant and susceptible lines, respectively. Plants containing genes that encode sequences of two assembly-deficient mutants of TEV-CPDelta1-63 exhibited resistance to infection, suggesting that self-assembly of the CP is not responsible for resistance. Highly resistant lines accumulated low levels of transgene mRNA and non-detectable amounts of protein, and tissues accumulated lower amounts of transgene mRNA following recovery than before infection. In addition, co-suppression of replication of a recombinant tobamovirus containing the TEV-CPDelta1-63 sequence was observed in several lines, suggesting homology-dependent degradation of RNA, most likely through induction of post-transcriptional gene silencing. Plants not exhibiting high resistance via gene silencing exhibited moderate levels of resistance that is attributed to and/or affected by the CP molecule.


Subject(s)
Capsid Proteins/genetics , Nicotiana/virology , Plants, Genetically Modified/virology , Potyvirus/pathogenicity , Capsid Proteins/analysis , Gene Expression , Plant Diseases/virology , Point Mutation , Potyvirus/genetics , RNA Interference , RNA Stability , RNA, Messenger/analysis , RNA, Viral/analysis , Sequence Deletion , Virus Assembly/genetics
9.
Arch Virol ; 149(4): 699-712, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15045558

ABSTRACT

The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli. To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CPDelta63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of alpha-helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.


Subject(s)
Capsid Proteins/metabolism , Potyvirus/metabolism , RNA, Viral/metabolism , Capsid Proteins/chemistry , Capsid Proteins/genetics , Escherichia coli/metabolism , Point Mutation , Potyvirus/chemistry , Potyvirus/genetics , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Nicotiana/virology
10.
Proc Natl Acad Sci U S A ; 101(5): 1415-20, 2004 Feb 03.
Article in English | MEDLINE | ID: mdl-14745003

ABSTRACT

The genome of tobacco mosaic virus (TMV) encodes replicase protein(s), movement protein (MP), and capsid protein (CP). On infection, one or more viral proteins direct the assembly of virus replication complexes (VRCs), in association with host-derived membranes. The impact of CP-mediated resistance on the structures of the replication complexes was examined in nontransgenic and transgenic BY-2 cell lines that produce wild-type CP, mutant CP(T42W), and Ds-Red, which was targeted to endoplasmic reticulum by using immunofluorescence and 3D microscopy. We developed a model of VRCs that shows a clear association of MP with and surrounding the endoplasmic reticulum. Replicase is located within the MP bodies, as well as isolated sites throughout the cell. CP surrounds the VRCs. CP enhances the production of MP and increases the size of the VRC; however, the mutant CP(T42W) reduces the amount of MP and interferes with the formation of VRCs. We propose a regulatory role of the CP in the establishment of the VRC. We suggest that the lack of formation of VRCs restricts the efficiency of virus replication and the formation of virus movement complexes, resulting in restriction of cell-cell spread of infection. This results in higher levels of plant CP-mediated protection provided by CP(T42W).


Subject(s)
Capsid Proteins/physiology , Tobacco Mosaic Virus/physiology , Virus Replication , Endoplasmic Reticulum/virology , Nicotiana/virology
11.
Science ; 302(5653): 2118-20, 2003 Dec 19.
Article in English | MEDLINE | ID: mdl-14684821

ABSTRACT

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C0t selection were sequenced to generate approximately 200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.


Subject(s)
Genes, Plant , Genome, Plant , Sequence Analysis, DNA/methods , Zea mays/genetics , Chromosomes, Plant/genetics , Cloning, Molecular , Computational Biology , Contig Mapping , DNA Methylation , DNA, Plant/genetics , Databases, Nucleic Acid , Expressed Sequence Tags , Gene Dosage , Gene Library , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid , Retroelements , Sequence Alignment , Transcription, Genetic
12.
Mol Plant Microbe Interact ; 14(7): 895-904, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11437263

ABSTRACT

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.


Subject(s)
Tobacco Mosaic Virus/physiology , Viral Proteins/physiology , Cucumis sativus/virology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microtubules/virology , Plant Diseases/virology , Plant Viral Movement Proteins , Plants, Genetically Modified , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Nicotiana/virology , Tobacco Mosaic Virus/genetics , Viral Proteins/genetics
13.
Proc Natl Acad Sci U S A ; 98(13): 7635-40, 2001 Jun 19.
Article in English | MEDLINE | ID: mdl-11390974

ABSTRACT

The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides -164 to +45, result in phloem-specific expression of beta-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2-20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.


Subject(s)
Badnavirus/genetics , Gene Expression Regulation, Viral/physiology , Glucuronidase/analysis , Nicotiana/genetics , Nicotiana/virology , Plants, Genetically Modified/virology , Plants, Toxic , Promoter Regions, Genetic , Trans-Activators/metabolism , Transcription Factors/metabolism , Agrobacterium tumefaciens/genetics , Basic-Leucine Zipper Transcription Factors , Caulimovirus/genetics , Cloning, Molecular , Escherichia coli , Genes, Reporter , Glucuronidase/genetics , Phenotype , Plant Leaves , Plant Proteins/metabolism , Recombinant Proteins/metabolism , Sequence Deletion , TATA Box , Nicotiana/physiology , Trans-Activators/genetics
14.
J Biol Chem ; 276(27): 25631-8, 2001 Jul 06.
Article in English | MEDLINE | ID: mdl-11342533

ABSTRACT

The minimal DNA binding domain of the replication-associated protein (Rep) of Tomato leaf curl New Delhi virus was determined by electrophoretic mobility gel shift analysis and co-purification assays. DNA binding activity maps to amino acids 1-160 (Rep-(1-160)) of the Rep protein and overlaps with the protein oligomerization domain. Transient expression of Rep protein (Rep-(1-160)) was found to inhibit homologous viral DNA accumulation by 70-86% in tobacco protoplasts and in Nicotiana benthamiana plants. The results obtained showed that expression of N-terminal sequences of Rep protein could efficiently interfere with DNA binding and oligomerization activities during virus infection. Surprisingly, this protein reduced accumulation of the African cassava mosaic virus, Pepper huasteco yellow vein virus and Potato yellow mosaic virus by 22-48%. electrophoretic mobility shift assays and co-purification studies showed that Rep-(1-160) did not bind with high affinity in vitro to the corresponding common region sequences of heterologous geminiviruses. However, Rep-(1-160) formed oligomers with the Rep proteins of the other geminiviruses. These data suggest that the regulation of virus accumulation may involve binding of the Rep to target DNA sequences and to the other Rep molecules during virus replication.


Subject(s)
DNA Helicases/genetics , DNA, Viral/metabolism , DNA-Binding Proteins , Geminiviridae/genetics , Trans-Activators/genetics , Viral Proteins/metabolism , Binding Sites , Chromosome Mapping , DNA Helicases/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Geminiviridae/physiology , Peptide Mapping , Polymers , Protein Conformation , Trans-Activators/metabolism , Virus Replication
15.
Structure ; 8(10): 1095-103, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11080631

ABSTRACT

BACKGROUND: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS: The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.


Subject(s)
Oryza/virology , Plant Viruses/chemistry , RNA Viruses/chemistry , Amino Acid Sequence , Binding Sites , Calcium/metabolism , Capsid/chemistry , Capsid/metabolism , Chromatography, Ion Exchange , Crystallography, X-Ray , Dimerization , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Plant Viruses/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary , RNA Viruses/metabolism , Sequence Alignment , Thermodynamics
16.
Proc Natl Acad Sci U S A ; 97(22): 12345-9, 2000 Oct 24.
Article in English | MEDLINE | ID: mdl-11050252

ABSTRACT

Despite its central role in virus infection, little is known about the mechanisms of intracellular trafficking of virus components within infected cells. In this study, we followed the dynamics of tobacco mosaic virus movement protein (MP) distribution in living protoplasts after disruption of microtubules (MTs) by cold treatment and subsequent rewarming to 29 degrees C. At early stages of infection, cold treatment (4 degrees C) caused the accumulation of MP fused to green fluorescent protein (GFP) in large virus replication bodies that localized in perinuclear positions, whereas at midstages of infection, the association of MP:GFP with MTs was disrupted. Rewarming the protoplasts to 29 degrees C reestablished the association of MTs with the replication bodies that subsequently spread throughout the cytoplasm and to the periphery of the cell. The role of MTs in the intracellular distribution of the MP also was analyzed by examining the distribution pattern of a nonfunctional mutant of MP (TAD5). Like MP:GFP, TAD5:GFP interacted with the endoplasmic reticulum membranes and colocalized with its viral RNA but did not colocalize with MTs. The involvement of MTs in the intracellular distribution of tobacco mosaic virus MP is discussed.


Subject(s)
Microtubules/physiology , Nicotiana/virology , Plants, Toxic , Viral Proteins/metabolism , Fluorescent Antibody Technique , Green Fluorescent Proteins , In Situ Hybridization , Luminescent Proteins/genetics , Microscopy, Confocal , Plant Viral Movement Proteins , RNA, Viral/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Nicotiana/ultrastructure , Viral Proteins/genetics
17.
J Mol Biol ; 303(2): 197-211, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-11023786

ABSTRACT

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.


Subject(s)
Cryoelectron Microscopy , Image Processing, Computer-Assisted , Plant Viruses/chemistry , Plant Viruses/ultrastructure , RNA Viruses/chemistry , RNA Viruses/ultrastructure , Amino Acid Sequence , Calcium/metabolism , Calcium/pharmacology , Capsid/chemistry , Capsid/drug effects , Capsid/ultrastructure , Cations, Divalent/metabolism , Cations, Divalent/pharmacology , Crystallography, X-Ray , Fabaceae/virology , Genome, Viral , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Oryza/virology , Plant Viruses/drug effects , Plant Viruses/genetics , Plants, Medicinal , RNA Viruses/drug effects , RNA Viruses/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Sequence Alignment , Virus Assembly/drug effects
18.
Virology ; 273(2): 341-50, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10915605

ABSTRACT

The DNA binding sites for the replication-associated protein (Rep) of two strains of tomato leaf curl virus from New Delhi (ToLCV-Nde) were identified using electrophoretic mobility shift assays (EMSAs). The Rep proteins of the two strains were found to exhibit sequence specificity in recognition of their cognate repeat motifs (iterons) in the origin, despite the fact that they share 91% sequence identity. Using a series of synthetic oligonucleotides as probes in EMSAs, the interaction of Rep protein with its binding site was found to be dependent on number, size, and sequence of the two iterons. Mutations in the sequence of the repeat motifs or alteration in the arrangement of the motifs compromised the ability of Rep protein to bind the DNA sequence and reduced accumulation of viral DNA in protoplasts, suggesting that binding of Rep protein to its cognate iterons is an essential step in virus replication. In addition, a difference in sequence of two base pairs in the binding site of two ToLCV-Nde strains was found to affect DNA binding by the corresponding Rep protein and replication of the virus DNA in protoplasts.


Subject(s)
DNA Helicases/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins , Geminiviridae/genetics , Geminiviridae/physiology , Trans-Activators/metabolism , Viral Proteins/metabolism , Virus Replication , Animals , DNA Helicases/chemistry , Electrophoresis, Polyacrylamide Gel , Replication Origin/genetics , Sequence Analysis, DNA , Spodoptera , Structure-Activity Relationship , Trans-Activators/chemistry , Viral Proteins/chemistry
19.
Proc Natl Acad Sci U S A ; 97(13): 7112-7, 2000 Jun 20.
Article in English | MEDLINE | ID: mdl-10840061

ABSTRACT

The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg/ml without aggregation and displayed approximately 70% alpha-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative alpha-helical transmembrane domains and a protease-sensitive carboxyl terminus.


Subject(s)
Membrane Proteins , RNA-Binding Proteins , Tobacco Mosaic Virus/genetics , Tobacco Mosaic Virus/metabolism , Viral Proteins , Amino Acid Sequence , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Plant Viral Movement Proteins , Protein Folding , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
20.
FEMS Immunol Med Microbiol ; 27(4): 291-7, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10727884

ABSTRACT

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.


Subject(s)
Bacterial Vaccines/immunology , Influenza A virus/genetics , Lung Diseases/prevention & control , Plant Viruses/genetics , Porins/immunology , Pseudomonas Infections/prevention & control , Pseudomonas aeruginosa/immunology , Animals , Antibodies, Bacterial/blood , Bacterial Vaccines/administration & dosage , Comovirus/genetics , Comovirus/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Influenza A virus/metabolism , Lung/microbiology , Lung Diseases/microbiology , Mice , Plant Viruses/metabolism , Porins/chemistry , Porins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Tobacco Mosaic Virus/genetics , Tobacco Mosaic Virus/metabolism , Vaccination , Vaccines, Combined/administration & dosage , Vaccines, Combined/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology
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