In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV). (1/317)

Biochemical and structural characterization studies on the p7 putative movement protein from a Spanish isolate of carnation mottle carmovirus (CarMV) have been conducted. The CarMV p7 gene was fused to a sequence coding for a six-histidine tag and expressed in bacteria, allowing the purification of CarMV p7 and the production of a specific antiserum. This antiserum led to the immunological identification of CarMV p7 in infected leaf tissue from the experimental host Chenopodium quinoa. Putative nucleic acid-binding properties of the CarMV p7 have been explored and demonstrated with both electrophoretic mobility shift and RNA-protein blot in vitro assays using digoxigenin-labeled riboprobes. CarMV p7 did not show preferential binding to any of the different regions of the CarMV genomic RNA tested, suggesting that RNA binding was sequence nonspecific. Quantitative analyses of the data allowed calculation of the apparent dissociation constant of the p7-RNA complex (Kd approximately 0.7 microM) and supported a cooperative type of binding. A small 19-amino-acid synthetic peptide whose sequence corresponds to the putative RNA-binding domain of CarMV p7, at the basic central part of the protein, was synthesized, and it was demonstrated that it binds viral RNA probes. Peptide RNA binding was as stable as p7 binding, although data indicated it was not cooperative, thus suggesting that this cooperative binding requires another motif or motifs within the p7 amino acid sequence. The peptide could be induced to fold into an alpha-helix structure in which amino acids that are conserved among carmovirus p7-like proteins are distributed on one side. This alpha-helix motif could define a new and previously uncharacterized RNA-binding domain for plant virus movement proteins.  (+info)

Tobacco mosaic virus: a pioneer of cell-to-cell movement. (2/317)

Cell-to-cell movement of tobacco mosaic virus (TMV) is used to illustrate macromolecular traffic through plant intercellular connections, the plasmodesmata. This transport process is mediated by a specialized viral movement protein, P30. In the initially infected cell, P30 is produced by transcription of a subgenomic RNA derived from the invading virus. Presumably, P30 then associates with a certain proportion of the viral RNA molecules, sequestering them from replication and mediating their transport into neighbouring uninfected host cells. This nucleoprotem complex is targeted to plasmodesmata, possibly via interaction with the host cell's cytoskeleton. Prior to passage through a plasmodesma, the plasmodesmatal channel is dilated by the movement protein. It is proposed that targeting of P30-TMV RNA complexes to plasmodesmatal involves binding to a specific cell-wall-associated receptor molecule. This protein, designated p38, also functions as a protein kinase, phosphorylating P30 at its carboxy-terminus and minimizing P30-induced interference with plasmodesmatal permeability during viral infection.  (+info)

Mutagenesis of amino acids at two tomato ringspot nepovirus cleavage sites: effect on proteolytic processing in cis and in trans by the 3C-like protease. (3/317)

Tomato ringspot nepovirus (ToRSV) encodes two polyproteins that are processed by a 3C-like protease at specific cleavage sites. Analysis of ToRSV cleavage sites identified previously and in this study revealed that cleavage occurs at conserved Q/(G or S) dipeptides. In addition, a Cys or Val is found in the -2 position. Amino acid substitutions were introduced in the -6 to +1 positions of two ToRSV cleavage sites: the cleavage site between the protease and putative RNA-dependent RNA polymerase, which is processed in cis, and the cleavage site at the N-terminus of the movement protein, which is cleaved in trans. The effect of the mutations on proteolytic processing at these sites was tested using in vitro translation systems. Substitution of conserved amino acids at the -2, -1, and +1 positions resulted in a significant reduction in proteolytic processing at both cleavage sites. The effects of individual substitutions were stronger on the cleavage site processed in trans than on the one processed in cis. The cleavage site specificity of the ToRSV protease is discussed in comparison to that of related proteases.  (+info)

Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2. (4/317)

The gene encoding the 36.5 kDa ('36K') nonstructural protein located on RNA3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichia coli pGEX-2T system and the purified protein used to raise a polyclonal antiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plants showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. In infected cells there were tubular structures, some containing virus-like particles, scattered in the cytoplasm or protruding from or penetrating the cell wall at the plasmodesmata. Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules. Leaf trichome cells of N. tabacum plants, transformed with a 36K-green fluorescent protein (GFP) fusion construct, revealed localized fluorescence in the cell walls, possibly due to association of the fusion protein with plasmodesmata. When the same 36K-GFP fusion protein was expressed in N. tabacum protoplasts, long tubular fluorescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is drawn that this protein is likely to be the OLV-2 movement protein, mediating cell-to-cell virus movement, and that movement is by a tubule-guided mechanism.  (+info)

Both RNA rearrangement and point mutation contribute to repair of defective chimeric viral genomes to form functional hybrid viruses in plants. (5/317)

The putative movement protein gene (p27) plus 5' and 3' flanking sequences of cucumber leaf spot aureusvirus (CLSV) was inserted into an infectious cucumber necrosis tombusvirus (CNV) cDNA clone containing a deletion in the cell-to-cell movement protein gene. Approximately 5% of plants inoculated with synthetic transcripts of two such defective chimeric CNV/CLSV cDNA clones developed systemic symptoms 7-19 days postinoculation. Reverse transcription-polymerase chain reaction and sequence analysis of virus obtained from systemically infected leaves indicated that both point mutation and RNA rearrangement (deletion) contributed to the formation of movement competent CNV/CLSV hybrid viruses. The hybrid viruses were found to accumulate to high levels in infected plants, to form stable virions, and to be mechanically transmissible. In addition, a hybrid virus that lacked 50 amino acids at the carboxyl-terminal region of CLSV p27 was still capable of facilitating CNV movement. These data provide experimental evidence for the role of CLSV p27 in viral cell-to-cell movement and demonstrate that p27 can enable efficient movement of the CNV genome. Moreover, the data show that RNA rearrangements known to occur during CNV RNA replication can contribute to rapid evolution of the CNV genome.  (+info)

The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread. (6/317)

The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.  (+info)

Effect of C-terminal deletions in the movement protein of cowpea chlorotic mottle virus on cell-to-cell and long-distance movement. (7/317)

In order to elucidate the function of the C-terminal region of cowpea chlorotic mottle bromovirus (CCMV) movement protein (MP) in cell-to-cell movement, a set of deletions ranging from 10 to 80 amino acids (deltaMP10, deltaMP20, deltaMP33, deltaMP43, deltaMP60 and deltaMP80) was engineered into the MP gene encoded by the biologically active clone C3/deltaCP-EGFP, a variant of CCMV RNA3 that contained wild-type (wt) MP and the enhanced green fluorescent protein (EGFP) gene in place of the coat protein (CP). The effect of each MP deletion on cell-to-cell movement was examined in three susceptible host plants: Chenopodium quinoa, Nicotiana benthamiana and cowpea (Vigno sinensis cv. Black Eye). The results indicate that, except for mutant deltaMP43, infections resulting from the deletion mutants remained subliminal. Interestingly, infections resulting from inoculating mutant deltaMP43, which lacked the 43 most C-terminal amino acids, spread rapidly between cells and the number of infected cells expressing EGFP approached that of control inoculations made with C3/deltaCP-EGFP. To verify whether the presence of wt CP altered the movement behaviour of these mutants, each MP deletion was also incorporated into the genetic background of wt CCMV RNA3 (pCC3) and inoculated independently to all three hosts. The results suggest that the overall movement process exhibited by each MP mutant is influenced profoundly by the presence of CP and the particular host plant tested.  (+info)

Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. (8/317)

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.  (+info)