J.G. Atabekov

Beet yellows closterovirus HSP70-like protein mediates the cell-to-cell movement of a potexvirus transport-deficient mutant and a hordeivirus-based chimeric virus.

It has been suggested that the beet yellows closterovirus (BYV)-encoded p65 protein, a homologue of HSP70 cell chaperones, plays a role as a virus movement protein (MP). To test this hypothesis, we used two types of complementation experiments with plant viruses containing the triple gene block (TGB) of MP genes. In one, the BYV p65 gene was cloned into a 35S promoter plasmid and introduced into Nicotiana benthamiana plants by microprojectile bombardment along with the 35S promoter-driven GUS gene-tagged cDNA of a transport-deficient potexvirus mutant. Transient expression of p65 complemented the mutant as visualized by the significant increase in the number of cells expressing the GUS reporter gene in the infection foci. In the other test, the p65 gene was inserted into the infectious cDNA of the hordeivirus RNA beta component to replace either the 58 kDa MP gene or the whole TGB. Inoculation of Chenopodium quinoa and Chenopodium amaranticolor plants with the T7 transcripts of the chimeric RNA beta, together with the hordeivirus RNA alpha and RNA gamma, caused symptomless infection in inoculated leaves detected by hybridization of the total leaf RNA with a specific cDNA probe. The ability of BYV p65 to substitute for the potexvirus or hordeivirus MPs provides direct evidence for its involvement in the cell-to-cell movement of closterovirus infection.

Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants

Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr‐TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3′‐terminal region of the cr‐TMV RNA preceding the tRNA‐like structure was shown to form six potentially stable pseudoknots.

Movement protein-derived resistance to triple gene block- containing plant viruses

Two mutant potato virus X (PVX) movement protein (MP) genes (m 12K-Sal and m 12K-Kpn) were obtained by inserting specific linkers at the boundary between the N-terminal hydrophobic and putative transmembrane segment, and the central invariant hydrophilic region of the respective 12 kDa, 12K, triple gene block (TGB) protein. Several transgenic potato lines which expressed m 12K-Sal or m 12K-Kpn to different degrees were resistant to infection by PVX, potato aucuba mosaic potexvirus and the carlaviruses potato virus M and S over a wide range of inoculum concentrations (3-300 micrograms/ml). However, they were not resistant to potato virus Y, which lacks a TGB protein. We suggest that the resistance of m 12K-Sal and m 12K-Kpn transgenic potato lines is MP-derived and not RNA-mediated.

Effects of sequence elements in the potato virus X RNA 5' non-translated αβ-leader on its translation enhancing activity

The 5′ non-translated αβ-leader sequence of potato virus X RNA consists of two regions: the α sequence (41 nucleotides with no G) and the β sequence (42 nucleotides upstream from AUG). The αβ-leader has been shown to enhance strongly the expression of adjacent genes in chimeric mRNAs. This phenomenon has been postulated to be due to the unpaired conformation of the 5′-terminal 30 nucleotides and/or to the presence within the α region of the CCACC pentanucleotide complementary to the 3′-terminal conserved structure of 18S rRNA. Different derivatives of αβ-leader have been constructed for use in determining the contribution of separate elements of the αβ sequence to translational enhancement. It was found that deletion of the α sequence large fragment which was supposed to be unfolded did not reduce the Δαβ-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the β sequence resulted in a considerable increase in activity of the α-leader showing that the β region was dispensable for translation. Disruption or ‘masking’ of CCACC led to inactivation of the αβ-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of αβ-leader.

Specificity of protein-RNA and protein-protein interaction upon assembly of TMV in vivo and in vitro

The possibility of genomic masking and phenotypic mixing was studied in vivo (in plants mixedly infected by two TMV strains). The combinations of strains were: (1) vulgare and U2, (2) temperature-sensitive coat protein mutant Ni118 and U2. n nIn the experiments on mixed reconstitution the conditions of TMV maturation were modeled in vitro using combinations of RNAs and proteins from the same strains. n nCoating of viral RNA by the coat protein of a heterologous strain (termed genomic masking in vivo and mixed particle production in vitro) could be definitely demonstrated only when a doubly infected plant (or reconstituted mixture, respectively) contained two types of viral RNA and only one type of active protein (the protein of the second, temperature-sensitive strain was inactive at 33°). n nOn the other hand, the genomic masking and mixed-particle production was absent or very limited (within the limitation of the assay) when RNAs and functional proteins of both strains were present in a mixedly infected plant (in vivo) or in a reconstitution mixture (in vitro). Thus, a high specificity of protein-RNA interaction exists when both RNA and protein have access to the homologous component upon TMV assembly. The absence of phenotypically mixed particles with a mosaic capsid in doubly infected plants in vivo implies that the specificity of protein-protein interactions between subunits should also be high. However, some mixed coat protein virus with mosaic capsid could be reassembled in vitro.

Site-specific enzymatic cleavage of TMV RNA directed by deoxyribo- and chimeric (deoxyribo-ribo)oligonucleotides

The TMV RNA molecule can be cleaved at a single site by RNase H directed by chimeric oligo(deoxyribo‐ribo)nucleotide with an internucleotide pyrophosphate bond

Serological study on barley stripe mosaic virus protein polymerization: II. Comparative antigenic analysis of intact virus and some stable protein intermediates

Abstract Aggregates of barley stripe mosaic virus protein (BSMVp) beginning at the level of 10 S aggregate (i.e., 10 S, 20 S, 30 S, 40 S, etc.) are antigenically identical to each other and to BSMV. The monomeric BSMVp unit is serologically related to, but not identical with, the intact BSMV. The influence of quaternary structure of BSMVp on the conformation of polypeptide chain is discussed. The multiple line formation, with antibody in excess, by the mixtures of antigenically identical BSMVp and BSMV was demonstrated in double-diffusion tests.

Anomalous stable aggregates in mixture of TMV and cucumber virus 3 proteins

Abstract The mixing of TMV and cucumber virus 3 (CV3) proteins in the form of 4 S or 20 S aggregates results in their being rearranged to new forms with sedimentation coefficients of 11–16 S (13 S aggregate), and 18–23 S (20 S aggregate). Under certain conditions, either 13 S or 20 S aggregates are formed. In contrast to normal aggregates of virus protein, the 13 S and 20 S aggregates produced in mixtures of TMV and CV3 proteins were stable under conditions which ensured complete dissociation of normal 20 S aggregates of TMV and CV3 proteins into 4 S trimers. The 13 S and 20 S aggregates are probably mono- and double-layered disks, respectively. The stable aggregates, may contain mixtures of the protein subunits of both viruses. The 13 S aggregate can be transformed into a stable 20 S aggregate in an excess of 4 S TMV protein but not of CV3 protein. The stable aggregates are not capable of polymerizing into rodlike macromolecular viruslike structures at pH 5.6, or in the presence of nucleic acid.

Increase of histidine content in Brassica rapa subsp. oleifera by over-expression of histidine-rich fusion proteins

An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons.

Host-dependent Suppression of Temperature-sensitive Mutations in Tobacco Mosaic Virus Transport Gene

SummarynTobacco mosaic virus (TMV) mutants Ls1 and Ni2519, temperature-sensitive (ts) in transport function in tobacco plants, were able to spread at high temperature (33 °C) in Amaranthus caudatus L. plants. On the other hand, TMV ts coat protein mutants retained the ts phenotype in both host plants. The ability of Ls1 and Ni2519 to spread systemically in A. caudatus at high temperature is probably due to the functional stabilization of the transport proteins by a factor(s) provided by the host plant. In accordance with this, Ls1 complemented the transport function of cucumber green mottle mosaic tobamovirus and red clover mottle comovirus; they acquired the ability to spread systemically at 33 °C in the A. caudatus plants preinfected with Ls1.