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How do plant virus nucleic acids move through intercellular connections?

In addition to their function in transport of water, ions, small metabolites, and growth factors in normal plant tissue, the plasmodesmata presumably serve as routes for cell-to-cell movement of plant viruses in infected tissue. Virus cell-to-cell spread through plasmodesmata is an active process mediated by specialized virus encoded movement proteins; however, the mechanism by which these proteins operate is not clear. We incorporate recent information on the biochemical properties of plant virus movement proteins and their interaction with plasmodesmata in a model for transport of nucleic acids through plasmodesmatal channels. We propose that only single stranded (ss) nucleic acids can be transported efficiently through plasmodesmata, and that movement proteins function as molecular chaperones for ss nucleic acids to form unfolded movement protein-ss nucleic acid complexes. These complexes are targeted to plasmodesmata. Plasmodesmatal permeability is then increased following interaction with movement protein and the entire movement complex or its nucleic acid component is translocated across the plasmodesmatal channel.

DNA, Viral↗

A dendrogram of plant viruses.

To facilitate the recognition of plant viruses with similar characteristics a dendrogram of characterized viruses was constructed. The sequence of criteria included: type of nucleic acid; single or double stranded; presence or absence of lipid envelope; helical or nonhelical symmetry; and divided or single genome. Nonhelical RNA viruses with divided genomes were further divided into viruses with one or more than one capsid size. Those with one capsid size were subdivided into viruses with one or more than one sedimenting component. Nonhelical RNA viruses with a single genome were divided according to their RNA size, and their sensitivity to sodium dodecyl sulfate and ethylenediaminetetraacetic acid.

Capsid↗

Plant viruses and the recent discovery of unforeseen basic cellular processes.

Given their small genome size, the biological cycle of plant viruses is tightly integrated with the cellular processes of their host plants, so that studies of the viral biology will often provide insights into basic cellular processes. In the last decade, two such unforeseen mechanisms were discovered. One concerns intercellular communications: for their movement in infected plants, viruses use channels (plasmodesmata, phloem) also used by the plant to exchange information-rich molecules (proteins, RNAs) between cells. The second phenomenon concerns the existence, in plants, of an anti-viral defence mechanism based on the specific degradation of RNA molecules in the cytoplasm. This same mechanism, also allowing the regulation of gene expression (post-transcriptional gene silencing, PTGS) now appears to be widespread in pluricellular organisms. Besides their general interest, these new results modify drastically our vision of interactions between plant and viruses and raise numerous new research questions.

Cell Communication↗

A PCR-microplate hybridization method for plant virus detection.

A sensitive nonradioactive method for detection of plant viruses was evaluated. A cDNA fragment from the coat protein coding region of the potato virus Y (PVY) RNA genome amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR), was directly adsorbed onto polystyrene microplate wells after heat denaturation. The adsorbed cDNA was hybridized with a digoxigenin (DIG)-labelled cDNA probe without a prehybridization step. The probe was also prepared by PCR using the same pair of primers to amplify the target sequence from a cDNA clone of part of the viral genome. The hybrid of the adsorbed cDNA and DIG-labelled probe was reacted with alkaline phosphatase-conjugated anti-DIG antibody. The enzyme activity was then detected by hydrolysis of a substrate, and the absorbance values were measured using a microplate reader. Highest absorbance values were obtained when the amplified DNA fragments were diluted 100 or 125-fold in 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate) for adsorption. Highly concentrated DNA gave lower absorbance values. The absorbance values differed depending on the microplates used, and the highest value was obtained using Nunc Immunoplate II-Maxisorp microplates. DNA fragments longer than 300 bp all gave similar absorbance values, which were twice as high as those obtained with shorter fragments. When the amplified DNA was diluted 100-fold, 10 fg of PVY genomic RNA could be detected by this method, which is called PCR-microplate hybridization. This is about 10,000 times more sensitive than enzyme-linked immunosorbent assay (ELISA). By this method, PVY was detected from five field potato samples showing to be free from PVY by ELISA. PCR-microplate hybridization is nucleic acid-based ELISA-like highly sensitive diagnostic method, and may be generally applicable for detection of plant viruses, viroids, and possibly other plant pathogens.

Adsorption↗

Plasmodesmata and plant viruses. A centenary story.

The passage of plant viruses from a cell to adjacent ones remained for a long time an unexplained event. Only during the thirties did Samuel and other plant virologists put forward the hypothesis that the passage occurred through plasmodesmata, i.e. those protoplasmic connections between plant cells described since the late 19th century. A direct relation between viruses and plasmodesmata was first demonstrated by electron microscopy during the late 1960s by Esau and co-workers, and then widely confirmed. The mechanism of the passage was investigated in depth starting from the 1970s, and research received a remarkable impulse after that a well-defined model of plasmodesmata had been obtained thank, in particular, to work of the Robards' and Gunning's groups. In this context, the discovery of the polycystronic functionality of the viral genomes was fundamental. A protein coded by tobacco mosaic virus, discovered in 1982 independently by the Soviet group of Atabekov and the American group of Zaitlin, was demonstrated to be indispensable for the transport of virus infection from cell to cell through plasmodesmata. Elegant investigations on this 'movement protein' demonstrated that it actually increases the permeability of plasmodesmata. The relation between viruses and plasmodesmata is one of the most interesting and investigated theme of research, which is receiving much attention from plant virologists, physiologists and molecular biologists. The current status of knowledge still presents unsolved questions, and the story is far from over.

History, 20th Century↗

Plant virus gene vectors for transient expression of foreign proteins in plants.

The development of plant virus gene vectors for expression of foreign genes in plants provides attractive biotechnological tools to complement conventional breeding and transgenic methodology. The benefits of virus-based transient RNA and DNA replicons versus transgenic gene expression include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. These characteristics are especially advantageous when very high levels of gene expression are desired within a short time, although instability of the foreign gene in the viral genome can present some problems. The strategies that have been tested for foreign gene expression in various virus-based vectors include gene replacement, gene insertion, epitope presentation, use of virus controlled gene expression cassettes, and complementation. Recent reports of the utilization of virus vectors for foreign gene expression in fundamental research and biotechnology applications are discussed.

Journal Article↗

Antiviral and antitumor antibiotics. XX. Effects of rotenone, deguelin, and related compounds on animal and plant viruses.

Rotenoids and related compounds were investigated for their effects on animal and plant viruses. Of 35 compounds examined, rotenone, rotenone norketone, acetylrotenone, acetylrotenone norketone, deguelin, deguelic acid, dehydrodeguelin, and isotubanol norketone, all used at low concentrations, suppressed the growth of Newcastle disease and herpes simplex viruses as determined by the agar diffusion, plaque inhibition method. Most of the compounds likewise decreased the number of necrotic spots on tobacco mosaic virus-infected leaf discs. Only derrisic acid completely inhibited the local lesion formation at subphytotoxic concentrations. Correlation of antiviral activity with respiratory inhibition of these compounds is discussed.

Animals↗

Isopycnic centrifugation of plant viruses in Nycodenz density gradients.

Isopycnic centrifugation of plant viruses in density gradients prepared from a new non-ionic medium (Nycodenz) was investigated. Particle density of luteo-, tymo-, nepo-, cocksfoot mild mosaic, tobamo-, hordei-like, potex- and potyviruses in buffered solutions of Nycodenz ranged between 1.23 and 1.28 g/ml and did not strictly reflect their nucleic acid and protein composition. Isopycnic centrifugation of several partially purified viruses yielded preparations which were pure by electron microscopy and, when used as immunogens in rabbit or hen, gave antisera with low titers of antibodies against host plant antigens, as required for enzyme immunoassays. A preparation of purified potato leafroll virus contained a single dominant protein corresponding to the viral coat protein. It is concluded that isopycnic centrifugation in Nycodenz density gradients is particularly useful for the purification and analysis of plant viruses which are unstable in cesium chloride solutions, such as the luteoviruses, and preferable to zonal centrifugation in sucrose density gradients for the purification of rod-shaped multipartite viruses or viruses which aggregate.

Centrifugation, Isopycnic↗

Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22 amino acid peptide of gp41 expressed on the surface of a plant virus.

A plant virus, cowpea mosaic virus, expressing a 22 amino acid peptide 731-752 of the gp41 glycoprotein of human immunodeficency virus type 1 (HIV-1 IIIB), was shown previously to stimulate HIV-1 cross reactive neutralizing antibodies in adult C57/BL6 mice. Here some parameters concerning the stimulation of HIV-1-specific neutralizing and ELISA antibody have been determined in adult C57/BL6, C3H/He-mg and BALB/c mice. Two injections per mouse of all CPMV-HIV/1 doses tested (100, 10 and 1 microgram chimera which contained, respectively, 1700, 170 and 17 ng HIV peptide per injection) stimulated a strong serum neutralizing antibody response in all mice. One hundred micrograms or 10 micrograms CPMV-HIV/1 per injection gave 99% neutralization of HIV-1 IIIB in C8166 cells at a serum dilution of 1/200, whereas sera from mice immunized with 1 microgram per injection neutralized virus to 97%, 79% and 63% at a 1/200 dilution of serum from C3H/He-mg, C57/BL6 and BALB/c mice, respectively. Restimulation of these mice with the same immunogen dose marginally increased the neutralization titres. The longevity of the neutralizing antibody response increased as the immunogen dose decreased, and was dependent on the strain of mouse, in the order C57/BL6C3H/He-mg BALB/c. Re-immunization with a third injection improved the longevity of the antibody response. All mice immunized with 100 micrograms CPMV-HIV/1 responded with ELISA antibody to the gp41 peptide bound in solid phase. Ten micrograms stimulated ELISA antibody in some but not all mice, while mice immunized with 1 microgram had no detectable ELISA antibody. This synthesis of ELISA antibody decreased > or = 230-fold over the range of immunogen doses tested but, in the same mice, the neutralizing antibody response decreased only twofold, showing an unusual bias to production of the latter. Neutralizing antibodies were thus stimulated at a lower immunogen dose than ELISA antibodies. Antibody which was affinity purified using the free gp41 peptide gave a good ELISA titre but did not neutralize HIV-1, suggesting that the neutralizing antibody is recognizing a conformational epitope on the gp41 oligomer.

Amino Acid Sequence↗

Plant viruses: master explorers of evolutionary space.

Viruses represent the epitome of genetic diversity, encompassing both DNA and RNA genomes. Their variability probably results from high mutation rates, extensive genetic recombination, gene duplication, large populations and ancient origins. In addition, at least a third of all viral genes may have de novo origins. There is also recent evidence that additional novelty may be generated by genetic recombination between viruses and their plant hosts. Viruses are indeed master explorers of evolutionary space.

Biological Evolution↗

Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses.

Some characteristics of a microplate method for the detection and assay of plant viruses using enzyme-labelled antibodies are described. The method enabled the highly sensitive detection of a number of morphologically different viruses in purified preparations and in unclarified extracts of herbaceous hosts and of infected crop plants. Virus concentrations were estimated by photometric measurement of the colour intensity of the hydrolysed substrate. The suitability of the tehcnique for various field and research applications is considered.

Enzyme-Linked Immunosorbent Assay↗

Comparative sequence and structure of viroid-like RNAs of two plant viruses.

A newly discovered group of spherical plant viruses contains a bipartite genome consisting of a single-strand linear RNA molecule (RNA 1, Mr 1.5 x 10(6) ), and a single-strand, covalently closed circular viroid-like RNA molecule (RNA 2, Mr approximately 125,000). The nucleotide sequences of the RNA 2 of two of these, velvet tobacco mottle virus and solanum nodiflorum mottle virus, have been determined. RNA 2 of solanum nodiflorum mottle virus consists of 377 residues whereas that of velvet tobacco mottle virus consists of two approximately equimolar species, one of 366 residues and the other, with a single nucleotide deletion, of 365 residues. There is 92-95% sequence homology between the RNA 2 species of the two viruses. The predicted secondary structures possess extensive intramolecular base pairing to give rod-like structures similar to those of viroids. The structural similarities between the RNAs 2 of velvet tobacco mottle virus and solanum nodiflorum mottle virus and viroids may reflect functional similarities.

Base Sequence↗

Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus.

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.

Amino Acid Sequence↗

RNA recombination in animal and plant viruses.

An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.

Base Sequence↗

Disease control and its selection for damaging plant virus strains in vegetatively propagated staple food crops; a theoretical assessment.

Viral diseases are a key constraint in the production of staple food crops in lesser developed countries. New and improved disease control methods are developed and implemented without consideration of the selective pressure they impose on the virus. In this paper, we analyse the evolution of within-plant virus titre as a response to the implementation of a range of disease control methods. We show that the development of new and improved disease control methods for viral diseases of vegetatively propagated staple food crops ought to take the evolutionary responses of the virus into consideration. Not doing so leads to a risk of failure, which can result in considerable economic losses and increased poverty. Specifically in vitro propagation, diagnostics and breeding methods carry a risk of failure due to the selection for virus strains that build up a high within-plant virus titre. For vegetatively propagated crops, sanitation by roguing has a low risk of failure owing to its combination of selecting for low virus titre strains as well as increasing healthy crop density.

Animals↗