Genome-associated proteins of comoviruses

Previously, a protein has been reported to be associated with the RNAs of cowpea mosaic virus. Here we present evidence for genome-associated proteins of other comoviruses: squash mosaic virus, Echtes Ackerbohnemosaik-Virus, and another strain of cowpea mosaic virus. Although the proteins and protein-oligonucleotide complexes derived from the RNAs of these viruses showed similar patterns of electrophoretic mobility, two-dimensional chromatograms of their tryptic peptides were distinct. Chromatograms of peptides from preparations of cowpea mosaic virus that were isolated from two hosts were very similar, indicating that the genome-associated protein may be virus specified. Upon digestion with ribonuclease T1, RNAs from the viruses gave rise to a distribution of large oligonucleotides that is consistent with the presence of polyriboadenylate.     

Development of cowpea mosaic virus-based vectors for the production of vaccines in plants

Plant viruses are emerging as an attractive alternative to stable genetic transformation for the expression of foreign proteins in plants. The main advantages of using this strategy are that viral genomes are small and easy to manipulate, infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants and the sequence inserted into a virus vector will be highly amplified. One use of these virus expression systems is for vaccine production. Among plant viruses, cowpea mosaic virus makes an ideal candidate for the production of such vaccines because it grows extremely well in host plants, is very stable, and the purification of virus particles, if required, is straightforward. In this article, the authors review the progress made in the development of cowpea mosaic virus-based vectors for vaccine production, making use of two main approaches: epitope presentation and polypeptide expression.     

Ultrastructure of mixed plant virus infection: bean yellow mosaic virus with cowpea severe mosaic virus or cowpea mosaic virus in bean

Ultrastructural responses of bean leaf cells simultaneously infected with two morphologically distinct RNA viruses, cowpea mosaic virus (CPMV) and bean yellow mosaic virus (BYMV), or cowpea severe mosaic virus (CSMV) and BYMV, were studied in situ. The major effects on cells infected with two viruses included: (1) association of virus group-specific cytoplasmic inclusions characteristic of each virus; (2) close association of virions into specifically arranged aggregates in which CPMV or CSMV icosahedra were aligned along the long axes of the BYMV rods; and (3) the induced formation of intranuclear inclusions, spheres (22-26 nm in diameter) and filaments (10-14 nm wide and of variable length) in mixed infections of CSMV and BYMV. Intracellular serological testing using ferritin conjugated with CSMV antibodies revealed no relationship between the spherical intranuclear inclusions and CSMV capsids. We conclude that the ultrastructure of mixed infections could be used as another tool for identifying related plant viruses.     

Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.

An automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. Using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immobilized on the dextran matrix present on the sensor chip. Compared to conventional solid phase immunoassays, in which immobilized proteins are usually partly denatured, the biosensor system presents several advantages for studying virus-antibody interaction.     

Biochemical and physical characterization of parvovirus minute virus of mice virus-like particles

The cowpea (Vigna unguiculata) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non-Comoviruses: Cherry leafroll virus and Southern bean mosaic virus. Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus.     

Towards a system for the identification and classification of potyviruses: I. Serology and amino acid composition of six distinct viruses

Antigenic relationships of six distinct potyviruses were studied by immunodiffusion tests using highly purified sonicated virus preparations and anti-intact virus sera devoid of detectable antibodies to host-plant antigens. Three variants of bean yellow mosaic virus (BYMV) including BYMV sensu stricto and two variants of pea mosaic virus (PMV and SPMV) were shown to be antigenically very similar and also relatively closely related to lettuce mosaic virus (LMV). Distant antigenic relationships were detected between the BYMV variants and bean common mosaic virus (BCMV); between BCMV and passionfruit woodiness virus (PWV); and between PWV and potato virus Y (PVY). No antigenic relationships were detected between any of these viruses and sugarcane mosaic virus (SCMV). Antibodies in anti-viral sera were very poor in recognizing coat proteins dissociated with LiCl from homologous viruses and failed altogether to recognise those dissociated with pyrrolidine. Attempts to prepare antisera in mice against isolated viral coat proteins dissociated with either LiCl or pyrrolidine were unsuccessful due to poor immunogenicity of the preparations.Electrophoretic mobilities of the viral coat proteins relative to marker proteins in the presence of sodium dodecyl sulphate suggest that the protein subunits of all the viruses studied have molecular weights of about 33,000. However, the coat proteins were prone to partial degradation. The amino acid compositions of the antigenically closely related viruses were very similar, but similarities of those distantly related were no greater than those of the apparently unrelated viruses.The problems in the use of serological and amino acid composition data obtainable with currently available techniques for the classification of potyviruses are discussed.     

Scope for using plant viruses to present epitopes from animal pathogens

Epitope presentation to the immune system for vaccination purposes can be achieved either via an inactivated or attenuated form of a pathogen or via its isolated antigenic sequences. When free, these peptides can adopt a variety of conformations, most of which will not exist in their native environment. Conjugation to carrier proteins restricts mobility of the peptides and increases their immunogenicity. A high local concentration of epitopes boosts the immune response further and can be generated by the use of self-aggregating carriers, such as the capsid proteins of viruses. In this regard plant viruses have in recent years started to make an impact as safer alternatives to the use of bacterial and attenuated animal viruses: the latter both require propagation in costly cell-culture systems where they can undergo reversion towards a virulent form and/or become contaminated by other pathogens. Plant virus-based vectors can be multiplied cheaply and to high yields (exceeding 1 mg/g plant tissue) in host plants. Both helical (tobacco mosaic virus, potato virus X, alfalfa mosaic virus) and icosahedral (cowpea mosaic virus, tomato bushy stunt virus) particles have been used to express a number of animal B-cell epitopes, whose immunogenic properties have been explored to varying degrees. © 1998 John Wiley & Sons, Ltd.     

Tobacco mosaic virus particles contain ubiquitinated coat protein subunits

Virions of tobacco mosaic virus (TMV) are composed of a single strand of RNA, encapsidated in about 2130 copies of a coat protein of MW 17,500. Asselin and Zaitlin [Virology 91, 173-181 (1978)] demonstrated that virion preparations also contained small amounts of a second protein of MW 26,500, which they termed "H protein." H protein, detectable to an average frequency of one per virion, was thought to be a protein of host origin. Subsequent studies [Collmer, Vogt, and Zaitlin, Virology 126, 429-448 (1983)] showed the H protein was comprised of a backbone of TMV coat protein, linked by a postulated isopeptide bond to a small protein that probably was of host origin. The host-derived moiety of H protein is shown here to be ubiquitin, most probably coupled to the coat protein at lysine 53. This finding is based on microsequencing of the H protein, and is substantiated by immunoblotting analysis with antibodies to human ubiquitin. Conjugated ubiquitin was detected in virions of all five strains of the virus tested. To our knowledge, this is the first report of a ubiquitinated viral structural protein.     

Electron microscopy of leaves infected with sowbane mosaic virus and other small polyhedral viruses

Leaves of Chenopodium amaranticolor were infected with sowbane mosaic virus (SMV), sectioned and examined by electron microscopy. Leaves of Brassica pekinensis and C. amaranticolor were infected with turnip yellow mosaic virus and cowpea mosaic virus, respectively, and similarly studied. With all three viruses it was difficult, in sections, to distinguish the small isometric virus particles from ribosomes though sometimes this was possible, especially when the viruses crystallized. Pretreatment of tissue with permanganate or EDTA appeared to destroy the ribosomes but resulted in excessive disorganization of the tissue. Although SMV did not normally crystallize, wilting the infected leaves caused it to do so. All three viruses were found free in the cytoplasm and were absent from nuclei, chloroplasts, and mitochondria. Some abnormal structures found in the infected tissues are described.     

Polyamine content of several RNA plant viruses

Three polyhedral viruses in the bromovirus group, brome mosaic virus, cowpea chlorotic mottle virus, and broad bean mottle virus, contain no detectable polyamines. Two other polyhedral viruses, turnip yellow mosaic virus and cowpea mosaic virus, contain roughly 1% spermidine by weight. The rod-shaped barley stripe mosaic virus contains no detectable polyamines.     

A comparative study of the cowpea and bean strains of southern bean mosaic virus

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3' termini have been determined. These include the 3' noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5' terminus-the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.     

Nonspecific binding of immunoglobulins to coat proteins of certain plant viruses in immunoblots and indirect ELISA

Nonspecific binding of immunoglobulins to coat protein of cucumber mosaic virus and several other plant viruses was observed in Western immunoblots, and to a much lesser extent in enzyme-linked immunosorbent assays. The binding appears to occur between immunoglobulin molecules and the basic domains of viral coat proteins which bind to RNA during encapsidation. In all cases tested, the nonspecific reactions could be prevented by addition of 5 U/ml of heparin to the incubation buffers.     

Melting of viral RNA by coat protein: assembly strategies for elongated plant viruses

The coat proteins of two rod-shaped plant viruses, papaya mosaic virus (PMV) and tobacco mosaic virus (TMV), have been tested for RNA melting activity. Under reconstitution conditions, PMV protein melts RNA in a noncooperative fashion. This activity is aspecific and is inhibited by low concentrations of NaCl as is virus reconstitution. TMV protein does not melt RNA either in the absence of NaCl or under reconstitution conditions at moderate ionic strength levels. The results suggest that elongated plant viruses have evolved at least two different assembly strategies in order to satisfy the requirement that the RNA within these viruses be in a melted configuration.     

Spatial analysis for exclusive interactions between subgroups I and II of Cucumber mosaic virus in cowpea

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.     

Evidence for intrastrand complementation in cowpea mosaic virus infection

In supplementation tests both middle component (M)-RNA and bottom component (B)-RNA of cowpea mosaic virus mutant N142 were shown to carry mutations affecting local symptoms in bean and cowpea. The M- and B-RNA mutations were separately incorporated into the two reciprocal hybrids of N142 and wild-type Sb, designated as M142BSb and MSbV142. Mixtures of the two hybrids induced normal local symptoms in bean and cowpea as a result of reassortment of components. This was also the case with a mixture of MSbV142 and mutant N123. The latter mutant was earlier shown to contain a M-RNA mutation for defective symptom expression in bean and cowpea. Although both M142BSb and N123 carried M-RNA mutations, mixtures of these isolates induced normal local symptoms. Subculturing showed no wild-type virus to be present in the inoculated leaves. Thus, intrastrand complementation was assumed to result in restoration of normal symptom production. We conclude that the results of genetic mixing experiments with multicomponent plant viruses must be interpreted cautiously. Subculturing experiments should be performed to determine whether restoration of wild-type symptoms results from exchange of nonmutated components or from intrastrand complementation.     

Cowpea golden mosaic disease in Gujarat is caused by a <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> isolate with a DNA B variant

It has long been assumed that cowpea golden mosaic disease (CGMD) in southern Asia is caused by a begomovirus distinct from those causing disease in other legumes. The components of a begomovirus causing CGMD in western India were isolated, cloned and sequenced. Analysis of the sequences shows the virus to be an isolate of Mungbean yellow mosaic India virus, but with a distinct DNA B component with greater similarity to components of a second legume-infecting begomovirus occurring in the region, Mungbean yellow mosaic virus. The clones of the virus were readily infectious to cowpea, mungbean, blackgram and French bean by agroinoculation. However, the wild-type isolate was shown to be easily transmissible by whiteflies between cowpea plants but not to blackgram and mugbean, suggesting that the insect vector plays a major role in determining the natural host range of these viruses.     

Antiviral activity in plants of a mycoviral double-strained RNA from Trichothecium roseum

Hexagonal virus-like particles (VLPs) measuring 45 nm across were detected in mycelial extracts from Trichothecium roseum Himachal strain, the source fungus for the production of T-poly (Trichothecium polysaccharide), a known inhibitor of plant viruses. VLPs were found to contain double-stranded RNA (ds RNA) and the purified ds RNA was capable of inhibiting tobacco mosaic virus infection in Nicotiana glutinosa plants. Active preparations of T-poly were found to contain traces of ds RNA, probably of mycoviral origin.     

Lack of specificity of virus-stimulated plant RNA-dependent RNA polymerases

RNA-Dependent RNA polymerase preparations from tobacco and cowpea plants, either uninfected, or infected with tobacco necrosis or cowpea mosaic virus, were tested for their capability to bind radioactive viral or other RNAs or polynucleotides. The limited binding of up to 30% of all RNAs tested was found to be completely nonspecific in regard to any infecting virus, and it was nonspecifically diminished by excess of other unlabeled RNAs. These data are in line with our previous conclusion that plant viral RNAs are replicated by host enzymes. The data also speak against the hypothesis that these enzymes might be modified as a consequence of viral infection.     

Mutational analysis of the cowpea mosaic virus movement protein

Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.     

Phylogenetic Analysis of Two Potyvirus Pathogens of Commercial Cowpea Lines: Implications for Obtaining Pathogen-derived Resistance

As a prelude to developing engineered resistance to two important potyvirus pathogens of cowpea, a phylogenetic analysis of strains of Cowpea aphid-borne mosaic virus (CAbMV) and Bean common mosaic virus--blackeye cowpea strain (BCMV-BlC) was undertaken. Nucleotide sequences for the coat protein genes and 3-untranslated regions of four CAbMV and one BCMV-BlC strains were determined and included in an analysis with published sequences. While all the newly sequenced viruses showed strong homology with the existing respective sequences in the database, the CAbMV group showed a divergence into two subgroups. These groups differed from each other by more than some CAbMV strains differed from the South African Passiflora virus (CAbMV-SAP), which has distinct biological characteristics. The implications of the sequence analyses are discussed with respect to a strategy for the generation of engineered resistance to both groups of viruses.