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.     

The genome-linked protein of cowpea mosaic virus is coded by RNA from the bottom component

It has recently been shown that RNA from the bottom component of cowpea mosaic virus is capable of self-replication in cowpea mesophyll protoplasts, providing a tool for the investigation of the contribution of each RNA strand toward viral multiplication. Evidence is presented here, using this system, to show that the genome-linked protein is coded by the RNA from the bottom component. As the replicase function is coded by this RNA, the results remain consistent with the involvement of the genome-linked protein in viral RNA replication.     

Mutational analysis of the putative catalytic triad of the cowpea mosaic virus 24K protease

To investigate the mechanism of action of the cowpea mosaic virus (CPMV) 24K protease, a full-length cDNA clone of bottom component (B) RNA has been constructed from which RNA can be transcribed in vitro using T7 RNA polymerase. Translation of the resulting RNA in rabbit reticulocyte lysate leads to the synthesis of a 200 kDa product (the 200K protein) which cleaves itself in a manner identical to that of the product translated from B RNA isolated from virions. Site-directed mutagenesis of the full-length clone was used to examine the effects of altering individual amino acids in the 24K protease on its activity. The results obtained are consistent with the prediction that the 24K protease is structurally similar to the trypsin-like family of serine proteases and suggest that His40, Glu76, and Cys166 comprise the active site. Substitution of Cys166 by a serine residue results in an enzyme with reduced catalytic activity.     

Crystalline cowpea mosaic virus

Crystals of Cowpea mosaic virus displaying a high degree of order have been grown in two different forms. Hexagonal crystals, space group P6(1)22 or P6(5)22 with a = 450 A and c = 1038 A containing six virus particles per cell, have been grown in sodium citrate buffer at pH 4.9. Each particle has four nearest neighbors at 284 A. Cubic crystals, with space group I23 and a = 308 A , containing two particles per cell were grown from ammonium sulfate at pH 4.9. In the cubic crystals, the tetrahedral subgroup of the icosahedral particle symmetry is expressed as part of the crystal lattice, with each particle occupying tetrahedral crystallographic positions. Contacts of 268 A occur between particles in contact along threefold icosahedral symmetry axes.     

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.     

Infection of cowpea mesophyll protoplasts with cowpea mosaic virus.

Mesophyll protoplasts were isolated from the primary leaves of cowpeas by a one-step procedure using a mixture of Macerozyme and Cellulase. The protoplasts were inoculated with cowpea mosaic virus and virus multiplication was shown to occur by measuring the virus infectivity at various times after inoculation. Eighty to ninety-six percent of the protoplasts were infected as shown by fluorescent antibody staining. More than 10⁸ progeny virus particles were produced per infected protoplast. A cytopathic structure similar to the structure found in CPMV infected leaf cells occurred in infected protoplasts. Poly-l-ornithine was not essential for CPMV infection of the cowpea mesophyll protoplasts, but it had a stimulating effect. This is the first protoplast system after the tobacco mesophyll protoplast system suitable for virus infection studies. As cowpeas belong to a plant family other than tobacco, the new protoplasts allow comparative infection studies with plant viruses.     

Location of the gene specifying the smaller protein of the cowpea mosaic virus capsid

Cowpea mosaic virus (CPMV) preparations contain two centrifugal nucleoprotein components, middle and bottom, both of which are required for infection. The capsid of the virus is constructed from equal numbers of large and small protein subunits. A mutant isolate, designated G3, was derived from nitrous acid-treated virus. Mutant G3 demonstrated three electrophoretic forms in contrast to the two electrophoretic forms found in wild-type preparations. As with wild-type virus, the slower forms of G3 were found to undergo an apparent in vivo conversion to the fast form. The conversion could be simulated in vitro by the action of chymotrypsin. This suggests that the G3 mutant carries an altered electronic charge on the labile carboxy-terminal portion of the smaller capsid protein. Examination of separated proteins by gel electrophoresis verified that wild-type and G3 capsids differed only in their smaller proteins. Complementation experiments with separated centrifugal components demonstrated that the recovery of G3 type progeny is dependent upon the presence of G3 middle component in the inoculum. This study indicates that the gene specifying the smaller capsid subunit is located in the middle component RNA of CPMV.     

Further genetic analysis of a temperature-sensitive mutant of cowpea mosaic virus

The two reciprocal hybrids of wild-type cowpea mosaic virus (Sb) and the temperature-sensitive mutant N168 were constructed. Symptoms of both hybrids differed from those of the parent strains in three differential hosts indicating that N168 carries mutations in both components and that symptoms result from interactions between components. The occurrence of one or more temperature-sensitive mutations in the middle-component RNA was confirmed. In vitro recombination of the hybrids yielded isolates indistinguishable from the parent strains.     

Intracellular distribution of cowpea mosaic virus movement protein as visualised by green fluorescent protein fusions

Summary. Cowpea mosaic virus (CPMV) derivatives expressing movement protein (MP) green fluorescent protein (GFP) fusions (MP:GFP) were used to study the intracellular targeting and localization of the MP in cowpea protoplasts and plants. In protoplasts, a virus coding for a wild type MP:GFP (MPfGFP) induced the formation of fluorescent tubular structures, which shows that subcellular targeting and tubule formation are not affected by fusion of GFP to the C-terminus of the MP. In plants, MPfGFP infections were mostly confined to single epidermal cells and failed to achieve a systemic infection, probably because the fusion of GFP to the MP interfered with MP-virion interaction. MP:GFP mainly accumulated in fluorescent spots in the cell wall of epidermal cells of inoculated leaves, which may represent short tubular structures in modified plasmodesmata. At the cuticle-side of epidermal cells tubular structures were detected indicating that tubule formation in plants, as in protoplasts, does not require the presence of functional plasmodesmata. Furthermore, results were obtained which indicate that CPMV MP:GFP is able to traffic from cell-to-cell by itself. The possible significance of this finding is discussed.Received January 3, 2003; accepted June 25, 2003 Published online August 18, 2003     

Factors influencing the infection of cowpea protoplasts by cowpea mosaic virus RNA.

Protoplasts were isolated from primary leaves of cowpeas (Vigna sinensis) by a slight modification of the procedure reported (Beier and Bruening, 1975). They were inoculated with cowpea mosaic virus RNA under various conditions. The highest yield of virus from the infected protoplasts was achieved when viral RNA was incubated in 0.45 M mannitol, 0.1 M potassium phosphate, 50 μg/ml of protamine sulfate, pH 6, at room temperature, and then was mixed with protoplasts at 10° for 15 min. Sixty-five percent of the protoplasts were infected at an RNA concentration of 50 μg/ml. When protoplasts were inoculated with virions and with RNA under the same conditions, the phase of rapid increase of protoplast-associated infectivity occurred sooner and was more steep with RNA as inoculum.     

Mutational analysis of the feline immunodeficiency virus matrix protein

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.     

A three-dimensional image reconstruction of cowpea mosaic virus

Three-dimensional image analysis from electron micrographs of cowpea mosaic virus shows that the virus possesses icosahedral symmetry. The reconstruction shows that the virus, although not capsomeric, does have protruding bumps at the 5-fold and 3-fold positions. In conjunction with the already available chemical evidence, this leads us to propose a model for the structure consisting of 12 pentamers at the 5-fold positions and 20 trimers at the 3-fold positions.     

Proteolytic activity of the cowpea mosaic virus encoded 24K protein synthesized in Escherichia coli

The function of the 24-kilodalton (24K) protein encoded by cowpea mosaic virus (CPMV) has been studied by constructing a bacterial expression plasmid that contained a cloned chimeric segment consisting of partial DNA copies of CPMV M-RNA (including sequences coding for both capsid proteins) and B-RNA (including sequences coding for the 24K protein). Viral sequences were transcribed from the phage T7 promoter phi 10 of plasmid pT7-6 using T7-RNA polymerase expressed from plasmid pGP1-2 present in the same cells. Upon inducing the synthesis of T7-RNA polymerase several new polypeptides that contained CPMV-specific sequences were expressed, as demonstrated by immunoprecipitation and immunoblotting. Furthermore a proteolytic activity was detected in induced cells which cleaved the viral protein sequences specifically at two glutamine-glycine sites. One of the cleavage products represented capsid protein VP23. The proteolytic activity was absent when an 87-bp deletion was introduced in the coding region for the 24K protein, indicating that this protein represented the protease involved in the proteolytic processing at those specific sites.     

Mutational analysis of the putative pipo of soybean mosaic virus suggests disruption of PIPO protein impedes movement

The presence of a small open reading frame embedded in the P3 cistron of potyvirus turnip mosaic virus, termed “pipo,” was recently discovered. We have now studied the putative pipo of soybean mosaic virus (SMV). Introduction of single, or multiple, stop codon mutations at different locations within pipo, without substitution in polyprotein amino acids, did not abolish replication, but restricted the virus to small cluster of cells within the inoculated leaves. Furthermore, extensive mutagenesis of the conserved GA₆ motif at the 5′ end of pipo also generated two out of five mutants that remained restricted to small foci of infected cells within the inoculated leaves. Long-distance movement function of the movement-defective PIPO-mutants was not restored following co-inoculation with competent SMV strains. Taken together, the data suggest that the putative pipo of SMV is essential for the virus movement; however, knock out of its expression does not abolish replication.     

Genetic analysis of cowpea mosaic virus mutants by supplementation and reassortment tests.

Three nitrous acid induced mutants were derived from the yellow strain isolate Sb of cowpea mosaic virus. The mutants, designated as N123, N163, and N140, were differentiated by atypical local lesions on “Pinto” beans and “Early Red” cowpeas and defective symptom development on the systemic hosts “Noordhollandse bruine” bean and “Blackeye” cowpea. Mutants N123 and N140 attained only low titers in “Blackeye” cowpeas and could not be purified in workable quantities.Three tests were applied to locate the mutations in either the middle or bottom components of the mutants: (1) In vitro recombination tests, involving the screening of hybrid isolates obtained from heterologous mixtures of components of wild and mutant strains; (2) supplementation tests, in which wild-type components were separately added to mutant preparations; restoration of a wild-type character to one of the mixtures indicated in which component the mutation (or mutations) occurred; (3) reassortment of components tests, based on the assumption that a mixture of two defective mutants may only show a wild-type character if the mutants have mutations in different components. With all mutants, the results of the different test methods agreed. In mutants N123 and N163, the mutations were located in the middle component. N140 appeared to be a bottom component mutant. The results suggest that changes in the reaction of all differential hosts to a specific mutant are due to the same mutational event. Since N123 and N140 carry mutations for defective symptom development in different components, this pair of mutants may be used to locate mutations of other defective mutants by means of simple reassortment tests.     

Polyadenylate sequences in the ribonucleic acids of cowpea mosaic virus

Polyadenylate sequences at the 3′-termini of cowpea mosaic virus middle and bottom component RNAs were revealed both by the sequential removal of nucleotide residues and by the release of polyadenylate during nuclease digestion. The released polyadenylates from the two RNAs were similar in the distributions of their sedimentation rates. A weight average degree of polymerization of about 200 was estimated. No evidence was found for internal polyadenylate sequences or for terminal sequences of 16 or fewer residues.