The cleavable carboxyl-terminus of the small coat protein of cowpea mosaic virus is involved in RNA encapsidation. (1/108)

The site of cleavage of the small coat protein of cowpea mosaic virus has been precisely mapped and the proteolysis has been shown to result in the loss of 24 amino acids from the carboxyl-terminus of the protein. A series of premature termination and deletion mutants was constructed to investigate the role or roles of these carboxyl-terminal amino acids in the viral replication cycle. Mutants containing premature termination codons at or downstream of the cleavage site were viable but reverted to wild-type after a single passage through cowpea plants, indicating that the carboxyl-terminal amino acids are important. Mutants with the equivalent deletions were genetically stable and shown to be debilitated with respect to virus accumulation. The specific infectivity of preparations of a deletion mutant (DM4) lacking all 24 amino acids was 6-fold less than that of a wild-type preparation. This was shown to be a result of DM4 preparations containing a much increased percentage (73%) of empty (RNA-free) particles, a finding that implicates the cleavable carboxyl-terminal residues in the packaging of the virion RNAs.  (+info)

Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus. (2/108)

A synthetic peptide (peptide 10) representing a surface-exposed, linear B cell epitope from outer-membrane (OM) protein F of Pseudomonas aeruginosa was shown previously to afford protection in mice from P. aeruginosa infection. This peptide was expressed in tandem with the protein F peptide 18 on each of the two coat proteins of cowpea mosaic virus (CPMV). The chimaeric virus particles (CVPs) expressing the peptides on the S (small) coat protein (CPMV-PAE4) and L (large) coat protein (CPMV-PAE5) were used to immunize mice. Following subcutaneous immunization in Freund's and QuilA adjuvants, CPMV-PAE4 induced antibodies predominantly against peptide 18, whereas CPMV-PAE5 produced antibodies exclusively against peptide 10, indicating that the site of peptide expression on CPMV influences its immune recognition. The anti-peptide antibodies elicited by CPMV-PAE5 were predominantly of the IgG2a isotype, indicating a highly polarized TH1-type response. The peptide-specific IgG2a strongly recognized the whole F protein, but more importantly, recognized protein F in all seven Fisher-Devlin immunotypes of P. aeruginosa. Furthermore, the peptide-specific IgG2a in CVP/QS-21 adjuvant-immunized mice was shown to bind complement and to augment phagocytosis of P. aeruginosa by human neutrophils in vitro. The ability of CPMV-PAE5 to induce P. aeruginosa-specific opsonic IgG2a gives it potential for further development as a protective vaccine against P. aeruginosa.  (+info)

A chimaeric plant virus vaccine protects mice against a bacterial infection. (3/108)

The plant virus cowpea mosaic virus (CPMV) is an efficient carrier of foreign peptides for the generation of strong humoral immune responses. Peptides derived from both viruses and bacteria are strongly immunogenic when displayed on the surface of CPMV and elicit high titres of peptide-specific antibody. However, the protective effects of antibodies generated using bacterial epitopes in this system have yet to be demonstrated. In this study the ability of chimaeric virus particles (CVPs) to afford protection against bacterial infection was assessed. Immunization of outbred mice with CPMV expressing a peptide derived from outer-membrane protein F of Pseudomonas aeruginosa (CPMV-PAE5) generated high titres of P. aeruginosa-specific IgG that opsonized the bacteria for phagocytosis by human neutrophils and afforded protection upon challenge with two different immunotypes of P. aeruginosa in a model of chronic pulmonary infection. When examined 8 d after challenge, CVP-immunized mice had fewer severe lung lesions and fewer bacteria in their lungs compared to mice immunized with wild-type virus. Different levels of protection were seen with CPMV-PAE5 when Freund's or alum adjuvants were used. These studies highlight the ability of CVPs to generate protective immunity against infectious disease agents.  (+info)

Studies on hybrid comoviruses reveal the importance of three-dimensional structure for processing of the viral coat proteins and show that the specificity of cleavage is greater in trans than in cis. (4/108)

A series of cowpea mosaic virus (CPMV)-based hybrid comoviral RNA-2 molecules have been constructed. In these, the region encoding both the large (L) and small (S) viral coat proteins was replaced by the equivalent region from bean pod mottle virus (BPMV). The hybrid RNA-2 molecules were able to replicate in cowpea protoplasts in the presence of CPMV RNA-1. Though processing of the hybrid polyproteins by the CPMV-specific 24K proteinase at the site between the 58/48K and L proteins could readily be achieved, no processing at the site between the L and S coat proteins could be obtained even when the sequence of amino acids between the two coat proteins was made CPMV-like. As a result, none of the hybrids was able to form functional virus particles, and they could not infect cowpea plants. Comparison with the processing of the L-S site in cis in reticulocyte lysates demonstrated that the requirements for processing are more stringent in trans than in cis. The results suggest that the L-S cleavage site is defined by more than just a linear sequence of amino acids and probably involves interactions between the L-S loop and the beta barrels of the viral coat proteins.  (+info)

Structural fingerprinting: subgrouping of comoviruses by structural studies of red clover mottle virus to 2.4-A resolution and comparisons with other comoviruses. (5/108)

Red clover mottle virus (RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 A. The overall structure of RCMV is similar to that of two other comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.  (+info)

The refined crystal structure of cowpea mosaic virus at 2.8 A resolution. (6/108)

Comoviruses are a group of plant viruses in the picornavirus superfamily. The type member of comoviruses, cowpea mosaic virus (CPMV), was crystallized in the cubic space group I23, a = 317 A and the hexagonal space group P6(1)22, a = 451 A, c = 1038 A. Structures of three closely similar nucleoprotein particles were determined in the cubic form. The roughly 300-A capsid was similar to the picornavirus capsid displaying a pseudo T = 3 (P = 3) surface lattice. The three beta-sandwich domains adopt two orientations, one with the long axis radial and the other two with the long axes tangential in reference to the capsid sphere. T = 3 viruses display one or the other of these two orientations. The CPMV capsid was permeable to cesium ions, leading to a disturbance of the beta-annulus inside a channel-like structure, suggesting an ion channel. The hexagonal crystal form diffracted X rays to 3 A resolution, despite the large unit cell. The large ( approximately 200 A) solvent channels in the lattice allow exchange of CPMV cognate Fab fragments. As an initial step in the structure determination of the CPMV/Fab complex, the P6(1)22 crystal structure was solved by molecular replacement with the CPMV model determined in the cubic cell.  (+info)

Immunogenic and antigenic dominance of a nonneutralizing epitope over a highly conserved neutralizing epitope in the gp41 envelope glycoprotein of human immunodeficiency virus type 1: its deletion leads to a strong neutralizing response. (7/108)

The Kennedy peptide, (731)PRGPDRPEGIEEEGGERDRDRS(752), from the cytoplasmic domain of the gp41 transmembrane envelope glycoprotein of HIV-1 contains a conformationally dependent neutralizing epitope (ERDRD) and a linear nonneutralizing epitope (IEEE). No recognized murine T cell epitope is present. The peptide usually stimulates virus-specific antibody, but this is not always neutralizing. Here we show that IEEE (or possibly IEEE plus adjacent sequence) is immunogenically and antigenically dominant over the ERDRD neutralizing epitope. Thus rabbits immunized in a variety of routes, doses, and adjuvants with a chimeric cowpea mosaic virus (CPMV) expressing the Kennedy peptide on its surface (CPMV-HIV/1) synthesized IEEE-specific serum antibody but no ERDRD-specific or HIV-1-neutralizing antibody. To test if this resulted from immunodominance or from a hole in the antibody repertoire, we immunized rabbits with chimera CPMV-HIV/29, which expresses the GERDRDR part of the Kennedy sequence. This chimera readily stimulated ERDRD-specific, neutralizing antibody. In mice the situation was less extreme, but individual animals with low neutralizing titers had a high ratio of IEEE-specific:ERDRD-specific antibody. Data are consistent with immunodominance of IEEE over ERDRD in the Kennedy peptide. IEEE-specific antibody was also antigenically dominant and prevented ERDRD-specific antibody from binding to its epitope and from neutralizing HIV-1. It may be that HIV-1 has evolved a nonneutralizing immunodominant epitope that allows it to possess a neutralizing epitope without suffering the consequences, and this idea is supported by the covariance of both epitope sequences. To our knowledge this is the first example of a defined sequence that controls the activity of an adjacent epitope.  (+info)

Complementation of the host range restriction of southern cowpea mosaic virus in bean by southern bean mosaic virus. (8/108)

Vigna unguiculata (cowpea) and Phaseolus vulgaris (common bean) are permissive hosts for southern cowpea mosaic virus (SCPMV) and southern bean mosaic virus (SBMV), respectively. Neither of these two sobemoviruses systemically infects the permissive host of the other. Although bean cells are permissive for SCPMV RNA synthesis, they do not support the assembly of this virus. Thus, the host range restriction of SCPMV in bean may occur at the level of movement and may involve the inability of SCPMV to assemble in this host. In this study, it was demonstrated that SCPMV accumulates in an encapsidated form in the inoculated and systemic leaves of bean plants following coinoculation with SBMV. No evidence was observed that the SCPMV that accumulated in coinoculated bean plants had an altered host range relative to wild-type SCPMV. These results suggested that SBMV complemented the host range restriction of SCPMV in bean. Additional experiments demonstrated that cowpea protoplasts are permissive for SBMV RNA synthesis and assembly. It was concluded from these results that the host range restriction of SBMV in cowpea occurs at the level of movement. In mixed infections of cowpea with SCPMV and SBMV, the latter was recovered from the inoculated but not the systemic leaves. Its recovery from the inoculated leaves, however, was not dependent on the presence of SCPMV in the inoculum. From these results, it was concluded that SCPMV did not complement the host range restriction of SBMV in cowpea.  (+info)

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