Infectivity of a mixture of cowpea mosaic virus ribonucleoprotein components

Bruening, George; Agrawal, Hari Om

doi:10.1016/0042-6822(67)90279-6

Cited by 61 publications

(17 citation statements)

References 21 publications

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“…1), suggesting that each virus component binds equally to the plates and that the components bind equal amounts of rabbit antibodies. These simple dilution curves support the earlier results of double-diffusion assays, which suggested that polyclonal sera did not distinguish between centrifugal components of CPMV (Bruening & Agrawal, 1967). However, binding of antigen to microtitre plates promotes substantial changes in conformation and antigenic character of proteins (Friguet et al, 1984;Halk, 1986;Muller et al, 1986).…”

supporting

confidence: 75%

“…The slowest sedimenting top component consists of empty capsids whereas the middle and bottom components are nucleocapsids containing one molecule of either RNA 2 (Mr 1"37 x 106) or RNA 1 (Mr 2.02 x 106), respectively. PAGE does not show differences between the protein composition of the three components (Geelen et al, 1972;Wu & Bruening, 1971) and rabbit antisera prepared against unfractionated virus reacts with all components in Ouchterlony double-diffusion assays (Bruening & Agrawal, 1967). This paper reports that liquid phase competition assays utilizing rabbit antisera reveal a difference between the binding of antibodies to empty capsids and to nucleoprotein virions.…”

mentioning

confidence: 94%

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Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Kalmar

Eastwell

1989

Journal of General Virology

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SUMMARYRabbit antisera produced against two comoviruses (cowpea mosaic virus and cowpea severe mosaic virus) were used in plate-trapped ELISA and liquid phase competition ELISA. In the latter, the top component competed against bound unfractionated virus more effectively than did nucleoprotein components. One murine monoclonal antibody elicited to cowpea mosaic virus and three monoclonal antibodies to cowpea severe mosaic virus exhibited differential binding to top component, relative to either middle or bottom components in plate-trapped or antibody-trapped ELISAs. Differential binding to centrifugal components of virus by two of these monoclonal antibodies was maintained in liquid phase competition assays, These data suggest that the encapsidation of RNA alters the configuration of the virion surface in the vicinity of the antibody epitopes. Ribonuclease digestion of intact or denatured virus did not affect the binding of monoclonal antibodies.Like other comoviruses, cowpea mosaic virus (CPMV) and cowpea severe mosaic virus (CPSMV) are resolved into three major fractions by density gradient centrifugation (Bruening, 1969). The slowest sedimenting top component consists of empty capsids whereas the middle and bottom components are nucleocapsids containing one molecule of either RNA 2 (Mr 1"37 x 106) or RNA 1 (Mr 2.02 x 106), respectively. PAGE does not show differences between the protein composition of the three components (Geelen et al., 1972;Wu & Bruening, 1971) and rabbit antisera prepared against unfractionated virus reacts with all components in Ouchterlony double-diffusion assays (Bruening & Agrawal, 1967). This paper reports that liquid phase competition assays utilizing rabbit antisera reveal a difference between the binding of antibodies to empty capsids and to nucleoprotein virions. Antigenic differences between top component and middle or bottom components were confirmed by assays with monoclonal antibodies.CPSMV-DG and CPMV-SB isolates were originally obtained from G. Bruening (Department of Plant Pathology, University of California, Davis, Ca., U.S.A.), propagated in cowpeas (Vigna unguiculata Walp. cv. California Black Eye) and isolated as previously described (Bruening, 1969). Approximately 5 mg virus was suspended in 0-5 ml gradient buffer (50 mM-KH2PO, pH 7.0, 2 mM-disodium EDTA) and layered onto 11 ml of 39 ~ (w/w) caesium chloride in gradient buffer. Gradients were centrifuged for 36 h at 36000 r.p.m, and 4 °C in an SW 40Ti rotor (Beckman) and fractionated by side-puncture. Fractions were diluted at least 10-fold in gradient buffer and pelleted by centrifugation at 45000r.p.m. for 2h. The pellets were resuspended in gradient buffer. Concentrations of virus components were calculated using A260 at 1 mg/ml of 6.2, 10-0 and 8.1 for middle component, bottom component and unfractionated virus and A2so at 1 mg/ml of 1.28 for top component (Geelen et al., 1972).The preparation of rabbit antisera and of the panel of monoclonal antibodies produced by hybridomas have been described (Kalmar & Eastwell,...

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supporting

confidence: 75%

mentioning

confidence: 94%

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Kalmar

Eastwell

1989

Journal of General Virology

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“…It was hypothesized that these forms could be derived from 1) the different electrophoretic forms resulting from processing of the S subunit, or 2) from the different nucleoprotein components of CPMV. Isopycnic ultracentrifugation in density gradients allows CPMV particles to be separated into three components (Figure 2) that have identical protein composition14 but differ in their RNA contents 15. The particles of the top (T) component are devoid of RNA, while the middle (M) and bottom (B) components each contain a single RNA molecule 3…”

Section: Resultsmentioning

confidence: 99%

Chemical Introduction of Reactive Thiols Into a Viral Nanoscaffold: A Method that Avoids Virus Aggregation

2007

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The use of viral nanoparticles (VNPs) as building blocks for material fabrication has received particular attention in recent years. In earlier studies we showed the applicability of native gel electrophoresis in an agarose matrix as a useful method for the characterization of chemically modified VNPs. Here, we extend these studies and analyze the observed band pattern of intact Cowpea mosaic virus (CPMV) VNPs in agarose gels and show the applicability of native agarose gels for monitoring interparticle linkage of thiol-containing CPMV mutant particles. In addition, we report a protocol that allows the introduction of acetate-protected thiols to CPMV by means of a chemical reaction (rather than genetic modification). The advantage of this approach is that, by incorporating protected thiol groups, the formation of disulfide bonds leading to interparticle linkage is prevented. The resulting thiol-modified CPMV-SH(n) particles are stable, and following deprotection, the introduced thiols are reactive and can be labeled with thiol-selective reagents. They therefore provide a useful additional building block in the CPMV toolbox.

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“…As a result of its ease of propagation, high yield and the stability of the viral particles, CPMV rapidly became an object of intense scientific research. Early studies revealed the bipartite nature of the viral genome (7,78), the structural similarities between CPMV and the animal picornaviruses (87) and the mechanism of gene expression (polyprotein processing; 53).Subsequent work resulted in the determination of the nucleotide sequences of both genomic segments (81, 43), a realisation of the genetic similarities between CPMV and picornaviruses (21), an atomic resolution structure of the virus particles (32, 33), and the creation of infectious cDNA clones (16, 25,84). A crucial step for the development of practical CPMVbased expression systems was the creation of vectors that could be inoculated by agroinfiltration (38), and this approach is now the method of choice for introducing CPMVbased constructs into plants.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Cowpea mosaicVirus: The Plant Virus–Based Biotechnology Workhorse

Sainsbury

Cañizares

Lomonossoff

2010

Annu. Rev. Phytopathol.

109

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INTRODUCTIONThe first plant viruses to be developed as expression vectors in the early 1980s were those with DNA genomes (for reviews, see 42, 56). This was due to the fact that, at the time, only the DNA genomes could be manipulated and the technology for creating infectious cDNA copies of viruses with RNA genomes did not exist. However, the vast majority of plant viruses have genomes that consist of one or more strands of positive-sense RNA. These viruses infect a wide range of hosts and some can reach extremely high titres. Following the construction of the first full-length cDNA clones shown to be infectious (1), the past 25 years has seen a large number of RNA viruses developed as vectors for the expression of foreign sequences and other uses, such as gene silencing (10, 31,42, 55, 56,70).Many proteins have been successfully expressed with virus vectors and significant progress in vector design has been driven by the demands of this application. Generally developed with the expression of fluorescent marker proteins, RNA virus-based vectors have become a highly effective means of producing recombinant heterologous protein in plant tissue within a short time frame (28, 35,44,67). In addition to their use as vectors for the production of heterologous polypeptides and as gene silencing vectors, plant RNA viruses have also provided a source of particles for various applications. Virus capsids provide nano-scale particles with consistent size and shape, which can be exploited for a number of chemical and biological applications (72). For example, a number of systems make use of the repetitive geometry of plant virus capsids to present multiple copies of antigenic sequences, to increase their potential as a source of novel vaccines (10,39, 58). Also, both wild type and genetically modified capsids of plant viruses are also being used as biotemplates for novel materials in nanotechnology (88). 3Cowpea mosaic virus (CPMV) has borne witness to most of the aforementioned biotechnological uses of RNA viruses. The year 2009 marks the 50 th anniversary of the first description of CPMV as a pathogen of cowpeas (Vigna unguiculata) in West Africa (13). As a result of its ease of propagation, high yield and the stability of the viral particles, CPMV rapidly became an object of intense scientific research. Early studies revealed the bipartite nature of the viral genome (7,78), the structural similarities between CPMV and the animal picornaviruses (87) and the mechanism of gene expression (polyprotein processing; 53).Subsequent work resulted in the determination of the nucleotide sequences of both genomic segments (81, 43), a realisation of the genetic similarities between CPMV and picornaviruses (21), an atomic resolution structure of the virus particles (32, 33), and the creation of infectious cDNA clones (16, 25,84). A crucial step for the development of practical CPMVbased expression systems was the creation of vectors that could be inoculated by agroinfiltration (38), and this approach is now the method of choice for intr...

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Infectivity of a mixture of cowpea mosaic virus ribonucleoprotein components

Cited by 61 publications

References 21 publications

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Chemical Introduction of Reactive Thiols Into a Viral Nanoscaffold: A Method that Avoids Virus Aggregation

Cowpea mosaicVirus: The Plant Virus–Based Biotechnology Workhorse

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