Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Abstract

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,...

Influence of the C terminus of the small protein subunit of bean pod mottle virus on the antigenicity of the virus determined using monoclonal antibodies and anti-peptide antiserum

“…In contrast to the results of Kalmar & Eastwell (1989b), none of the 10 MAbs raised against BPMV was able to detect an antigenic difference between coat protein derived from different centrifugal components of the virus. However, all anti-BPMV MAbs were able to distinguish virions containing only the cleaved form of the small subunit from virions that also contained the uncleaved form.…”

“…In antibody-trapped ELISA, three anti-neotope MAbs reacted better with middle and bottom components than with top component, whereas in a liquid-phase competition assay two of these antineotope MAbs could not differentiate the various components. On the other hand the one anti-metatope MAb which differentiated between the centrifugal components reacted better with top component than with the middle and bottom components in antigentrapped ELISA (Kalmar & Eastwell, 1989b).…”

“…Recently, Kalmar & Eastwell (1989 a) prepared monoclonal antibodies (MAbs) against cowpea mosaic virus (CPMV) and cowpea severe mosaic virus (CPSMV), and reported that in various immunoassays four of the MAbs bind differentially to the top component of the viruses (Kalmar & Eastwell, 1989b). Of the 15 MAbs raised against the two viruses, six were directed against neotopes (epitopes specific for intact virions), three were directed against cryptotopes (epitopes specific for dissociated viral subunits) and six were directed against metatopes (epitopes recognizing both virions and subunits).…”

Differentiation and Antigenic Characterization of Closely Related Alfalfa Mosaic Virus Strains with Monoclonal Antibodies

“…However, MAb 8 recognized an epitope on all three AMV antigens used in ELISA but only on fixed virus particles in immunodiffusion tests. It seems that MAb 5 may have reacted with a conformationally altered epitope resulting from the binding of native virus particles to polyclonal antibodies adsorbed to the ELISA microtitre wells (Heinz et al, 1984;McCullough et al, 1985;Getzoff et al, 1987;Dekker et al, 1989;Kalmar & Eastwell, 1989). It has been suggested that such antibodies may be generated as a result of presenting antigen-antibody complexes to the lymphoid systems of immunized animals during later stages of immunization (Heinz et al, 1984).…”

“…Although not recognized in immunodiffusion tests, this epitope may have been stabilized in some way by binding to polyclonal antibodies and was therefore detectable by ELISA (Heinz et al, 1984;McCullough et al, 1985;Getzoffet al, 1987;Kalmar & Eastwell, 1989;Dekker et al, 1989). The inability of MAbs 5 or 8 to precipitate AMV coat protein preparations in immunodiffusion tests is probably due to a lack of repeated epitopes on each coat protein subunit which are necessary to form a three-dimensional lattice.…”