Biomedical subjects
Publications and source records attributed to G Lomonossoff.
As with animals, plants are susceptible to viral diseases. Until recently, plant viruses had no direct bearing on animal health, though the crop losses they can cause may have indirect effects. However, recent advances in recombinant DNA technology make it possible to adapt plant viruses so that they can produce pharmaceutically important proteins in plants.
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Capsid proteins of a number of plant viruses are permissive to genetic modifications in which foreign polypeptides are inserted in exposed loops or at their C termini. Plant viruses with these genetic alterations often grow at wild-type levels, providing gram quantities of modified viruses. Presented polypeptides studied most extensively correspond to antigenic epitopes of animal viruses and in some cases appropriately altered plant viruses generate neutralizing antibodies to the cognate animal virus when the plant virus is used as a vaccine. Structure-based analyses of these animal-plant virus chimeras have led to rational alterations to the presentation in efforts to increase the efficacy of the presented peptide.
BACKGROUND: We employed a genetically engineered icosahedral plant virus, cowpea mosaic virus (CPMV), as an expression and presentation system to display a 14 amino acid linear antigenic epitope found in a capsid protein of human rhinovirus 14 (HRV14). RESULT: Gram quantities of the CPMV/HRV 14 chimera were made in plants and purified particles were crystallized in a form isomorphous with wild-type CPMV. The 2.8 A resolution structure of the chimera shows that the inserted loop is well ordered and that if the loop were intact, a phenylalanine residue of CPMV would be placed in a hydrophilic environment. The resultant strain may make the loop an attractive substrate for endogenous plant proteases, as roughly 80% of the inserted polypeptides are cleaved, allowing the phenylalanine to be partially buried. Altering the phenylalanine to an arginine could relieve the stress, reducing the propensity for cleavage and increasing the likelihood that the peptide will assume a structure closely similar to its structure in HRV14. CONCLUSIONS: Successful crystallization of other CPMV chimeras in forms isomorphous with the native virus suggests that this is a viable system for structure-based design of peptide presentation.
The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.
An in vitro murine T cell proliferation assay was used to determine whether an antigenic epitope(s) recognized by enterovirus-immune T cells is held in common between plant comoviruses and human enteroviruses. Splenocytes isolated from C3H/HeJ mice infected with coxsackievirus B3 (CVB3) proliferated in vitro not only against a variety of enterovirus (CVB2, CVB3, CVB6, CVA16, PV1) antigens, but against comovirus (CPMV, BPMV) antigens as well. Splenocytes from mice inoculated with bean pod mottle virus (BPMV) also proliferated in response to comoviral and enteroviral antigens in vitro. However, if the viral inocula were highly purified prior to inoculation, then the splenocyte response was generated only against the group used to inoculate, suggesting that the epitope shared between the comoviruses and the enteroviruses resided in the nonstructural region. B (nonstructural) and M (structural) genomic segments of CPMV were translated in rabbit reticulocyte lysates and used as in vitro antigens. Splenocytes from mice inoculated with live CVB3 proliferated in response to the B-RNA-encoded but not the M-RNA-encoded polypeptides, confirming the nonstructural coding region location of the common epitope. Comparison of predicted amino acid sequences in the nonstructural coding regions of the comoviruses and picornaviruses suggested a potentially immunogenic linear epitope in protein 2C. The consensus peptide LEEKGI was synthezized and shown to be immunogenic for both BPMV- and CVB3-immune splenocytes.
Nearly 20 percent of the packaged RNA in bean-pod mottle virus (BPMV) binds to the capsid interior in a symmetric fashion and is clearly visible in the electron density map. The RNA displaying icosahedral symmetry is single-stranded with well-defined polarity and stereochemical properties. Interactions with protein are dominated by nonbonding forces with few specific contacts. The tertiary and quaternary structures of the BPMV capsid proteins are similar to those observed in animal picornaviruses, supporting the close relation between plant comoviruses and animal picornaviruses established by previous biological studies.
Computer analyses have revealed sequence homology between two non-structural proteins encoded by cowpea mosaic virus (CPMV), and corresponding proteins encoded by two picornaviruses, poliovirus and foot-and-mouth disease virus. A region of 535 amino acids in the 87-K polypeptide from CPMV was found to be homologous to the RNA-dependent RNA polymerases from both picornaviruses, the best matches being found where the picornaviral proteins most resemble each other. Additionally, the 58-K polypeptide from CPMV and polypeptide P2-X from poliovirus contain a conserved region of 143 amino acids. Based on the homology observed, a genetic map of the CPMV genome has been constructed in which the 87-K polypeptide represents the core polymerase domain of the CPMV replicase. These results have implications for the evolution of RNA viruses, and mechanisms are discussed which may explain the existence of homology between picornaviruses (animal viruses with single genomic RNAs) and comoviruses (plant viruses with two genomic RNAs).
A partial amino acid sequence of cowpea mosaic virus (CPMV) VPg radiochemically modified by chloramine-T and Bolton-Hunter reagent has been determined. VPg covalently bound to viral RNA chains (VPg-RNA) was iodinated with chloramine-T and Bolton-Hunter reagent to label tyrosine and lysine residues, respectively. [I]VPg-RNA was digested with nuclease P1 and the resulting [I]VPg-pU was purified by SDS-polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Control experiments with chemically synthesized poliovirus VPg showed the feasibility of radiochemical microsequence analysis of protein that had been radiochemically modified by chloramine-T and Bolton-Hunter reagent. Analysis of CPMV [I]VPg-pU revealed the presence of tyrosine residues at position 12 and 14, and of lysine residues at position 3 and 20, respectively. In combination with Edman degradation of unlabeled CPMV VPg, which showed serine and arginine residues to be present at position 1 and 2, respectively, the data obtained allow the precise positioning of VPg within the 200 000 dalton (200 K) polyprotein encoded by CPMV B RNA and the prediction of its entire amino acid sequence. VPg is located at the COOH terminus of its 60 K, membrane-bound,precursor and proximal to the amino terminus of the protease-polymerase domain of the polyprotein. A processing scheme for the 200 K polyprotein is discussed in which Gln-Ser amino acid pairs act as the major signal for proteolytic cleavage.