We propose to extend our studies of the use of a plant virus as a vector for expressing antigenic polypeptide epitopes of animal viruses. The chimeric viruses will be tested as potential vaccines and analyzed by X- ray crystallography to establish the most effective way to introduce heterologous peptides into a virus capsid protein. Cowpea mosaic virus (CPMV) can be grown to high titer in blackeye peas. CPMV is an icosahedral plant virus with biological and structural similarity to animal picornaviruses. The 2.8 angstroms X-ray structure has been refined to 18.1% with the Hendrickson-Konnert procedure. An infectious clone of the virus has been developed and both site-directed and insert mutants have been prepared and shown to infect cowpea protoplasts. Two of the insertional mutants and two site-directed mutants grow in whole plants. Yields of 1-2 grams of wild type virus per kilogram of leaves (the mass of leaves from approximately 100 pots of plants occupying 50 square feet of space in a greenhouse) can be obtained in a preparation requiring less than two days and costing less than $50.00. We propose to create a highly infectious chimeric plant virus with the immunogenic regions known as the """"""""kennedy epitope"""""""" of gp41 of HIV, the """"""""V3 loop"""""""" from gp120 of HIV and the """"""""NIM-1A antigenic determinant"""""""" in rhinovirus 14 expressed as part of the capsid protein of CPMV. Crystallographic analyses of the chimeras will be performed to learn the factors affecting the antigenicity of linear epitopes and to improve their presentation. Crystallographic analysis of the gp120 protein of HIV will be initiated with material supplied by Cambridge Biotech, Worchester, MA. The protein is expressed in a baculovirus system and characterization of the glycosylation shows that the carbohydrate chains are on average two thirds of the length of gp120 isolated from mammalian. Experiments will also be performed with the T=3 icosahedral plant virus cowpea chlorotic mottle virus (CCMV) to determine if insertional mutagenesis is feasible with this system.
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