STUDY OF NUCLEOTIDE SEQUENCES ENCODING ANTIGENIC SITES

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The continued study of rabies virus glycoprotein antigenic sites will concentrate on the location of single amino acid changes in the glycoprotein of antigenic variants which differentiate epitopes involved in the binding of virus-neutralizing antibodies. Comparative nuecleotide sequence analysis will identify mutations in variant glycoprotein genes which express antigenically altered glycoproteins. This will be accomplished by using synthetic DNA primers in primer extension experiments to obtain complementary DNA transcripts from virion RNA templates isolated from the antigenic variant viruses. Amino acid substitutions deduced from the altered nucleotide sequences will be correlated with the grouping of epitopes in the functional epitope maps of two fixed rabies virus strain glycoproteins. This study will be extended to compare antigenic determinants of fixed virus with street rabies virus glycoproteins. The topographic relationship of amino acids associated with the different epitopes in the rabies virus glycoprotein will be determined based on (a) the linear distribution of residue changes deduced from nucleotide sequence analysis, and (b) the effect of substituted residues induced by site-directed mutagenesis of the cloned glycoprotein gene. The significance of the induced change as a local perturbation of the antigenic structure will be evaluated. Conformational and/or linear alterations in the structure of the glycoprotein derived from the mutated gene will be characterized by its ability to bind monoclonal antibodies. Finally, the nucleotide sequence of a cloned rabies virus nucleoprotein double-stranded complementary DNA will be determined, and the sequences encoding antigenic sites will be investigated. Antigenic variations reflected by differences in chemical structure of the two major antigens (nucleoprotein and glycoprotein) will define limitations of effective vaccine prophylaxis and post-exposure treatment.