The long term goals of the experiments outlined in this proposal are to investigate the proteolytic processing of vaccinia virus (VV) core proteins with regard to: (i) identification of the endo- and/or exoproteolytic pathways required for the maturation of VV core proteins; (ii) analysis of the precursor protein determinants which specify the correct site(s) of proteolytic processing; (iii) identification and molecular examination of the protease(s) responsible for these reactions; (iv) elucidation of whether, and how, the substrate-protease interaction is regulated in vivo; and, (v) determination of the role processed VV core proteins play in assembly of progeny viral particles. Three of the major VV core constituents proteins 4a, 4b, and 25K are late viral proteins produced as precursor polypeptides, p4a, p4b and p25K which are subjected to proteolytic cleavage during viral morphogenesis. Since previous work in our has indicated this proteolysis step plays a very central and important role in the assembly of infectious virions, we now propose to directly analyze this reaction. The map location of these loci and their nucleotide sequences have recently been determined. This information will be used to design fusion protein and synthetic peptide antigens with which to generate epitope-specific antisera for use in concert with immuoprecipitation, western blotting, and immunofluorescence procedures to establish the sequence, kinetics, and cytolocalization of the processing reactions which occur within the infected cell. Furthermore, comparison of amino acid sequences predicted from the derived nucleotide sequences of p4a, p4b, and p25K with the N-terminal amino acid sequences of mature 4a, 4b, and 25K proteins (determined by microsequencing procedures) should identify the portions of the precursors which contain potential cleavage sites. This information in concert with site-directed mutagenesis and marker transfer techniques will be used to dissect the important cis recognition features of these cleavage sites. Similarly, plasmid expression vectors will be utilized to preparatively produce precursor proteins for use in in vitro and in vivo cis and trans processing assays designed to identify the proteinase(s) which catalyze these reactions. If, as is likely, these functions are virus-encoded, the genes will be mapped and subjected to detailed molecular genetic analyses. It is likely that the results of these experiments will provide considerable insight regarding proteolytic processing during VV replication. Given the ability to readily carry out directed genetics on VV-encoded gene products, this information should enable detailed structure-function studies on the molecular mechanisms of proteolysis to be undertaken.
