The experiments described in this proposal are designed (1) to elucidate the mechanism of homologous DNA recombination in the orthopoxvirus vaccinia virus, and (2) to construct a novel type of recombinant vaccinia virus vector that provides """""""" runaway"""""""" expression of foreign genes. As well as increasing our understanding of the mechanism of a fundamental genetic process. these experiments will permit the design of safer and more effective vaccines based on recombinant vaccinia viruses. Vaccinia virus represents one of the very few systems in which DNA recombination in higher eukeryotic cells is accessible to both a genetic and a biochemical approach. We therefore plan to isolate recombination-deficient (rec-) mutants of the virus and to examine the structures and functions of the rec gene products using both infected cells in culture and cell-free systems. Deletion of viral rec genes that are not essential for virus replication will enhance the safety of potential vaccines that are based on vaccinia virus recombinants. In the second part of the proposal, plans are described for the construction of a vaccinia virus recombinant that expresses the gene for an RNA-dependent RNA polymerase from a nodavirus. This enzyme normally catalyzes the replication of its own mRNA and should thus be expressed at very high levels from vaccinia virus recombinants. The possibility will be explored that RNA sequences that encode proteins of general interest and importance can be rendered competent templates for replication by the nodavirus replicase by being sandwiched between sequences that function as origins of nodavirus RNA replication. It is hoped that this approach will head to the development of new high-level expression vectors and potential vaccines based on vaccinia virus recombinants.
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