The long term goals of this project are to understand the functional organization of the vaccinia virus genome, the mechanisms by which vaccinia virus gene expression is regulated, and the interactions between vaccinia virus and the host cell. The approach taken to achieve these goals is the isolation and functional analysis of conditionally lethal mutants of the virus. Specifically, this application proposes to characterize in detail previously isolated temperature sensitive mutations which seem to affect three distinctly different aspects of vaccinia biology. Viral transcription will be addressed by studying several temperature sensitive mutations affecting two of the viral RNA polymerase subunits. Post transcriptional regulation of RNA stability will be addressed by studying temperature sensitive, drug resistant, and drug dependent mutants all related to an "abortive late" mutant phenotype. Virion morphogenesis will be studied by analyzing a large group of temperature sensitive mutants which confer a "normal" phenotype on an infection done at the non- permissive temperature. This application also proposes to continue the development of methods for site directed in vitro mutagenesis of vaccinia genes. Using a recently developed protocol for efficient insertion of unselected markers into the vaccinia genome, attempts will be made to construct temperature sensitive mutations in specific viral genes after chemical mutagenesis of defined viral DNA's in vitro. Experiments will also be performed to test the usefulness of a newly constructed nonsense suppressing cell line for the isolation of nonsense mutants of vaccinia virus.