The long term goal of this project is to understand the mechanisms employed in the expression of early and late vaccinia virus genes, and in the regulation of their expression. In this proposal, we focus our attention on one viral enzyme, the vaccinia capping enzyme complex. This enzyme has been shown to catalyze the first three steps in the pathway of cap formation. In addition, this remarkable complex is required for DNA sequence specific transcription termination of early genes. In this proposal we will first employ immunological and biochemical techniques to investigate the synthesis and assembly of the virus capping enzyme complex, in vivo, and to determine if the capping enzyme employed late in infection is the same as the virion encapsidated enzyme. Secondly, we will combine genetic and biochemical approaches to study this enzyme complex both in vivo and in vitro. We will first characterize the capping enzyme, and capt formation in vivo, in mutants which are resistant to the S-adenosyl methionine analogue sinefungin. Next, we will isolate conditional lethal mutations to determine the role of mRNA cap formation and early gene transcription termination in the vaccinia life cycle. Finally, we will construct expression vectors to produce large quantities of each capping enzyme subunit in E. coli. We will develop methods to purify the subunits and to reconstitute capping activity and transcription termination activity in vitro. We will localize the active site of each enzyme activity to one subunit and then employ specific substrate analogues to identify the amino acids present in each active site.
