Vaccinia virus is a complex DNA virus that exhibits a significant degree of physical and genetic autonomy from the host cell. Its 192 kb genome encodes most of the functions required for gene expression, genome replication, and virion morphogenesis, as well as many proteins that intersect with the apoptotic, antiviral and immune/inflammatory response of the host. The basis of this proposal is the recognition that vaccinia virus also encodes two protein kinases and a protein phosphatase that are essential for productive viral infection. It is now well recognized that reversible phosphorylation is a profoundly important and universal mechanism for biological regulation. At the level of an individual protein, phosphorylation can alter stability, catalytic activity, intracellular trafficking, and protein/protein interactions as well as associations with nucleic acids or membranes. Vaccinia virus represents a biomedically important and genetic tractable system for functional analysis of protein kinases and phosphatases. This proposal represents our continuing interest in understanding how the F10 kinase and H1 phosphatase regulate the vaccinia virus life cycle. The F10 kinase, which the applicant has shown to be a dual specificity kinase, plays an essential role in virion morphogenesis, where as the H1 phosphatase, which is also a dual specificity enzyme, is essential for ensuring the stability, transcriptional competence and infectivity of nascent virions. The three aims of this proposal concern: 1. Analysis of the F10 kinase: 2. Morphogenesis of the virion membrane, and 3. Understanding the role of the H1 phosphatase in the maturation of the nucleoprotein template and the virion core. She will use genetic, biochemical and cell biological approaches to investigate the structure and function of these enzymes in vivo and to identify and characterize their substrates. She will also test our hypothesis that the F10 kinase intersects with the membranes of pre-Golgi compartment, causing the diversion of transport vesicles, which are then remodeled into viral membranes with the participation of key viral proteins such as A14, A17 and H5. The applicant will also test the hypothesis that the transcriptional defect in H1 phosphatase-deficient virions is primarily due to the hyperphosphorylation of the F18 DNA-binding protein, and she will use substrate-trapping mutants of H1 to identify additional viral and cellular substrates for the phosphatase.
