The long-term goal of this proposed basic research is designed to contribute substantially to a deeper understanding of the mechanism of viral morphogenesis by employing vaccinia virus (VV) as a model eukaryotic system. The morphogenesis of VV begins with the appearance of """"""""virus factories"""""""" within the cytoplasm of infected cells. These factories are the sites of viral transcription and DNA replication. Nearby these regions, and concomitant with the synthesis of late proteins, crescent-shaped cisternal membranes emanate. From these crescents the spherical immature viruses (IV) are made. IVs, which are non-infectious, are the precursors of the first infectious form of the virus: the intracellular mature virus (IMV). The transition from IV to IMV is an extremely complex event in which membrane assembly of the viral envelope, proteolytic processing of structural proteins, and packaging of the genome into the viral core, are all necessarily coordinated. The molecular details of these events are largely unknown. This proposal involves a focused study on an essential VV phosphoserine protein called VP11, which is encoded by the F17R gene. The function of VP11, which remains elusive, is known to be required for the IV to IMV transition. VV encodes two ser/thr protein kinases and a dual specificity phosphatase. All of these enzymes are essential. However, the precise function of dynamic cycles of phosphorylation in the VV life cycle is unknown, although recent studies have pointed to a correlation between phosphorylation and the progression of viral morphogenesis. Using a combination of genetic, virological, ultrastructural, and biochemical techniques, we will test the hypothesis that phosphorylation is required for the in vivo function of VP11. Towards this end, we will determine the precise serine residues of VP11 that are phosphorylated. Finally, we will extend and complement this knowledge by determining to what degree the function of vaccinia's VP11 can be substituted for with heterologous poxviral VP11 counterparts, including that from the genome of molluscum contagiosum virus (MCV). MCV is a specifically human poxvirus that causes benign neoplasms in children and opportunistic infections in AIDS patients.
