Poxviruses are highly unusual among DNA viruses in replicating solely within the cytoplasm of the infected cell. This physical autonomy from the nucleus is accompanied by genetic complexity: ~200 viral gene products regulate diverse aspects of the viral life cycle. Despite this genetic autonomy, the close relationship between cell biological processes and the progression of the viral life cycle is increasingly clear. Nowhere is this interplay more evident than during the morphogenesis of nascent virions in the cytoplasm. The biogenesis of the poxvirus membrane is one of the most intriguing and puzzling questions in the field. Genetic analyses have identified the F10 protein kinase, and the A6, A11, A30.5, H7 and L2 regulatory proteins as essential mediators of crescent biogenesis. In their absence, membrane diversion and crescent formation are defective, but little is known about how they coordinate and induce membrane egress from the ER. Likewise, when the major membrane proteins A14 or A17 are repressed, crescents do not form, but large numbers of ~25 nm vesicles accumulate in the cytoplasm. Thus, they are essential for the shaping, stability and elongation of nascent membranes. Their role in mediating these processes remains unknown. Our long-term goal is to fully understand poxviral membrane biogenesis. Our current objectives are to identify host proteins that enable the assembly and egress of viral ER exit sites, to understand the mechanism by which the viral regulatory proteins mediate this process, and to evaluate the dynamic interaction of A14 and A17 with membranes. We will accomplish these objectives by using genetic, cell biological and biochemical approaches to pursue three complementary Aims.
Aim I : Analysis of the role of host proteins and membrane trafficking systems in viral morphogenesis. We will identify and analyze host proteins that are either substrates of the F10 protein kinase, associated with viral membrane intermediates, or important for viral membrane biogenesis.
Aim II : Analysis of the A6, A11, A30.5, H7 and L2 proteins and their contributions to crescent biogenesis.
This aim i s focused on characterizing the biochemical properties of these 5 regulatory proteins, assessing their interaction with each other, with membranes, and with host proteins, and gaining insight into how they direct and facilitate membrane biogenesis.
Aim III : Assessment of the association of A14 and A17 with membranes in vivo and in vitro. Using a variety of innovative in vitro and in vivo techniques, we will probe the topology of these proteins within membranes and test the hypothesis that they mediate membrane tubulation or heterotypic fusion.
The fear that smallpox might be used as a bioterrorist weapon, the recognition that monkeypox virus is a cause of human morbidity and mortality, and the promise of using poxviruses as therapeutic vaccines reinforce the need to study the poxviral life cycle in depth. Our investigations of virion morphogenesis will define new targets for the development of rational antiviral therapies and provide fundamental insights into the membrane dynamics that are integral to many vital biological processes.
|Greseth, Matthew D; Carter, Dominique C; Terhune, Scott S et al. (2017) Proteomic Screen for Cellular Targets of the Vaccinia Virus F10 Protein Kinase Reveals that Phosphorylation of mDia Regulates Stress Fiber Formation. Mol Cell Proteomics 16:S124-S143|
|Unger, Bethany; Mercer, Jason; Boyle, Kathleen A et al. (2013) Biogenesis of the vaccinia virus membrane: genetic and ultrastructural analysis of the contributions of the A14 and A17 proteins. J Virol 87:1083-97|