The long term objective of this project is to elucidate the molecular mechanism controlling the assembly of icosahedral virus capsids. The key questions that we wish to address are: 1) What is the pathway by which viral protein subunits assemble into icosahedral capsids, and 2) What controls the conformation switching of the subunits that is required for proper assembly. Our approach is to study the in vitro assembly of the procapsid of wild type and temperature sensitive bacteriophage P22 strains. In this application I propose: 1) To dissect the initiation complex for the in vitro polymerization of procapsids. the presence of the phage encoded pilot protein increases the rate of assembly by stabilizing the initiation complex I will (a) characterize, and isolate the complex of pilot protein with coat protein responsible for stabilizing the initiation complex and (b) I will purify assembly competent chemically crosslinked coat protein oligomers and directly test their ability to initiate the assembly reaction, and interact with the scaffolding protein. 2) To detect, characterize and isolate the """"""""building blocks"""""""" for procapsid assembly. The two potential candidates are (a) an oligomer of scaffolding protein, and (b) a coat/protein scaffolding protein hetero- oligomer. The nature of the scaffolding oligomer will be defined by analytical ultracentrifugation. Mixed oligomers will be described by fluorescence, analytical ultracentrifugation, and cryo-electron microscopy. The use of bis-ANS to trap assembly intermediates will be examined. 3) To detect the conformation changes in the coat proteins subunits accompanying assembly. These conformation changes are required for successful polymerization of coat protein subunits into procapsids. The changes in secondary structure will be detected by circular dichroism, and changes in side chain environment by both CD and fluorescence. The stabilization afforded to the subunit upon polymerization will be assessed by thermal studies, and the origin of the morphology change following in vitro polymerization determined by cryoelectron microscopy and image reconstruction. Development of therapeutic agents targeted at direct inhibition of subunit assembly during viral morphogenesis is a promising though relatively unexplored arena. The increasing research effort devoted to the development of viruses as delivery vehicles for therapeutics, be they DNA, protein, or chemotherapeutic agents suggests that the ability to assemble virions i vitro from their proteins subunits in a controlled fashion will ultimately to be essential. It is expected that these studies will contribute to the conceptual framework required to realized these medically important goals.
