Morphogenesis of the Bacillus subtilis bacteriophage phi29 will be studied in the most efficient in vitro viral assembly system known. Protein conformational change and movement in particle assembly and DNA encapsidation will be investigated by the use of an integrated genetic, biochemical and biophysical approach. Cryo-electron microscopy and X-ray crystallography will provide a structural basis for direct measurement of the dynamic events of particle assembly. A complete model of phi morphogenesis will serve as a model for animal virus assembly and aid in the search for new antiviral therapy. Specifically, the structure of phi29 DNA packaging intermediates and intermediates in neck/tail assembly will be studied by 3D cryo-EM reconstruction; high resolution structures of the phi29 pro-head, scaffolding protein and head-tail connector will be obtained by X-ray crystallography; the phi29 pro-head will be assembled in vitro from purified proteins and chaperonins; the interaction of the phi pro-head with the super-coiled DNA-gp3-gp16 complex and the initiation and progression of packaging will be visualized by electron microscopy; the rotational motion of the head- tail connector in DNA-gp3 packaging will be measured by the use of polarized absorption relaxation after photobleaching (PARAP) and by direct visualization; and the rate of DNA-gp3 packaging in real time and the force exerted by the DNA packaging machine will be measured by the use of the laser optical trap.
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