We have described a single morphogenetic pathway for the Bacillus subtilis bacteriophage Phi 29 in which the 12 megadalton genome is packaged into a relatively simple prohead constructed with the aid of a recyclable core-scaffolding protein. We will study Phi 29 structure and morphogenesis to define the molecular events of morphogenesis in vitro. During in vitro assembly with a yield of 200 phage/cell equivalent, more than 30% of the proheads are converted to phage, and 20% of the exogenous DNA-gp3 can be repackaged in an oriented, quantized fashion. With purified proheads and DNA-gp3, 30% of exogenous DNA-gp3 is packaged with the aid of the single viral protein pg16. We have crystallized intermediate particles and DNA-filled heads for X-ray and optical diffraction analysis in order to correlated the surface lattice structures with conformational changes that occur during prohead maturation and DNA packaging. Inactive purified proheads can regain their ability to package DNA-gp3 when complemented with an extract lacking viral structural proteins. We will study the conformation and composition of the head proximal vertex that serves as the connector for neck/tail assembly. Using purified DNA-gp3, proheads, gp16 and ATP we will study the process of substrate recognition in in vitro DNA packaging. We will correlated left-end DNA fragment packaging with changes in particle electrophoretic mobility, morphology, and lattice structure, and with the exit of the core-scaffolding protein gp7. We will interrupt DNA packaging with DNase and precisely measure the lengths of packaged molecules to advance our understanding of quantized packaging. We will continue studies of initiation of prohead assembly by using protein affinity chromatography and electron microscopy. We hope to assemble proheads more efficiently in vitro with proteins from dissociated proheads and new mutant combinations in complementations. We are studying ts mutants that make unusual intermediates in nonpermissive infections. We are continuing to identify cistron products and to add detail to the genetic map. We are studying the noncovalent hydrophobic interactions of dynamic proteins that reveal new reactive sites on nascent particles. The Phi 29 DNA-protein encapsidation is a model for studies of the genome packaging of poliovirus, SV40, foot and mouth virus and oncogenic adenovirus. We are studying the initiation of prohead assembly, DNA encapsidation, and the mechanisms of form determination in the most efficient in vitro DNA packaging and viral assembly system known.
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