Three unsolved problems in understanding the structure and assembly of double-standed DNA viruses are the control of the polymerization of precursor capsid subunits into icosahedral shells, the mechanism of the precise coiling of the DNA within the precursor shell, and the nature of the later transport of the chromosome out of the capsid into the cell. By purifying precursor forms of the gene 5 coat protein and gene 8 scaffolding protein of Phage P22, we have been able to assemble procapsids in vitro. Taking advantage of the in vitro assembly systems, and of the well-developed genetics and physiology of P22, we plan to dissect the detailed pathway by which the scaffolding protein directs the assembly of the coat protein into closed double shells. This will involve identification of coat/scaffolding intermediates in the in vitro reaction, as well as purification from extracts of mutant-infected cells of the complex containing the gene 1 portal protein that initiates procapsid assembly in vivo. In parallel with the pathway analysis, we are collaborating in systematic efforts to solve the protein structures in the precursor shells and mature virions at atomic resolution; procapsids, empty capsids, virions, and scaffolding subunits will be purified to grow crystals for X-ray diffraction. To support the structure analysis, the nucleotide sequence of gene 5 will be determined. To understand the coiling of the newly packaged DNA within the capsid, the procapsid to capsid transformation associated with this process will be studied in vitro. Raman spectroscopy will be used to characterize the in vitro transformation of wild type procapsids, and procapsids formed of mutant coat or scaffolding subunits, in an effort to identify the regions of the protein which functionally interact during scaffolding recycling and capsid expansion. To elucidate the mechanism by which the chromosome is released from the virion and injected in to the host cell, the minor virion gene products which carry out these functions wil be purified and their interactions with the chromosome, each other, and the host cell envelope characterized. These experiments should provide a first order understanding of the role of every virion protein in the infectious process.
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