9600574 Casjens The long term objective of this project is the furtherance of our knowledge of the life cycle of dsDNA viruses by gaining an understanding of the molecular mechanisms by which these viruses package DNA and assemble progeny virions within infected cells. The particular virus chosen for this study, bacteriophage P22, serves as a model for similar processes in less experimentally accessible eukaryotic viruses such as the dsDNA Herpesviruses and Adenoviruses. The P22 system is one of the most highly studied virus systems, both genetically and biochemically. This type of virus assembles protein shells, called procapsids, first and then inserts the dsDNA chromosomes into these shells. The icosahedral P22 procapsid contains three critical polypeptides, an external coat protein, an internal scaffolding protein and the portal protein. In addition, two non-structural P22 encoded proteins are critical in the DNA recognition/entry process. The coat protein forms the exterior shell of the virus particle, the scaffolding protein is required to build the procapsid but leaves the structure before DNA enters, and the portal protein forms a ring-shaped structure at one vertex of the icosahedral protein shell. DNA is thought to enter into this preformed capsid through the portal structure. The procapsid is a central feature of this viral assembly strategy and few details are known about its assembly and function. We propose to examine the mechanism of assembly of the P22 procapsid. In particular, the function of the scaffolding protein, which guides the coat protein assembly by co-assembling with it, will be studied in detail. After co-assembly with coat, scaffolding protein is released, and so functions catalytically in virion assembly. Questions will be addressed from genetic, biochemical, biophysical and structural directions. Knowledge gained will contribute to the understanding of the life cycle of the dsDNA viruses, and will also be relevant to all studies of the catalysis of macromolecular assembly. %%% The objective of this project is to understand the molecular mechanisms by which dsDNA viruses package DNA and assemble progeny virions within infected cells. Knowledge gained will contribute to the understanding of the life cycle of dsDNA viruses and to the catalysis of macromolecular assembly. Bacteriophage P22 is chosen as a model for this investigation. P22 assembles protein shells first and then inserts the dsDNA chromosomes into these shells. The icosahedral P22 procapsid shell contains three critical structural polypeptides namely an external coat protein, an internal scaffolding protein which is required to build the procapsid but leaves the structure before DNA enters, and a ring-shaped portal protein through which DNA is thought to enter into this preformed capsid. Two additional non-structural proteins that are critical for the DNA recognition/entry process are also present in the procapsid. The procapsid is a central feature of this viral assembly strategy, yet few details are known about its assembly and function. We propose to examine the detailed catalytic function of the scaffolding protein which guides the coat protein assembly and co-assembles with it and is followed by its own release using genetic, biochemical, biophysical and structural approaches. ***
