The long term objective of this project is to increase our knowledge of the life cycle of dsDNA viruses through study of the molecular mechanisms by which their virions assemble and function. The viruses chosen for this study, the tailed-bacteriophages, have direct medical importance since they are the most abundant `life form' on Earth and have huge importance in bacterial pathogenicity and microbial ecology. They also serve as very important models for similar processes in less experimentally accessible eukaryotic viruses such as Herpesviruses, Iridopoxviruses and Adenoviruses. Thus the proposed research is relevant to human health. The P22 bacteriophage system utilized in this project is one of the most genetically and biochemically well-developed of all virus systems, and so is ideal for obtaining new knowledge about the assembly of virus particles. This type of virus first assembles a protein shell, called a procapsid, and then inserts the dsDNA chromosome into this shell to form a virion. The P22 procapsid contains three critical polypeptides, a coat protein that forms the external shell, an internal scaffolding protein which is required to build the procapsid but leaves before DNA enters, and a portal protein which forms a ring through which DNA enters the coat protein shell. A P22-encoded `terminase' interacts with procapsids and is critical to the DNA recognition and entry processes, and three additional proteins bind to stabilize the packaged DNA. The procapsid with its scaffold and terminase are central features of the assembly strategy of most if not all large dsDNA viruses, but many features of their assembly and function remain unknown. The role of the scaffolding protein in procapsid assembly is one focus of this proposal. It guides coat protein's assembly into a closed shell by co-assembling with it, and it recruits other proteins into the structure. After co-assembly with coat protein to form procapsids, scaffolding protein is released from the procapsid before the DNA insertion step, and so it functions catalytically in virion assembly. The other focuses of the proposal are aimed at understanding the structure and function of the components of the DNA packaging/injection molecular machine which include the terminase DNA packaging motor and proteins that are injected into cells with the DNA.
The aims of the project will be pursued through a combination of genetic, biochemical, biophysical and structural analysis strategies.
The proposed work on virus particle assembly and function focuses on the study of the proteins that are part of the portal vertex molecular machine of bacteriophage P22. The roles of these proteins, which form the motor that packages DNA and releases DNA during injection, are studied by genetic, biochemical and structural analysis ? ? ?
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