Role of scaffolding protein in bacteriophage P22 morphogenesis

The scaffolding protein of the dsDNA bacteriophage P22 was studied in order to better understand the molecular basis of procapsid assembly. The procapsid is a structural precursor to the capsid, a protein shell that encloses and protects a viral chromosome. The phage P22 procapsid is composed of icosahedrally arranged coat protein subunits encasing a core of scaffolding protein and contains a ring of 12 portal protein subunits at one of its 12 icosahedral vertices. The scaffolding protein activates the assembly of coat protein subunits, directs the positioning of those subunits such that a closed shell of the correct dimension is formed, and is required for the inclusion of portal and injection proteins into the procapsid. DNA enters the procapsid through the portal vertex and the scaffolding protein exits the procapsid intact before DNA packaging is completed. How scaffolding protein accomplishes these processes is the focus of the work presented in this dissertation. The scaffolding protein was first examined by a functional domain analysis in vivo. Scaffolding protein truncation mutants were cloned and expressed in P22's host, Salmonella enterica serovar Typhimurium, during infection with a P22 strain defective in scaffolding protein synthesis. Mutants were tested for their ability to complement multiple rounds of infection, exit from the procapsid, recruit portal and injection proteins, and form procapsid-like particles. Successive N-terminal deletions defined the boundaries of amino acids necessary for these functions, including a 15 amino acid minimal coat protein binding domain. Scaffolding protein's ability to bind coat protein and promote assembly of procapsid-like particles both was also studied. Point mutations within the coat protein binding domain were generated and mutant scaffolding proteins were expressed in vivo during scaffolding deficient P22 infection. Fifteen different single amino acid substitutions failed to abrogate coat protein binding in vivo. Scaffolding protein mutants were subsequently overexpressed, purified, and assayed, in vitro, for the abilitiy bind coat protein as well as assemble procapsid-like particles from purified monomeric coat protein. Mutations that affected both processes were found.