The long-term objective is to elucidate the regulatory interactions of all macromolecules involved in the virus assembly processes. The project includes (1) establishment of a map of the virus assembly pathway that represents the processes occuring in infected cells; (2) development of an in vitro system that mimics the in vivo situati0n and synthesizes biologically active viruses from purified viral and host proteins; and (3) elucidation of the regulatory interactions of macromolecules involved in the virus assembly processes. With this sequence as the research program, the bacteriophage phi X174 system is chosen as a model system because physiology and genetics of the phage assembly in vivo have been well characterized. Furthermore, an in vitro phage synthesizing system from purified viral and host components has been established. The mechanisms involved in the formation of infectious phage in the in vitro system mimic those that occur in vivo during phage morphogenesis. This project has entered the last stage where the regulatory interactions of the macromolecules involved in the virus assembly can be investigated. The in vitro phage synthesizing system will be utilized extensively. Intermediates of assembly processes will be isolated by manipulating the in vitro system. DNA-protein and protein-protein interactions will be analyzed by foot printing, sequencing, cross-linking, two-dimensional gels, FPLC (HPLC) and Western blot/ELISA, etc. The phi X174 system will provide an excellent model system for the assembly process of the other spherical viruses, including animal and plant viruses. The linkage of DNA replication and packaging in phi X174 Stage III provides a unique opportunity to investigate in vitro a complex protein-DNA system on a molecular level.
