Our long-range goal is to understand virus assembly at the molecular level. DNA packaging is a critical step in the assembly of many double-stranded DNA viruses, including poxvirus, adenovirus, herpesviruses, and many bacteriophages. Terminase enzymes are common to these viruses and function to package viral DNA into the capsid. Common mechanisms for genome packaging have been proposed for these all viruses. The process of genome packaging is unique to viruses, and thus represents an ideal target for antiviral therapy; however, the lack of mechanistic detail precludes a rational approach to drug design. Bacteriophage lambda presents an ideal system to study DNA packaging. Lambda terminase is a central component of an ordered series of packaging intermediates, and is an integral part of the packaging motor. The goal of this project is to define a coherent physical and kinetic model for the assembly of a viral DNA packaging motor. A mechanistic model for any complex biological process requires a description of the physical nature of the macromolecular complexes involved, a kinetic dissection of the catalytic activities required for the process, and a mechanism that clearly describes the linkage between catalysis, structure, and function. Unfortunately, virtually nothing is known about the physical properties of the packaging intermediates in lambda. Moreover, a mechanistic link between catalytic activity and function remains elusive, due in part to the dearth of structural information. This project seeks to (i) characterize the interactions responsible for specific assembly of the packaging motor on viral DNA, (ii) define the assembly state of the functional packaging motor, to (iii) characterize critical packaging intermediates, and (iv) characterize the activity of defined enzyme species to provide a direct link between stucture and function of termiase. A combined structural, biophysical, and kinetic approach will define critical aspects central to our understanding of the mechanism and enzymology of DNA packaging.
