Retroviruses cause numerous diseases in humans and other animals. During the life cycle of all retroviruses, the budded viral particles undergo a carefully timed morphogenesis into their mature morphology. This morphogenesis process is driven by the activation of a viral protease, but it is not known for any retrovirus what factors control this timed event. While several researchers have succeeded in replicating the process of retroviral particle formation in in vitro systems, because of inherent difficulties in purifying proteins with an active protease, none have attempted to reconstitute the process of retroviral particle maturation in vitro. The experiments outlined in this proposal are designed to: 1) identify a source of wild type Rous sarcoma virus (RSV) Gag protein, the major structural protein which encodes the protease; 2) optimize a system for solubilizing and purifying RSV Gag; 3) assemble RSV Gag in Vitro and determine the conditions required for protease activation and 4) assess whether RSV's enzymatic protein, Gag/PR/Pol, can be incorporated and properly matured in an in vitro system. This work has two health related implications: 1) the knowledge of what activates retroviral proteases could lead to new strategies for fighting infections such as inhibiting or prematurely initiating protease activation; and 2) developing a technology encompassing the ability to assemble and mature retroviral particles in vitro is an obligatory prerequisite to the production of safe retroviral gene therapy vectors in vitro.
