I propose to develop the cell-free reticulocyte lysate translation system with the aim of synthesizing active encephalomyocarditis(EMC) viral replicase in vitro. This will be achieved by setting up two different translation-replication reactions, called simultaneous and sequential, respectively. The two systems will be used to test various aspects of the picornavirus protease regulation hypothesis, such as the mechanism of VPg addition, proteolytic interconversion, and the roles of peptides C, D and E. In the event that the in vitro approach proves unfeasible, I propose to isolate EMC polymerase proteins from infected cells, taking into consideration the internal protease, and autocatalytic cleavage of these proteins. The proteins will then be tested according to the predictions of the protease regulation hypothesis.