MAPPING THE CLEAVAGE SITE IN PROTEIN-SYNTHESIS INITIATION FACTOR-EIF-4-GAMMA OF THE 2A PROTEASES FROM HUMAN COXSACKIEVIRUS AND RHINOVIRUS

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by in itiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4gamma. Infection of cells with picorn aviruses of the rhino- and enterovirus groups causes a shut-off in tra nslation of cellular mRNAs but permits viral RNA translation to procee d. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease. In this report we have u sed highly purified recombinant 2A protease from either human Coxsacki evirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4gamma in vi tro in the eIF-4 complex purified from rabbit reticulocytes. Neither t he rate of cleavage nor fragment sizes were affected by addition of eI F-3. The NH2- and COOH-terminal fragments of eIF-4gamma were separated by reverse phase HPLC and identified with specific antibodies, and th e NH2-terminal sequence of the COOH-terminal fragment was determined b y automated Edman degradation. The cleavage site for both proteases is 479GRPALSSR down GPPRGGPG494 in rabbit eIF-4gamma, corresponding to 4 78GRTTLSTR down GPPRGGPG493 in human eIF-4gamma.