MAPPING OF FUNCTIONAL DOMAINS IN EUKARYOTIC PROTEIN-SYNTHESIS INITIATION-FACTOR 4G (EIF4G) WITH PICORNAVIRAL PROTEASES - IMPLICATIONS FOR CAP-DEPENDENT AND CAP-INDEPENDENT TRANSLATIONAL INITIATION

Cap-dependent binding of mRNA to the 40 S ribosomal subunit during tra nslational initiation requires the association of eukaryotic initiatio n factor 4G (eIF4G; formerly eIF-4 gamma and p220) with other initiati on factors, notably eIF4E, eIF4A, and eIF3. Infection of cells by pico rnaviruses results in proteolytic cleavage of eIF4G and generation of a cap-independent translational state. Rhinovirus 2A protease and foot -and-mouth-disease virus L protease were used to analyze the associati on of eIF4G with eIF4A, eIF4E, and eIF3. Both proteases bisect eIF4G i nto N- and C-terminal fragments termed cp(N) and cp(C). cp(N) was show n to contain the eIF4E-binding site, as judged by retention on m(7)GTP -Sepharose, whereas cp(C) was bound to eIF3 and eIF4A, based on ultrac entrifugal co-sedimentation. Further proteolysis of cp(N) by L proteas e produced an 18-kDa polypeptide termed cp(N2) which retained eIF4E bi nding activity and corresponded to amino acid residues 319-479 of rabb it eIF4G. Further proteolysis of cp(C) yielded several smaller fragmen ts. cp(C2) (similar to 887-1402) contained the eIF4A binding site, whe reas cp(C3) (similar to 480-886) contained the eIF3 binding site. Thes e results suggest that cleavage by picornaviral proteases at residues 479-486 separates eIF4G into two domains, one required for recruiting capped mRNAs and one for attaching mRNA to the ribosome and directing helicase activity. Only the latter would appear to be necessary for in ternal initiation of picornaviral RNAs.