Mapping of Functional Domains in Eukaryotic Protein Synthesis Initiation Factor 4G (eIF4G) with Picornaviral Proteases

• Published in: The Journal of biological chemistry 1995-09, Vol.270 (37), p.21975-21983
• Main Authors: Lamphear, Barry J., Kirchweger, Regina, Skern, Tim, Rhoads, Robert E.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.37.21975

Cap-dependent binding of mRNA to the 40 S ribosomal subunit during translational initiation requires the association of eukaryotic initiation factor 4G (eIF4G; formerly eIF-4γ and p220) with other initiation factors, notably eIF4E, eIF4A, and eIF3. Infection of cells by picornaviruses 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 association of eIF4G with eIF4A, eIF4E, and eIF3. Both proteases bisect eIF4G into N- and C-terminal fragments termed cpN and cpC·. cpN was shown to contain the eIF4E-binding site, as judged by retention on m7GTP-Sepharose, whereas cpC· was bound to eIF3 and eIF4A, based on ultracentrifugal co-sedimentation. Further proteolysis of cpN by L protease produced an 18-kDa polypeptide termed cpN2 which retained eIF4E binding activity and corresponded to amino acid residues 319-479 of rabbit eIF4G. Further proteolysis of cpC· yielded several smaller fragments. cpC2 (∼887-1402) contained the eIF4A binding site, whereas cpC3 (∼480-886) contained the eIF3 binding site. These 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 internal initiation of picornaviral RNAs.