THE PROTEOLYTIC CLEAVAGE OF EUKARYOTIC INITIATION-FACTOR (EIF) 4G IS PREVENTED BY EIF4E BINDING-PROTEIN (PHAS-I-4E-BP1) IN THE RETICULOCYTELYSATE

A common feature of viral infection is the subversion of the host cell machinery towards the preferential translation of viral products. In some instances, this is partly mediated by the expression of virally e ncoded proteases which lead to the cleavage of initiation factor eIF4G . The foot-and-mouth disease virus encodes two forms of a cysteine pro teinase (L protease) which bisects the eIF4G polypeptide into an N-ter minal fragment containing the eIF4E binding site, and a C-terminal fra gment which contains binding sites for eIF4A and eIF3 and which associ ates with the 40S ribosomal subunit. Previously, we have demonstrated that the cleavage of eIF4G by L protease stimulates the translation of uncapped transcripts encoding cellular proteins and supports internal initiation driven by picornavirus internal ribosome entry segment (IR ES) elements. Use of reticulocyte lysates manipulated to deplete them of eIF4E and the N-terminal fragment suggests that the C-terminal frag ment of eIF4G is responsible for these effects, and we have now confir med this by purifying the C-terminal fragment and analysing its effect s directly in the absence of L protease. Interestingly, we find that p re-incubation of reticulocyte lysates or ribosomal salt wash fractions with the specific eIF4E binding protein, PHAS-I (eIF4E-BP1), blocks t he proteolytic cleavage of eIF4G by L protease. This effect can be rev ersed by addition of recombinant eIF4E. These data are consistent with a model whereby the L protease cleavage site in eIF4G is inaccessible until a change in conformation is induced by the binding of eIF4E. Th is may have implications for a role for eIF4E binding in triggering ch anges that expose other domains in the eIF4G molecule during initiatio n of translation.