Activity of the hepatitis A virus IRES requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G

The question of whether translation initiation factor eIF4E and the complet e eIF4G polypeptide are required for initiation dependent on the IRES (inte rnal ribosome entry site) of hepatitis A virus (HAV) has been examined usin g in vitro translation in standard and eIF4G-depleted rabbit reticulocyte l ysates. In agreement with previous publications, the HAV IRES is unique amo ng all picornavirus IRESs in that it was inhibited if translation initiatio n factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In additi on, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, whic h binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m(7)GpppG cap a nalogue, irrespective of whether the RNA tested was capped or not. Thus, in itiation on the HAV IRES requires that eIF4E be associated with eIF4G and t hat the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction betwee n an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interac tions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the e IF4E unoccupied.