Fig. 4. Inhibition of translation observed with HIV-2 PR is due to cleavage of eIF4GI (A and B). (A) RRL (10 µl) or (B) HeLa lysate (127.5 µg) was incubated with buffer (lane 1) or increasing amounts of recombinant HIV-2 PR (lanes 2–5: 2.5, 5, 10 and 25 ng/µl) for 1 h at 30°C in a final volume of 20 µl. Aliquots were resolved on SDS–PAGE, proteins transferred to PVDF and the membranes were incubated with antibodies specific to eIF1, eIF1A, eIF3, eIF4A, eIF4B, eIF4E and the C-terminal part of eIF4GI, as indicated on the left side of each panel. For eIF4GI, resulting fragments and molecular weight markers (in kDa) are indicated on the figure. (C) RRL under full translation conditions was pre-incubated without (lanes 1 and 2) or with (lanes 3 and 4) 7 ng/µl HIV-2 PR. After 30 min at 30°C, Palinavir (10 µM final concentration) was added to the reactions with 1 ng/µl recombinant p100 fragment (lanes 2 and 4) or buffer (lanes 1 and 3). Uncapped XL–EMCV mRNA (10 ng/µl) was then translated under these conditions. Samples were processed on 15% SDS–PAGE, submitted to autoradiography, the relative intensities of the bands were quantified and the results are presented at the bottom of the figure.