FIG. 3.
4E-BP1 and eIF4E have no effect on EMCV protein synthesis and replication in nuclease-treated Krebs-2 cell extract. (A) Products of EMCV mRNA translation. Reaction mixtures contained the nuclease treated Krebs-2 cell extract but otherwise were identical to that described in Fig. 2A. Reaction mixtures were preincubated with control buffer, GST-4E-BP1 (60 μg/ml), or eIF4E (12 μg/ml) where indicated. Translation was performed at 32°C for 3 h. Aliquots (5 μl) of the translation reaction mixtures were analyzed by SDS-PAGE. An autoradiogram of the dried gel is shown. [35S]methionine incorporation into EMCV-specific protein 3Dpol was quantified using a Fuji BAS2000 phosphorimager. The value obtained from the reaction performed in the absence of added protein (lane 2, control) was defined as 100%. (B) EMCV yields. Reaction mixtures (30 μl, unlabeled) were preincubated with control buffer, GST-4E-BP1, or eIF4E and programmed with EMCV mRNA, as specified above. Plaques were scored following incubation for 20 h at 32°C and RNase treatment. Data represent the average of three determinations. Error bars indicate the standard deviation from the mean. (C) Coaddition of capped mRNA competitors rescues the regulation of translation of EMCV mRNA by 4E-BP1 and eIF4E in nuclease-treated extract. EMCV mRNA (4 μg/ml) was translated at 32°C for 90 min in the absence (lanes 2 and 9) or presence of either total Krebs-2 cell poly(A)+ mRNA (40 μg/ml, lanes 3 to 6) or globin mRNA (10 μg/ml, lanes 10 to 13). Reaction mixtures were preincubated with control buffer, GST-4E-BP1 (60 μg/ml), eIF4E (12 μg/ml), or m7GDP (0.5 mM) where indicated. Products of translation of Krebs-2 cell poly(A)+ mRNA or globin mRNA alone are shown in lanes 1 and 8, respectively. No mRNA was added to the reaction mixture analyzed in lane 7. Relative values for [35S]methionine incorporation into EMCV-specific protein 3Dpol were determined as in panel A. On panels A and C, the assignment of EMCV polypeptides was as described previously (55). The positions of the 14C-methylated protein molecular weight markers (GE Healthcare) are also shown. An asterisk on panel C indicates the position of globin.