FIGURE 3.

Effects of PABP depletion on EMCV genome expression in nuclease-treated Krebs-2 S10 extract. (A) Extracts were treated with glutathione-Sepharose-bound GST or GST-Paip2. Equal aliquots of these extracts (4 μL, 50 μg of protein) were analyzed by Western blotting for PABP or actin, as indicated. Signals were detected by ECL and quantified. The degree of PABP depletion was 99.5%. (B) Cap-dependent translation of a reporter mRNA is strongly reduced by PABP depletion. Capped poly(A+) luciferase mRNA (1 μg/mL) was translated in extracts that were either depleted of PABP or mock-depleted (Control). The reaction mixtures were supplemented with recombinant human PABP where indicated. (C) EMCV IRES-mediated translation of a reporter mRNA is less inhibited than cap-dependent translation by PABP depletion. Control and PABP-depleted extracts were programmed with EMCV IRES-containing poly(A+) luciferase mRNA (1 μg/mL) in the absence or presence of PABP as indicated. For panels B and C, relative luciferase units (RLU) are given for 1-μL aliquots of translation samples. Data are averages of three independent assays (with standard deviation from the mean). (D) EMCV RNA translation is slightly reduced by PABP depletion. Control and PABP-depleted extracts were programmed with the indicated concentrations of EMCV RNA in the presence of [³⁵S]methionine as described for Fig. 2A. Labeled proteins were analyzed by SDS-PAGE and fluorography. (E) RNA synthesis in control and PABP-depleted extracts programmed with the indicated concentrations of EMCV RNA. [α-³²P]CTP pulse labeling of RNA and its analysis were as described for Fig. 2B. (Arrow) Position of single-stranded EMCV RNA. (F) EMCV RNA dose response of virus yields as affected by PABP depletion. Control and PABP-depleted extracts were programmed with the indicated concentrations of EMCV RNA as described for Fig. 2C. EMCV titers were determined in triplicates. Average titer values (with standard deviation from the mean) are shown. (G) EMCV synthesis directed by the poly(A+) and poly(A−) EMCV RNA in PABP-depleted extract. The RNAs were used at a 10 μg/mL concentration; other conditions were same as described for panel F.