FIG. 5.

Effect of eIF4G cleavage on the inducible expression of luciferase. (A) Schematic representation of the protocol followed. HeLa clone X1/5 cells cultured in the presence of tetracycline (20 ng/ml) were incubated with purification buffer or with purified proteins in the presence of CELO virus for 15 h. Afterwards, cells were washed and tetracycline-free medium was added to induce luciferase gene expression (time zero). (B) At the indicated times postinduction (pi) (4, 8.5, and 12 h after tetracycline removal), luciferase activity (relative luciferase units [rlu]) was determined as described in Materials and Methods. (C) [³⁵S]methionine was added 1 h before each time point, the medium was incubated for 1 h, and protein synthesis was analyzed; HeLa clone X1/5 cells were incubated with purification buffer (lanes 5, 9, and 13), with 100 μg of MBP–β-gal-α and 10 μl of CELO virus (lanes 6, 10, and 14), with 100 μg of MBP-2A and 10 μl of CELO virus (lanes 7, 11, and 15), or with 100 μg of MBP–PE-III+2A and 10 μl of CELO virus (lanes 8, 12, and 16). pi, postinduction; P, extract from poliovirus-infected HeLa cells; Ac, actin.