FIG. 1.

Time course of protein synthesis and eIF4G cleavage in HeLa cells incubated with MBP-2A or MBP–PE-III+2A plus CELO virus. HeLa cells grown in 24-well dishes were incubated with column purification buffer (lanes 1, 5, 9, and 13), with 100 μg of MBP–β-Gal-α and 10 μl of CELO virus (lanes 2, 6, 10, and 14), with 100 μg of MBP-2A and 10 μl of CELO virus (lanes 3, 7, 11, and 15), or with 100 μg of MBP–PE-III+2A and 10 μl of CELO virus (lanes 4, 8, 12, and 16). [³⁵S]methionine was added to the medium 1 h before each time point, and cells were incubated for 1 h. Cells were harvested at 5 h (lanes 1 to 4), 10 h (lanes 5 to 8), 15 h (lanes 9 to 12), and 20 h (lanes 13 to 16). Cell extracts were separated by SDS-PAGE. (A) Western blot analysis with anti-eIF4G polyclonal antibodies. Intact eIF4G-1 and the amino-terminal (cp amino) and carboxy-terminal (cp carboxy) fragments of eIF4G are indicated. H, extract from mock-infected HeLa cells; P, extract from poliovirus-infected HeLa cells. (B) Labeled proteins were analyzed as described in Materials and Methods. The migration of some poliovirus proteins is indicated. Ac, cellular actin. (C) Cleavage of eIF4G-2 by hybrid proteins and CELO virus, determined by Western blot analysis using specific antibodies against human eIF4G-2. The intact protein and the cleavage products are shown. Lane 1, incubation with column purification buffer; lane 2, incubation with 100 μg of MBP–β-Gal-α and 10 μl of CELO virus; lane 3, 40 μg of MBP-2A and 10 μl of CELO virus; lane 4, 60 μg of MBP-2A and CELO virus. Cells were harvested 15 h after incubation, and the proteins were analyzed as described in Materials and Methods.