FIG. 6.

Effect of tsO82 mutant virus on host cell protein synthesis and eIF4E phosphorylation. (A) HeLa cells were mock infected (M) or infected with tsO82 virus for the indicated times and then labeled with [³⁵S]methionine for 10 min. Lysates (10 μl) were electrophoresed on a 12% gel, a phosphorescence image of which is shown. Viral proteins are indicated to the right of the image. (B) Quantification of host protein synthesis inhibition following tsO82 virus or VSV infection. The rate of host protein synthesis was determined from experiments like that shown in panel A by quantitation of the radioactivity between the viral L and G bands, between the P and M bands, and in the region below the M band. Cells infected with wt VSV are represented by filled squares, and cells infected with tsO82 virus are represented by open squares. (C) Extracts from mock- and VSV-infected cells were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting with antibodies to eIF4E phosphorylated at serine 209 [eIF4E-P (Ser 209)]. (D) Quantification of eIF4E phosphorylation following VSV or tsO82 infection. The levels of eIF4E phosphorylation were determined by densitometry of phospho-eIF4E-specific Western blots and normalized to total eIF4E. Cells infected with wt VSV are represented by filled circles, and cells infected with tsO82 virus are represented by open circles. The data shown are averages ± standard deviations of three separate experiments.