FIG. 3.

Dose-response effect of diphtheria toxin, ricin A chain, and puromycin on viral protein synthesis and RNA replication. Protein synthesis was measured in HeLa S10 translation-RNA replication reactions containing [³⁵S]methionine and diphtheria toxin, ricin A chain, or puromycin at the indicated concentrations. The reactions were incubated at 30°C as indicated in Materials and Methods. The incorporation of [³⁵S]methionine was determined after 30 min (A and B) or 5 h (C) of incubation and was plotted versus the concentration of each drug used. For RNA synthesis, preinitiation RNA replication complexes were resuspended in reaction mixtures containing fresh HeLa S10 extract (method 3; see Materials and Methods). The individual reactions also contained the indicated concentrations of diphtheria toxin, ricin A chain, or puromycin. The reactions were incubated at 30°C for 90 min, and the labeled RNA products were characterized by CH₃HgOH-agarose gel electrophoresis and visualized by autoradiography. The amount of labeled genome-length viral RNA present in each lane was quantitated by using a Molecular Dynamics PhosphorImager and was plotted versus the concentration of each protein synthesis inhibitor.