FIG. 5.

Effect of protein synthesis inhibitors on positive-strand RNA synthesis. Normal RNA replication complexes were isolated from 50 μl of HeLa S10 translation-RNA replication reactions that were incubated at 30°C for 5 h in the absence of 2 mM guanidine HCl (method 5 in Materials and Methods). The RNA replication complexes were resuspended in reactions containing HeLa S10 extract and 15 μCi of [α-³²P]CTP. The individual reactions also contained 2 mM guanidine HCl (lanes 2 to 7), 200 μg of cycloheximide per ml (lane 3), 8 μg of anisomycin per ml (lane 4), 20 μg of diphtheria toxin plus 20 μg of NAD⁺ per ml (lane 5), 100 μg of puromycin per ml (lane 6), or 2 μg of ricin A chain per ml (lane 7). The reactions were incubated at 30°C for 90 min, and the labeled RNAs were separated by CH₃HgOH-agarose gel electrophoresis and visualized by autoradiography.