Fig. 4.
Kinetics experiments reveal severely diminished ability of 2Apro to inhibit translation after polysomes form. (A) Immunoblot shows rapid cleavage of eIF4GI in HeLa translation lysates incubated with excess CVB3 2Apro. (B) Schematic depicting ribosome loading of capped/polyadenylated luciferase RNA after addition of RNA to translation lysates at timepoint zero. Luciferase enzymatic activity takes 8 min to appear, marking the time when fully loaded polysomes first appear on Luc RNA. Ribosome recycling cannot occur until after 8 min. (C) Effect of adding 2Apro to lysates at various times before or after Luc RNA. 2Apro was preincubated with lysate 5 min, or added at 0, 4 or 8 min after Luc RNA (depicted in panel B by yellow arrows). The graph shows the accumulation of LUC relative light units plotted as percentage of the translation in mock-treated control lysate. Continued efficient polysome translation after eIF4G cleavage is likely due to ribosome recycling since de novo initiation is blocked. (D) Effect of addition of 2Apro or 3Cpro to translation lysate at 11 min after RNA was added (arrow). 3Cpro inhibited translation more effectively than 2Apro when added late. Drastic translation inhibition requires both 2Apro and 3Cpro.