FIG 4.
The 3B3T>K amino acid substitution drives trans-mediated precursor proteolysis. Plasmids expressing proteolytically inactive versions of WT or 3B3T>K polyproteins (3CC163A and 3B3T>K3CC163A, respectively) were used to prime coupled [35S] labeled transcription/translation assays in the presence of 3AB1,2,3CproD, a proteolytically active precursor with all cleavage boundaries mutated to prevent self-proteolysis (+3AB123CproD). At regular intervals, samples were taken and reactions stopped by the addition of 2× Laemmli buffer. (A) Proteins were separated by SDS-PAGE and visualized by autoradiography. The relative proportion of uncleaved 3AB1,2,3CD (B) or 3CD (C) was quantified as a percentage of 3Dpol containing products (n = 2 ± SD).