FIG 3.
The 3B3T>K amino acid substitution drives trans-mediated precursor proteolysis. Plasmids expressing proteolytically inactive polyproteins with or without the 3B3T>K substitution (termed 3B3T>K, 3CC163A, and 3CC163A, respectively) were used to prime coupled [35S] labeled transcription/translation assays, followed by unlabeled amino acid chase. To a duplicate set of reactions 10 μM purified 3Cpro was added (+3C) immediately after chase. As controls, reactions were set up alongside the WT or 3B3T>K polyproteins without an inactivated 3Cpro. 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).