FIGURE 3.

Leucine or methionine can be incorporated at the amino terminus when AUG is replaced by CUG. (A) Amino-terminal amino acid sequences of wt C protein and corresponding mutants, marked in red. (B) BHK cells were transfected with in vitro synthesized replicons rep C + luc (AUG), rep C + luc (CUG), or rep C + luc (GUG), all containing the R to V mutation in C. After 8 h, cultures were collected and extracted proteins were separated by SDS-PAGE, visualized by Coomassie blue staining, and then protein bands from the different samples were excised, trypsin-digested and analyzed by LC–MS/MS. The MS/MS spectra of amino-terminal peptides of C detected are displayed. (C) BHK cells were mock-transfected or transfected with in vitro synthesized replicons rep C (one AUG) or rep C (no AUGs). After 7 h, cells were treated or not with sodium arsenite (Ars) for 15 min and then protein synthesis was detected with [³⁵S] Met/Cys labeling for 45 min. Samples were immunoblotted with anti-C antibodies (western blot). Then, peroxidase activity was inactivated by heating the membrane at 120°C for 15 min before exposure to an X-ray film to detect radioactive signals ([³⁵S] Met/Cys.) (D) The proportion obtained by immunoblotting or radioactive labeling of C protein in cells transfected with both replicons was calculated and the radioactive signal was normalized to the amount of C by immunoblotting; the graph shows the percentage incorporation of [³⁵S] Met/Cys in C protein by rep C (no AUGs) relative to rep C (one AUG). The results are shown as mean ± SD of three experiments. Statistical significance in panel D was calculated compared to control using Student's t-test, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.