FIG. 7.

Time course of synthesis of HCV polypeptides in Krebs-2 S10. (A) HCV RNA (20 μg/ml) was translated in a 100-μl total reaction volume in either the absence (−) or presence (+) of CMMs, as indicated. At the time points indicated, aliquots (10 μl) of the translation samples were withdrawn, supplemented with the SDS sample buffer, and analyzed by SDS-15% PAGE and autoradiography. (B) HCV RNA was programmed into a CMM-supplemented reaction (100 μl) as described above. At the time points indicated, two aliquots (10 μl each) were withdrawn. One aliquot (p, pulse) was immediately supplemented with the SDS sample buffer, while another (ch, chase) was additionally incubated in the presence of cycloheximide (0.6 mM final concentration). The total incubation time for all chased samples was 180 min. HCV-specific proteins and the positions of the prestained protein markers are indicated. (C) Alkaline calf intestinal phosphatase (CIP) treatment of HCV RNA translation products. HCV RNA wastranslated in a 40-μl total volume reaction in the presence of CMMs for 3 h as described for panel A. After the adjustment of the MgCl₂ concentration to 10 mM, the reaction mixture was divided into two portions. One portion was incubated with CIP (40 U; New England BioLabs) for 30 min at 37°C, while another was incubated with the control buffer. The samples were analyzed by SDS-PAGE as described above. The position of dephosphorylated NS5A [NS5A (−P)] is indicated by an arrow.