FIG. 4.

Effects of actinomycin D and micrococcal nuclease on HCV replication. (A) HCV replication assay of lysates from transiently transfected Huh-7 cells with HCV subgenomic replicon RNA (BM4-5) and FCA4 cell lysates treated with micrococcal nuclease. Agarose gel electrophoresis of the products of a replication assay carried out as described in Materials and Methods with lysates prepared from Huh-7 cells (lane 1), Huh-7 cell lysates transiently transfected with BM4-5 RNA subgenome (lane 2), FCA4 cell lysates (lane 3), FCA4 lysates treated with Ca²⁺ and subsequently with EGTA in a mock reaction (lane 4), and FCA4 cell lysates treated with micrococcal nuclease and Ca²⁺ and subsequently with EGTA (lane 5) is shown. (B) Replication of HCV RNA is resistant to actinomycin D. Agarose gel electrophoresis of the products of a replication assay with Huh-7 cell lysates (lane 1), FCA4 cell lysates (lane 2), and FCA4 cell lysates in the presence of actinomycin D (Act. D) (lane 3) is shown. (C) Analysis of the products synthesized in the replication assay. The RNAs labeled in Huh-7 (lane 1) or FCA4 (lane 2) cell lysates were fractionated with 2 M LiCl as described in Materials and Methods. The insoluble and soluble fractions were analyzed by native agarose gel electrophoresis. Lanes 3 and 4, LiCl-soluble forms of the replication assay products; lanes 5 and 6, nucleic acid products that are insoluble in LiCl.