FIG. 2.
The variable region in the 3′ NTR enhances RNA replication. (A) Representative result of a transient-replication assay with luciferase replicons carrying given engineered restriction sites in the variable region of the 3′ NTR. Cells were lysed at 4, 24, 48, and 72 h posttransfection, and luciferase activities were measured and corrected for transfection efficiency as determined from the 4-h value (set at 100%). Bars, means of quadruplicate determinations and error ranges. (B) Transient replication of luciferase replicons lacking part or the complete variable region (Δvar-9401 and Δvar-9415, respectively). Transfected cells were analyzed as for panel A. (C) Effects of mutations in the variable region of selectable replicons on the number of Geneticin-resistant colonies. Huh-7 cells were transfected with neo replicons carrying the given mutations in the 3′ NTR, subjected to Geneticin selection, and, after about 3 weeks, fixed and stained. The CFU per microgram of RNA (mean ± error range) as determined by transfection of serial dilutions of a given in vitro transcript are given below each culture dish. Representative results obtained after transfection of 100 ng of each RNA are shown. Wt, wild-type replicon (rep5.1); GND and Δ5B, inactive replicons with a single amino acid substitution in motif C of the NS5B RdRp (for luciferase replicons) or a 10-amino-acid deletion in the same motif (for neo replicons), respectively.