FIG. 3.
Determination of the minimal length of the poly(U/UC) tract required for RNA replication. (A) Result of a transient-replication assay using luciferase replicons that carry a homopolymeric uridine tract of 1, 6, 26, or 46 nucleotides. Wt and GND are as defined for Fig. 2. (B) Number of Geneticin-resistant colonies obtained after transfection of selectable replicons with the same modifications in the 3′ NTR as in panel A. Representative results obtained after transfection of 100 ng of in vitro transcript are shown. For further details see the legend to Fig. 2. (C) Sequence analysis of the poly(U/UC) tracts of replicons isolated from Huh-7 cells that had been transfected with U6 replicons and subjected to selection with Geneticin. From six independent colonies replicon RNA was amplified by RT-PCR and cloned, and two clones from each colony were sequenced. The sequence of only one clone of each colony is shown below the poly(U/UC) tracts of the wild-type replicon and the U6 mutant. Dashes, deletions; boldface, sequences of VSL2 and SL1.