FIG. 5.

I432V rescues replication of a GT1b replicon from the lethal effect of P540A mutation. (A) Colony formation assay for Rep1b and derivative replicons. One microgram of the replicon RNA generated by in vitro transcription was electroporated into either Huh-7.5 or GS5 IFN-cured cells. G418-resistant cells were selected with 500 μg/ml of G418 for 3 weeks. Cell colonies were stained with crystal violet at the end of the selection. (B) Transient replication assay. Equivalent amounts (1 μg) of the RNA of the three replicons generated by in vitro transcription (left) were used in the electroporation of Huh-7.5 cells. Total RNA was then extracted 4 and 8 days postelectroporation and subjected to RT-PCR for the detection of HCV RNA. Primers that amplify the NS5B coding region of GT1b were used to detect HCV RNA, and the cDNA for RNA helicase A (RHA), a cellular gene, was amplified as a control for the input of total cellular RNA.