FIG. 1.
(A) Schematic presentation of the basic replicon constructs used in this study. The 5′ and 3′ NTRs (only shown for the selectable replicon) are given with their secondary structures (solid line, EMCV IRES; open bars, coding regions for neomycin phosphotransferase (neo) and firefly luciferase (Ffl-luc), as well as the HCV nonstructural proteins). ∗, positions of the three cell culture-adaptive mutations. (B) Potential structures of the 3′ NTR from references 7 and 29 with two stem-loops in the variable region (VSL1 and VSL2), the 83-nucleotide poly(U/UC) tract, and the three stem-loops in 3′X. The positions of the engineered recognition sequences of restriction enzymes StuI and BamHI as well as the authentic recognition site for NheI are indicated. To generate runoff transcripts with authentic 3′ ends, the two substitutions in SL3 (∗) were introduced. They result in a change of an A:U to a U:A base pairing required to generate a ScaI restriction site. The stop codon of the polyprotein ORF is boxed. Numbers refer to the nucleotide positions of the HCV Con1 isolate (EMBL database accession no. AJ238799).