FIG. 1.

Replication of the AGP_int-GP_ext constructs. (A) Schematic representation of the replication system. For a detailed description, see Results. NoP and PE refer, respectively, to the Northern blot riboprobe and to the primer extension oligonucleotide, both of positive polarity, complementary to the replicated RNA, depicted in diagram e. (B) Schematic representation of the AGP_int-GP_ext series constructs. 0, Partial scheme of the pSP65 plasmid harboring the basic copyback minireplicon pSV-DI-H4Δ96 flanked by the T7 RNA polymerase promoter (T7p) and the hepatitis delta virus ribozyme (Rbz). 1, T7 RNA transcript produced from 0, carrying at its 5′ and 3′ ends the complementary sequence of the antigenomic promoter (AGP^L_C) and the antigenomic promoter sequence (AGP^R), respectively. 2, Minireplicon T7 transcript harboring at its 3′ end the genomic promoter (GP) instead of the AGP. 3, Minireplicon T7 transcript harboring at its 3′ end an internal AGP sequence adjacent to an external GP. 4 to 9, Series of minireplicon T7 transcripts harboring at their 3′ ends a double promoter, as in construct 3, but separated by increasing intervening sequences as indicated (nt, nucleotides). Note that all the constructs harbor the same 5′ end sequence (AGP^L_C). (C) Northern blot analysis (see Materials and Methods) of the encapsidated minireplicon RNAs of negative polarity produced upon transfection of A549 cells with the various plasmids described for panel B in replication assays made in the absence of the C proteins (P/C^stop conditions; see Materials and Methods). Open and solid arrowheads, external and internal initiation, respectively. (D) As for panel C, but the replication assays were performed in HeLa cells in the presence of the C proteins (C^wt conditions; see Materials and Methods).