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. 2004 Feb;78(3):1393–1402. doi: 10.1128/JVI.78.3.1393-1402.2004

FIG. 1.

FIG. 1.

IRES stem-loops IV, V, and VI are dispensable for negative-strand RNA synthesis. (A) DNVR2 helper mRNA. DNVR2 is a chimeric HCV-PV helper mRNA. This mRNA possesses the 5′ NTR of HCV, an ORF encoding the PV replication proteins, and the 3′ NTR-poly(A) tail of PV. (B) PV template RNAs. Diagrams of DJB14 and DNVR10 RNAs. DJB14 RNA possesses a wild-type PV IRES, a small ORF encoding a COOH-terminal fragment of 3DPol (Δ3DPol), and the PV 3′ NTR-poly(A) tail. DNVR10 RNA possesses a deletion of IRES nt 220 to 742. (C) Cotranslation of DNVR2 helper mRNA with DJB14 and DNVR10 PV template RNAs. Proteins were synthesized in reaction mixtures containing [35S]methionine, DNVR2 RNA, and DJB14 RNA (lane 1) or DNVR10 RNA (lane 2). (D) Negative-strand RNA synthesis. Preinitiation RNA replication complexes formed during cotranslation reactions were assayed for the ability to synthesize negative-strand RNA as described in Materials and Methods. Preinitiation RNA replication complexes contained either DJB14 (lanes 1 and 2) or DNVR10 (lanes 3 and 4) RNA templates. Negative-strand RNA synthesis was assayed in the presence (lanes 1 and 3) and absence (lanes 2 and 4) of 2 mM guanidine HCl. Radiolabeled negative-strand RNAs fractionated by electrophoresis in a denaturing methyl mercury hydroxide agarose gel were detected by phosphorimaging. Mobilities of DJB14 and DNVR10 negative-strand RNAs are indicated.