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

FIG. 6.

FIG. 6.

The IRES is dispensable for positive-strand RNA synthesis. (A) Cotranslation of helper mRNA and template RNAs. PV proteins were synthesized in reaction mixtures containing [35S]methionine, DNVR2, and either DJB1 RNA (lane 2), DNVR36 RNA (lane 3), or DNVR38 RNA (lane 4). Radiolabeled proteins fractionated by SDS-PAGE were detected by phosphorimaging. Arrows mark the mobility of frameshifted protein fragments from DJB1 and DNVR38 RNAs. (B) Positive-strand RNA synthesis. PV RNA was synthesized in reaction mixtures containing [α-32P]CTP by preinitiation RNA replication complexes containing either a DJB1 template (lanes 6 and 7), a DNVR36 template (lanes 8 and 9), or a DNVR38 template (lanes 10 and 11). Reactions were performed in the presence (lanes 7, 9, and 11) and absence (lanes 6, 8, and 10) of 2 mM guanidine HCl (GuHCl). Products from the reactions were fractionated by electrophoresis in a nondenaturing 1% agarose-TBE gel. Radiolabeled RNA was detected by phosphorimaging. An RNA ladder (lane 1), DNVR2 helper mRNA (lane 2), and positive-strand template RNAs were used as markers in the gel. Lanes: 3, DJB1 RNA; 4, DNVR36 RNA; 5, DNVR38 RNA. The mobilities of positive-strand, replicative-form (RF), and replicative-intermediate (RI) RNAs are indicated.