FIG. 4.

Negative-strand RNA synthesis with a minimally sufficient RNA template. (A) Diagram of DNVR22 RNA. DNVR22 RNA possesses the 5′-terminal 123 nt (cloverleaf) and the 3′ NTR-poly(A) tail of PV. (B) Cotranslation of DNVR2 helper mRNA with PV template RNAs. Proteins were synthesized in reaction mixtures containing [³⁵S]methionine, DNVR2 RNA, and DNVR19 RNA (lane 1) or DNVR22 RNA (lane 2). (C) 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 DNVR19 (lanes 1 and 2) or DNVR22 (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 (GuHCl). Radiolabeled negative-strand RNAs were fractionated by electrophoresis in a denaturing methyl mercury hydroxide agarose gel and detected by phosphorimaging. The mobilities of DNVR19 and DNVR22 negative-strand RNAs are indicated.