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. 2003 Apr;77(8):4739–4750. doi: 10.1128/JVI.77.8.4739-4750.2003

FIG. 5.

FIG. 5.

Poliovirus negative-strand RNA synthesis does not require the CRE in cis or in trans. Poliovirus protein synthesis, negative-strand RNA synthesis, and VPg uridylylation were assayed as described in Materials and Methods. Preinitiation RNA replication complexes containing DJB14 RNA were prepared by cotranslating DNVR2 or DNVR21d (Fig. 5A) with DJB14 RNA as previously described (30). (A) Diagrams of DNVR2, DNVR21d, wild-type (WT) RNA2, and DJB14 RNAs. The * in the 2C coding region of DNVR2, DNVR21d, and wild-type RNA2 indicates the approximate location of the CRE. The CRE is not present within DJB14 RNA. (B) SDS-PAGE. Poliovirus proteins were synthesized in reaction mixtures containing [35S]methionine, DJB14 RNA, and DNVR2 RNA (duplicate samples in lanes 3 and 4) or mutant CRE DNVR21d RNA (duplicate samples in lanes 1 and 2). Radiolabeled proteins were fractionated by SDS-PAGE and detected by phosphorimaging. (C) Negative-strand RNA synthesis. Preinitiation RNA replication complexes formed during cotranslation reactions were assayed for their ability to synthesize negative-strand RNA when the helper virus possessed either a wild-type (lanes 1 and 2) or a mutant (lanes 3 and 4) CRE as indicated in the figure. Reactions were performed in the presence (lanes 1 and 3) and absence (lanes 2 and 4) of 2 mM guanidine HCl (GuHCl). Products from the reactions were fractionated by electrophoresis in a denaturing methylmercury hydroxide agarose gel. Radiolabled negative-strand RNA in the gel was detected by phosphorimaging. (D) VPg uridylylation. Preinitiation complexes formed during cotranslation were evaluated for their ability to synthesize VPgpUpUOH when the helper virus contained either a wild-type CRE (lanes 3 and 4) or a mutant CRE (lanes 5 and 6). Wild-type RNA was used as a positive control (lanes 1 and 2). Reactions were performed in the presence (lanes 1, 3, and 5) or the absence (lanes 2, 4, and 6) of 2 mM guanidine HCl. Products from the reactions were fractionated by polyacrylamide-Tris-Tricine gel electrophoresis and detected by phosphorimaging.