FIG. 3.

CRE-dependent VPg uridylylation is not required for negative-strand RNA synthesis. Poliovirus VPg uridylylation and negative-strand RNA synthesis were assayed as described in Materials and Methods. Poliovirus RNA replicons with wild-type (WT) or mutant CREs and two nonviral guanosine residues at their 5′ termini were used as illustrated in Fig. 1D. (A) VPg uridylylation. VPgpUpU_OH was synthesized in reaction mixtures containing [α-³²P]UTP by preinitiation RNA replication complexes containing wild-type poliovirus RNA templates (lanes 1 and 2) or mutant CRE RNA templates (lanes 3 and 4). 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 polyacrylamide-Tris-Tricine gel electrophoresis and detected by phosphorimaging. (B) Negative-strand RNA synthesis. Poliovirus RNA was synthesized in reaction mixtures containing [α-³²P]CTP by preinitiation RNA replication complexes containing wild-type poliovirus RNA templates (lanes 1 and 2) or mutant CRE RNA templates (lanes 3 and 4). Reactions were performed in the presence (lanes 1 and 3) and absence (lanes 2 and 4) of 2 mM guanidine HCl. Products from the reactions were fractionated by electrophoresis in a denaturing methylmercury hydroxide agarose gel. Radiolabeled negative-strand RNA in the gel was detected by phosphorimaging.