The poliovirus type 3-based in vitro uridylylation assay. (A) CRE-mediated uridylylation of VPg requires the presence of 3CDpro and 3Dpol. The assay was constituted as described in Materials and Methods, with the exclusion of individual components as indicated in the table. Activity was quantified by measuring 32P-UMP incorporation onto VPg using a Bio-Rad phosphorimager and is shown normalized to the activity observed in the presence of all three proteins. (B) Formation of VPg-pU(pU) requires the CRE template and 3CDpro. The production of uridylylated VPg is dependent upon the presence of a template containing an unpaired A triplet (CRE, poly[A] or the 3′NCR) and CDpro, whereas polyadenylated RNA templates (poly[A] and the 3′NCR) yield VPg-poly(U) in the presence or absence of 3CDpro (indicated above the column labels). The reaction products of the 3Dpol terminal transferase activity are indicated with an asterisk. (C) CRE-templated uridylylation of VPg requires the A triplet in the A1A2A3CA motif and a base-paired stem. VPg-pU(pU) was quantified by Bio-Rad phosphorimager and normalized to the levels observed with an unmodified CRE template (WT). The mutations present in the CRE RNA template are indicated above the autoradiogram, and relate to the numbering scheme shown in Figure 1A. The G19A/C22U mutations disrupt Stem 3 (Figs. 1A, 2B; Goodfellow et al. 2000).
