FIG. 7.
Effect of substitutions at residue M79 in PV protein 3A on VPg uridylylation and negative-strand RNA synthesis. Translation-replication extracts were programmed with full-length RNA transcripts as indicated. (A) Synthesis and processing of viral proteins were measured in reactions carried out in the presence of [35S]methionine, and labeled viral proteins were analyzed as described in the legend to Fig. 4. (B and C) VPg uridylylation (B) and negative-strand RNA synthesis (C) were measured in reactions containing preinitiation RNA replication complexes isolated from translation reactions containing 2 mM GuHCl and the indicated RNAs after sedimentation and resuspension in the absence of guanidine (lanes 1, 3, 4, 5, and 6). GuHCl was added to the samples in lane 2. Negative-strand RNA synthesis and VPg uridylylation assays were performed as described in Materials and Methods; the data were analyzed with a PhosphorImager and are expressed as a percentage of the product observed with wild-type PV RNA transcript. The major uridylylated VPg band in panel B is thought to be diuridylylated, but it has not been rigorously characterized.