FIG. 8.

Initiation of negative-strand RNA synthesis does not necessarily precede CRE-dependent VPg uridylylation. (A) PV RNA2 and 3′ΔGUA₃ protein synthesis. [³⁵S]methionine-labeled proteins were separated by electrophoresis in an SDS-9 to 18% polyacrylamide gel. The mock-treated control lacked PV RNA. (B) Negative-strand RNA synthesis. PV RNA2 (lanes 1 and 2) and PV 3′ΔGUA₃ (lanes 3 and 4) RNA products labeled with 40 μCi of [α-³²P]UTP in 40-μl reaction mixtures (final concentration of 1.25 μM UTP [800 Ci/mmol UTP]) in the presence (+) or absence (−) of 2 mM guanidine HCl (GuHCl) were separated by electrophoresis in a 1% agarose-TBE gel. (C) VPgpUpU_OH synthesis. VPgpU_OH and VPgpUpU_OH products from PV RNA2 and PV 3′ΔGUA₃ were separated by electrophoresis in a Tris-Tricine gel. All gels were dried, and radiolabel was detected with a PhosphorImager. The mobility of VPgpUpU_OH is indicated. VPgpU_OH products migrate slightly faster than VPgpUpU_OH products in the gel.