Fig. 4.

Recombinant p33 promotes plus-strand initiation on (−)RNA templates by the RdRp. (A) Schematic presentation of the ssDNA oligo/ssRNA template duplex used to program the TCV p88C RdRp preparation in vitro. The cPR promoter sequence used for plus-strand initiation by the TCV RdRp is shown with an empty arrowhead. (B) Representative denaturing gel of ³²P-labeled RNA product synthesized by TCV p88C RdRp in vitro in the presence of 0, 1 and 2 μg of purified recombinant p33 is shown. The level of RNA synthesis was compared to that of the RdRp activity obtained in the absence of ssDNA and p33 (100%). The samples were treated with S1 nuclease to exclude terminal transferase-based labeling of DI-72(−)RNA, which might be present in the affinity-purified TCV p88C or p33 preparations. Each experiment was repeated three times.