FIG. 5.
Kinetics and amounts of negative-strand RNA synthesis and VPgpUpUOH synthesis measured by pulse-labeling. (A) Negative-strand RNA synthesis. PIRCs containing PV RNA2 templates were incubated in the absence (lanes 1 to 4 and 6 to 9) or presence (lanes 5 and 10) of 2 mM guanidine HCl (GuHCl) in 40-μl reaction mixtures containing 1× S10 buffer along with 1 mM ATP, 250 μM GTP, 10 μM UTP, and endogenous CTP (lanes 1 to 5) or 200 μM 3′-dCTP (lanes 6 to 10). Forty microcuries of [α-32P]UTP was added to each 40-μl reaction mixture for 15-min periods of time as indicated. Reactions were terminated after the indicated pulse-labeling period, and RNA products of the reactions were separated by electrophoresis in a nondenaturing 1% agarose-TBE gel. Phosphorimaging revealed the incorporation of radiolabel into RF RNA (lanes 1 to 4) along with faint labeling of RNA products comigrating with PV RNA2 template [lanes 6 to 9, VPg-poly(U) 3′-dCMP]. (B) VPgpUpUOH synthesis. Reactions were performed in duplicate with those in panel A. VPgpUOH and VPgpUpUOH products were separated on a polyacrylamide-Tris-Tricine gel and detected by phosphorimaging. The mobility of VPgpUpUOH is indicated. VPgpUOH products migrate slightly faster than VPgpUpUOH products in the gel.