FIG. 5.

Inhibitory effect of 3′ dNTPs on RNA synthesis directed by HCV RCs. (A) Effects of 3′ dGTP, 3′ dCTP, and 3′ dATP on RNA synthesis. Standard reactions were performed at 30°C for 2 h in the presence of increasing concentrations (0 to 600 μM) of the indicated 3′dNTPs. The labeled RNA products were recovered by ethanol precipitation, separated on a 1% agarose gel, and visualized by autoradiography. Incorporation of [³³P]-UMP was measured by phosphorimaging, and the concentrations resulting in a 50% reduction in labeled RNA were determined (IC₅₀). (B) 3′ dGTP and 3′ dCTP prevent further elongation of the labeled ss RNA product. RNA products were pulse-labeled for 60 min in standard reactions containing 10 μCi of [α-³³P]-UTP. The labeled products were chased for 40 min in the presence of excess cold UTP and increasing concentrations of the indicated 3′dNTP. Lanes 1, products from the standard pulse-labeling reaction prior to the chase step; lanes 2 to 6, products obtained with addition of 3′ dGTP at concentrations ranging from 0 to 200 μM; lanes 7 to 11, products obtained with addition of 3′ dCTP at concentrations ranging from 0 to 200 μM. The positions of ss and ds RNA are indicated with arrows at the left. The small arrowhead indicate the positions of the extended products.