FIG. 3.

NERT is inhibited by nucleoside analogs and nonnucleoside RT inhibitors. (A) NERT reaction mixtures containing 0.25 mU of virion RT activity were preincubated for 30 min with RT inhibitors (PAA, PFA, NVP, ddATP, and ddTTP) at the concentrations indicated (micromolar), or with dimethyl sulfoxide (DMSO) (solvent vehicle for NVP). Then 200 μM dNTP was added to each NERT reaction mixture except as indicated (panel A, -dNTP), and all the mixtures were incubated at 37°C for 90 min. The reactions were terminated, and the products were assayed for (−)ssDNA by PCR using primers P1 and P2 (Fig. 1). (B) RT inhibitors or DMSO was added to PCR mixtures containing 1,000 copies of proviral plasmid DNA at concentrations slightly higher than were carried over from any of the corresponding NERT assays. All PCR results were within the linear range of the assay (r² > 0.99). Experiments were performed at least three times, and typical results are shown.