Quantifying HIV-1 RT Inhibitor Effects

The cross-linked miniRTIC, RT, and vRNA–tRNA used in ³²P activity assays were prepared as described above. miniRTIC (50 nM) was preincubated in 50 mM Tris-HCl, pH 8.0, 50 mM KCl, and 6 mM MgCl₂ in a 37 °C water bath for 5 min. For drug conditions, nevirapine (50 nM) and efavirenz (50 nM) were separately incubated with miniRTIC (50 nM) under the same conditions. Free vRNA–tRNA (50 nM) and RT (250 nM) was also preincubated under the same conditions. Incorporation reactions were initiated by adding a mixture of α-³²P-dCTP (40 nM) and dCTP (50 μM). Reactions were quenched at various time points between 5 s and 4 h with the addition of EDTA and SDS loading buffer. The reaction sets for each condition (free, x-link, x-link + nevirapine, x-link + efavirenz) were run on a 12% SDS-PAGE gel, dried, and exposed for 18 h on a phosphoimager screen (Molecular Dynamics) and each gel was individually scanned with a Storm 860 (Molecular Dynamics)^{12 (link),13 (link)}. Bands were quantified using ImageQuant. The intensity was normalized to the highest intensity for the individual time-course assays after background subtraction (set to 1). The miniRTIC and free vRNA–tRNA with RT were repeated five times. Nevirapine and efavirenz conditions were each repeated three times. Plotting and curve fitting was performed using GraphPad Prism8.