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        Identification of HIV-1 Reverse Transcriptase Inhibitors Using a Scintillation Proximity Assay

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        The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) converts the viral single-stranded RNA into double-stranded DNA. The inhibition of reverse transcription in the viral life cycle has proven its efficacy as a clinically relevant antiviral target, but the appearance of resistance mutations remains a major cause of treatment failure and stresses the continuous need for new antiviral compounds. In this chapter, we describe an HIV-1 RT scintillation proximity assay (SPA) to identify inhibitors of the RT. The assay uses an RNA/DNA (poly(rA)/oligo(dT)) template/primer bound to SPA beads, which contain scintillant. Reverse transcriptase extends the primer by incorporating [3 H]dTTP and dTTP, which results in light production by the scintillant in the bead. Compounds that inhibit reverse transcriptase will prevent the incorporation of tritiated dTTP resulting in a reduction of emitted light compared to the untreated controls.
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