HIV Reverse Transcriptase (RT) Assay

Determination of HIV RT activity was adapted from Willey et al. (21). In brief, collected culture supernatant (10 μl) was added to a cocktail containing poly (A) (GE Healthcare, Mickleton, NJ), oligo (dT) (USB, Cleveland, OH), MgCl₂, and ³²P dTTP (PerkinElmer, Boston, MA) and incubated for 20 h at 37°C. The cocktail (30 μl) was spotted onto DE81 paper, dried, and washed five times with 2 × saline-sodium citrate buffer and once with 95% ethanol. The filter paper was then air-dried. Radioactivity was counted in a liquid scintillation counter (PerkinElmer, Boston, PA).