For the assay with human PBMCs, PHA stimulated cells from at least two pooled healthy were infected with HIV-1 strains in 96-well plates. Test drug dilutions, which were prepared at a 2X concentration in microtiter tubes and 100 μL of each concentration (nine total concentrations), were placed in appropriate wells using the standard format. 50 μL of a predetermined dilution of virus stock was placed in each test well (final MOI ≅ 0.1). The PBMC cultures were maintained for 7 days following infection at 37 oC, 5% CO2. After this period, cell-free supernatant samples were collected for analysis of reverse transcriptase (RT) activity. All assays were conducted in triplicates. For RT assay, a microtiter plate-based reverse transcriptase (RT) reaction was utilized (Buckheit et al., AIDS Research and Human Retroviruses 7:295–302, 1991). Tritiated thymidine triphosphate (3H-TTP, 80 Ci/mmol, NEN) is received in 1:1 dH2O: Ethanol at 1 mCi/mL. Poly rA:oligo dT template:primer (GE Healthcare) was prepared as a stock solution by combining 150 μL poly rA (20 mg/mL) with 0.5 mL oligo dT (20 units/mL) and 5.35 mL sterile dH2O followed by aliquoting (1.0 mL) and storage at—20°C. The RT reaction buffer was prepared fresh on a daily basis and consisted of 125 μL 1.0 M EGTA, 125 μL dH2O, 125 μL 20% Triton X100, 50 μL 1.0 M Tris (pH 7.4), 50 μL 1.0 M DTT, and 40 μL 1.0 M MgCl2. The final reaction mixture was prepared by combining 1 part 3H-TTP, 4 parts dH2O, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer. Ten microliters of this reaction mixture were placed in a round bottom microtiter plate and 15 μL of virus containing supernatant is added and mixed. The plate was incubated at 37°C for 60 mins. Following incubation, the reaction volume was spotted onto DE81 filter-mats (Wallac), washed 5 times for 5 mins each in a 5% sodium phosphate buffer or 2X SSC (Life Technologies). Next they were washed 2 times for 1 min each in distilled water, 2 times for 1 min each in 70% ethanol, and then dried. Incorporated radioactivity (counts per min, CPM) was quantified using standard liquid scintillation techniques. These PBMC analysis was conducted by fee to service through Southern Research Institute (Frederick, MD, USA). The IC50 were analyzed using GraphPad Prism (Version 9) and presented as means ± S.D. of the triplicates.

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