Quantifying HIV-1 mRNA in Resting CD4+ T Cells

Human primary PBMCs were obtained via peripheral phlebotomy as described above and resting CD4⁺ T cells were isolated using a magnetic bead negative-selection kit from StemCell Technologies according to the manufacturer’s instructions. Ex vivo cell culture assays with resting CD4⁺ T cells from HIV-1-infected aviremic participants were performed as previously described (38 (link), 39 (link)). Briefly, cells were cultured at a concentration of four million cells/ml of culture medium per condition. Cells were exposed to IL-15 (100 ng/ml), followed by the addition of benzotriazine analogues (100 μM), and then cultured for 48 h. Cells then underwent centrifugation, and cell-associated RNA was extracted. RNA isolation and purification were performed using TRIzol RNA isolation according to the manufacturer’s instructions. For the detection of HIV-1 mRNA transcripts, qPCR was performed on cell-associated RNA isolated from cultured cells, as described previously (38 (link), 39 (link)).